Production of dendritic cell vaccines using different methods with equivalent results: Implications for emerging centers

Introduction Dendritic cell (DC) vaccines have demonstrated good efficacy in preventing relapse and in increasing survival of patients affected by a variety of both solid and hematological tumors. Most protocols used to generate these cells involve the automated separation of peripheral blood monocytes from patients. This approach requires specialized equipment, which elevates the cost of this type of therapy, potentially limiting the widespread access to patients. Method: In this study, we compare the yield and quality of dendritic cells generated from monocytes and isolated by an automated method or by manual methods using gradient centrifugation. Results The results demonstrate the equivalence of the 3 methods in relation to the yield and final quality of the product, however with considerable differences between the costs of these procedures. In addition, this study also demonstrates the feasibility of the antigenic pulse with autologous tumor cell lysates, constituting a source of antigens, not only easily obtained and manipulated, but also specific to the patient's tumor. Conclusion These findings may have important implications for emerging centers interested in using this medical approach and potentially increase the access of a greater number of patients to this therapeutic option.

Novas formulações de Anexina A1 como estratégia terapêutica para a colite experimental / New formulations of Annexin A1 as a therapeutic strategy to experimental colitis

A Anexina A1 (AnxA1) é uma proteína de 37 kDa que controla o desenvolvimento da reação inflamatória inata, e favorece a eferocitose e o reparo tecidual. Em doenças inflamatórias intestinais (DIIs), tanto a AnxA1 endógena, como a sintética e o peptídeo sintético mimético ao N-terminal da proteína (Ac2-26) inibem o desenvolvimento de doença e induzem a cicatrização. O presente projeto teve o objetivo de obter novas formulações para carrear a AnxA1 recombinante (rAnxA1) ou o Ac2-26 e testar suas eficácias no modelo de colite experimental induzida pelo dextram sulfato de sódio (DSS, 0-6 dias) em camundongos C57Bl/6 machos. A rAnxA1 foi funcionalizada em nanocápsulas de núcleo lipídico de parede múltipla (MLNC) pela ligação Zn2+, com alta eficência de incorporação (92%) e adminsitrada pelas vias oral, intravenosa ou intraperitoneal durante a fase latente da doença (6º-9º dia). Somente o tratamento intraperitoneal com MLNC-AnxA1 (12,5 µg/mL) reduziu significativamente os sinais clínicos da doença, restaurou a integridade da estrutura colônica e a proliferação celular, bem como aumentou expressão de junções celulares da barreira intestinal. Ainda, MLNC-AnxA1 induziu a polarização de macrófagos para o fenótipo M2 in vivo no tecido inflamado e in vitro após estímulo com lipopolissacarídeos (LPS) bacteriano. Na tentativa de obter uma formulação terapêutica com atividade por vial oral, o peptídeo Ac2-26 foi incorporado em sílica mesoporosa ordenada SBA-15 e revestidos com Eudragit® L30-D55. A incorporação do peptídeo foi efetiva (88%) e a administração oral de Eudragit-SBA15-Ac2-26 (6º-9º dia; 200 µg/camundongo; 8 mg/kg) reduziu significativamente os sintomas clínicos e inflamação. De fato, ensaios de PET-SCAN mostraram que o SBA-15 permaneceu no intestino por até 16 horas após a administração e promoveu a liberação do peptídeo no intestino inflamado. Em cultura celular de epitélio (Caco-2), Eudragit-SBA15-Ac2-26 favoreceu a internalização de Ac2-26. Em conjunto, as duas estratégias expermentais de entrega do rAnxA1 ou Ac2-26 foram eficientes e os resultados obtidos sugerem que mais estudos devem ser realizados para a confirmação das estratégias de tratamento. Com o intuito de buscar ferramentas para ampliar estes estudos, durante estágio BEPE foram realizados estudos em cultura de células epiteliais baseado em células-tronco adultas diferenciadas in vitro. Os resultados mostraram que rAnxA1 ou Ac2-26 protegeram a integridade epitelial após desafio com LPS, pela regulação positiva da expressão das junções oclusivas e aderentes e redução da expressão de claudina-2, responsável pelo aumento da permeabilidade intercelular; pela modulação negativa decitocinas pró-inflamatórias CXCL-1 e MCP-1, e positiva de citocina antiinflamatória IL-10. Desta forma, padronizamos um novo modelo de cultura celular ainda não testada para a AnxA1 ou Ac2-26, que poderá ser empregada para desvendar os mecanismos da MLNC-AnxA1 e do Eudragit-SBA15-Ac2-26

Annexin Al (AnxA1) is a 37 kDa protein that controls the development of the innate inflammatory reaction, and favors efferocytosis and tissue repair. In inflammatory bowel diseases (IBDs), both endogenous and synthetic AnxA1 and the synthetic peptide mimetic to the N-terminal of the protein (Ac2-26) inhibit the development of disease and induce healing. The present project aimed