Scalable manufacture of therapeutic mesenchymal stromal cell products on customizable microcarriers in vertical wheel bioreactors that improve direct visualization, product harvest, and cost.

BACKGROUND AIMS: Human mesenchymal stromal cells (hMSCs) and their secreted products show great promise for treatment of musculoskeletal injury and inflammatory or immune diseases. However, the path to clinical utilization is hampered by donor-tissue variation and the inability to manufacture clinically relevant yields of cells or their products in a cost-effective manner. Previously we described a method to produce chemically and mechanically customizable gelatin methacryloyl (GelMA) microcarriers for culture of hMSCs. Herein, we demonstrate scalable GelMA microcarrier-mediated expansion of induced pluripotent stem cell (iPSC)-derived hMSCs (ihMSCs) in 500 mL and 3L vertical wheel bioreactors, offering several advantages over conventional microcarrier and monolayer-based expansion strategies. METHODS: Human mesenchymal stromal cells derived from induced pluripotent cells were cultured on custom-made spherical gelatin methacryloyl microcarriers in single-use vertical wheel bioreactors (PBS Biotech). Cell-laden microcarriers were visualized using confocal microscopy and elastic light scattering methodologies. Cells were assayed for viability and differentiation potential in vitro by standard methods. Osteogenic cell matrix derived from cells was tested in vitro for osteogenic healing using a rodent calvarial defect assay. Immune modulation was assayed with an in vivo peritonitis model using Zymozan A. RESULTS: The optical properties of GelMA microcarriers permit noninvasive visualization of cells with elastic light scattering modalities, and harvest of product is streamlined by microcarrier digestion. At volumes above 500 mL, the process is significantly more cost-effective than monolayer culture. Osteogenic cell matrix derived from ihMSCs expanded on GelMA microcarriers exhibited enhanced in vivo bone regenerative capacity when compared to bone morphogenic protein 2, and the ihMSCs exhibited superior immunosuppressive properties in vivo when compared to monolayer-generated ihMSCs. CONCLUSIONS: These results indicate that the cell expansion strategy described here represents a superior approach for efficient generation, monitoring and harvest of therapeutic MSCs and their products.

[Marjolin's ulcer associated with verrucous carcinoma in an immunocompromised patient]. / Úlcera de Marjolin asociada a carcinoma verrugoso en paciente inmunocomprometido.

Background: Marjolin's ulcer is the malignant degeneration of any chronic wound, with a latency period from tissue injury to variable malignant transformation that may occur up to 30 years later. Among the associated neoplasms, squamous cell carcinoma (SCC) is the predominant lineage in up to 71% of cases. The verrucous carcinoma variant has been estimated to have a low presentation, being described in the literature as 2% of all SCC and reported anecdotally in immunosuppressed patients, which justifies the objective of this publication. Clinical case: 65-year-old female patient with a history of being a carrier of human immunodeficiency virus (HIV) infection, who presented a verrucous carcinoma associated to a Marjolin ulcer secondary to herpes zoster and infection of soft tissues in the right leg, with a latency period of 10 years from the initial infectious process to histopathological confirmation. Conclusions: The finding of a verrucous carcinoma on a Marjolin ulcer has been little described in literature, with a lower incidence in the context of a patient with a history of being a carrier of HIV infection, finding 7 case reports, the oldest from 1998. For this reason, it is important to have diagnostic suspicion, to carry out an adequate study protocol and always making clinical-pathological correlation, in order to establish timely and individualized treatment.

Introducción: la úlcera de Marjolin es la degeneración maligna de cualquier herida crónica, con un periodo de latencia desde la lesión tisular a la transformación maligna variable que puede presentarse hasta 30 años después. De las neoplasias asociadas, el carcinoma espinocelular es la estirpe predominante hasta en 71% de los casos. La variante de carcinoma verrugoso se ha estimado con una presentación baja, pues ha sido descrito en la literatura como el 2% de todos los carcinomas espinocelulares y reportado de manera anecdótica en pacientes inmunosuprimidos, lo que justifica el objetivo de esta publicación. Caso clínico: mujer de 65 años con el antecedente de ser portadora de infección por virus de inmunodeficiencia humana (VIH), que presentó un carcinoma verrugoso asociado a una úlcera de Marjolin secundaria a herpes zóster e infección de tejidos blandos en pierna derecha, con un periodo de latencia de 10 años desde el proceso infeccioso inicial hasta la confirmación histopatológica. Conclusiones: el hallazgo de un carcinoma verrugoso asentado sobre una úlcera de Marjolin ha sido poco descrito en la literatura, con una menor incidencia en el contexto de un paciente con antecedente de ser portador de infección por VIH, ante lo cual encontramos 7 reportes de caso, el más antiguo de 1998. Por este motivo es importante contar con la sospecha diagnóstica, para poder hacer un protocolo de estudio adecuado y siempre haciendo correlación clínico-patológica, con la finalidad de instaurar un tratamiento oportuno e individualizado.

Volumetric imaging of human mesenchymal stem cells (hMSCs) for non-destructive quantification of 3D cell culture growth.

The adoption of cell-based therapies into the clinic will require tremendous large-scale expansion to satisfy future demand, and bioreactor-microcarrier cultures are best suited to meet this challenge. The use of spherical microcarriers, however, precludes in-process visualization and monitoring of cell number, morphology, and culture health. The development of novel expansion methods also motivates the advancement of analytical methods used to characterize these microcarrier cultures. A robust optical imaging and image-analysis assay to non-destructively quantify cell number and cell volume was developed. This method preserves 3D cell morphology and does not require membrane lysing, cellular detachment, or exogenous labeling. Complex cellular networks formed in microcarrier aggregates were imaged and analyzed in toto. Direct cell enumeration of large aggregates was performed in toto for the first time. This assay was successfully applied to monitor cellular growth of mesenchymal stem cells attached to spherical hydrogel microcarriers over time. Elastic scattering and fluorescence lightsheet microscopy were used to quantify cell volume and cell number at varying spatial scales. The presented study motivates the development of on-line optical imaging and image analysis systems for robust, automated, and non-destructive monitoring of bioreactor-microcarrier cell cultures.

Label-free optical imaging of cell function and collagen structure for cell-based therapies.

Cell-based therapies harness functional cells or tissues to mediate healing and treat disease. Assessment of cellular therapeutics requires methods that are non-destructive to ensure therapies remain viable and uncontaminated for use in patients. Optical imaging of endogenous collagen, by second-harmonic generation, and the metabolic coenzymes NADH and FAD, by autofluorescence microscopy, provides tissue structure and cellular information. Here, we review applications of label-free nonlinear optical imaging of cellular metabolism and collagen second-harmonic generation for assessing cell-based therapies. Additionally, we discuss the potential of label-free imaging for quality control of cell-based therapies, as well as the current limitations and potential future directions of label-free imaging technologies.

Quantifiable Intravital Light Sheet Microscopy.

Live imaging of zebrafish embryos that maintains normal development can be difficult to achieve due to a combination of sample mounting, immobilization, and phototoxicity issues that, once overcome, often still results in image quality sufficiently poor that computer-aided analysis or even manual analysis is not possible. Here, we describe our mounting strategy for imaging the zebrafish midbrain-hindbrain boundary (MHB) with light sheet fluorescence microscopy (LSFM) and pilot experiments to create a study-specific set of parameters for semiautomatically tracking cellular movements in the embryonic midbrain primordium during zebrafish segmentation.

A scalable system for generation of mesenchymal stem cells derived from induced pluripotent cells employing bioreactors and degradable microcarriers.

Human mesenchymal stem cells (hMSCs) are effective in treating disorders resulting from an inflammatory or heightened immune response. The hMSCs derived from induced pluripotent stem cells (ihMSCs) share the characteristics of tissue derived hMSCs but lack challenges associated with limited tissue sources and donor variation. To meet the expected future demand for ihMSCs, there is a need to develop scalable methods for their production at clinical yields while retaining immunomodulatory efficacy. Herein, we describe a platform for the scalable expansion and rapid harvest of ihMSCs with robust immunomodulatory activity using degradable gelatin methacryloyl (GelMA) microcarriers. GelMA microcarriers were rapidly and reproducibly fabricated using a custom microfluidic step emulsification device at relatively low cost. Using vertical wheel bioreactors, 8.8 to 16.3-fold expansion of ihMSCs was achieved over 8 days. Complete recovery by 5-minute digestion of the microcarriers with standard cell dissociation reagents resulted in >95% viability. The ihMSCs matched or exceeded immunomodulatory potential in vitro when compared with ihMSCs expanded on monolayers. This is the first description of a robust, scalable, and cost-effective method for generation of immunomodulatory ihMSCs, representing a significant contribution to their translational potential.

Enhanced detection of oral dysplasia by structured illumination fluorescence lifetime imaging microscopy.

We demonstrate that structured illumination microscopy has the potential to enhance fluorescence lifetime imaging microscopy (FLIM) as an early detection method for oral squamous cell carcinoma. FLIM can be used to monitor or detect changes in the fluorescence lifetime of metabolic cofactors (e.g. NADH and FAD) associated with the onset of carcinogenesis. However, out of focus fluorescence often interferes with this lifetime measurement. Structured illumination fluorescence lifetime imaging (SI-FLIM) addresses this by providing depth-resolved lifetime measurements, and applied to oral mucosa, can localize the collected signal to the epithelium. In this study, the hamster model of oral carcinogenesis was used to evaluate SI-FLIM in premalignant and malignant oral mucosa. Cheek pouches were imaged in vivo and correlated to histopathological diagnoses. The potential of NADH fluorescence signal and lifetime, as measured by widefield FLIM and SI-FLIM, to differentiate dysplasia (pre-malignancy) from normal tissue was evaluated. ROC analysis was carried out with the task of discriminating between normal tissue and mild dysplasia, when changes in fluorescence characteristics are localized to the epithelium only. The results demonstrate that SI-FLIM (AUC = 0.83) is a significantly better (p-value = 0.031) marker for mild dysplasia when compared to widefield FLIM (AUC = 0.63).

AbobotulinumtoxinA for Equinus Foot Deformity in Cerebral Palsy: A Randomized Controlled Trial.

BACKGROUND: Although botulinum toxin is a well-established treatment of focal spasticity in cerebral palsy, most trials have been small, and few have simultaneously assessed measures of muscle tone and clinical benefit. METHODS: Global, randomized, controlled study to assess the efficacy and safety of abobotulinumtoxinA versus placebo in cerebral palsy children with dynamic equinus foot deformity. Patients were randomized (1:1:1) to abobotulinumtoxinA 10 U/kg/leg, 15 U/kg/leg, or placebo injections into the gastrocnemius-soleus complex (1 or both legs injected). In the primary hierarchical analysis, demonstration of benefit for each dose required superiority to placebo on the primary (change in Modified Ashworth Scale from baseline to week 4) and first key secondary (Physician's Global Assessment at week 4) end points. RESULTS: Two hundred and forty-one patients were randomized, and 226 completed the study; the intention to treat population included 235 patients (98%). At week 4, Modified Ashworth Scale scores significantly improved with abobotulinumtoxinA; mean (95% confidence interval) treatment differences versus placebo were -0.49 (-0.75 to -0.23; P = .0002) for 15 U/kg/leg and -0.38 (-0.64 to -0.13; P = .003) for 10 U/kg/leg. The Physician's Global Assessment treatment differences versus placebo of 0.77 (0.45 to 1.10) for 15 U/kg/leg and 0.82 (0.50 to 1.14) for 10 U/kg/leg were also significant (both Ps < .0001). The most common treatment-related adverse event was muscular weakness (10 U/Kg/leg = 2; placebo = 1). CONCLUSIONS: AbobotulinumtoxinA improves muscle tone in children with dynamic equinus resulting in an improved overall clinical impression and is well tolerated.

Goma de mascar con efecto anticaries / Bubble gum with anti caries effect

Con el propósito de impulsar una nueva línea de investigación y de comprobar el efecto anticariogenénico de un producto natural con poder antimicrobiano comprobado, se desarrolló un estudio piloto cuasi experimental en jóvenes de 18 25 años matriculados en los tres últimos semestres del Programa de Odontología de la Universidad del Valle a un subgrupo de los cuales se les suministró el producto incorporado a una gomas de mascar comercial. El estudio fue aprobado por el Comité de Ética en Humanos de la Facultad de Salud de la Universidad del Valle que lo catalogó como ®sin riesgo¼ de acuerdo a la resolución 8430 de 1.993 emitida por el Ministerio de Salud de Colombia. En el grupo experimental los resultados del estudio mostraron una reducción significativa en el contenido de Streptococo Mutans en la saliva recolectada después del uso de goma de mascar comercial adicionada con propoleo. El estrptococo Mutans es la bacteria que genera el mayor riesgo de desarrollar caries dental Se considera necesario realizar un estudio similar que involucre grupos mayores de población con el fin de validar el resultado obtenido.

Factores de ajuste para producción de leche en bovinos Holstein colombiano / Adjustment factors for milk yield in colombian holstein cattle

El objetivo de este trabajo fue estimar factores multiplicativos de ajuste de la producción de leche a 305 días de lactación (PC305) y a edad adulta (PCEA), para rebaños de la raza Holstein colombiano.Fueron utilizadas 116.818 lactaciones de 48.707 vacas, provenientes de los archivos de la Asociación Holstein de Colombia. Los análisis estadísticos fueron realizados mediante la utilización de modeloslineales mixtos usando un modelo animal y el método de máxima verosimilitud restricta (REML). Para producción de leche a 305 días los efectos fijos considerados en el modelo fueron: grupo contemporáneo(rebaño-año), grupo genético de la vaca, mes de parto, número de partos y clases de duración de la lactación en dos grupos de edad de la vaca (<36 y ³ 36 meses) y la covarible edad de la vaca al parto (efecto lineal y cuadrático). Para producción de leche a edad adulta los efectos fijos considerados en el modelo fueron: grupo contemporáneo (rebaño-año), grupo genético de la vaca y clases de edad, número de partos y época de parto. Los efectos aleatorios para las dos variables fueron: efecto genético aditivo, ambiente permanente y residuo. Posteriormente fueron realizados análisis de variancia entre producciones de leche corregidas y no corregidas para determinar la efectividad de la aplicación de losfactores, indicando que la aplicación de los factores de corrección si ajustaron eficientemente producciones de leche para los efectos de duración de la lactación, edad de la vaca, número de partos y época de parto. La mayor producción de leche fue alcanzada por vacas de tres o más partos con edad de 76 a 83 meses de edad y partos ocurridos entre agosto y noviembre.

Aplicación de motores de traccion trifasicos en locomotoras Diesel Electricas de ENFE

El objetivo del presente trabajo es determinar la posibilidad de implementar mejoras en el sistema de tracción cambiando el sistema actual(motores de corriente contínua) por motores de corriente alterna trifasicos alimentados por medio de inversores y controlados por modulacion de ancho de pulso, de manera tal que se mejore la habilidad de transporte locomotor haciendolo mas competitivo. Para este estudio se realizo la simulacion del motor de induccion trifasico jaula de ardilla, por medio de un programa en turbopascal, aplicando la modulacion de ancho de pulso como tecnica de control y relaciones voltaje-frecuencia a momento constante y potencia constante como criterios de control. Teniendo como premisa mantener las caracteristicas de generacion: potencia constante, se realiza la simulacion para obtener las caracteristicas de transporte de carga.Se concluye que el sistema propuesto presenta caracteristicas de transporte con ventajas sobre el sistema antiguo, como ser : la habilidad de transporte de carga a plena potencia y bajas velocidades, buena regulacion de velocidad. Otras ventajas son : la factibilidad en el mantenimiento, disponibilidad de partes en el mercado local.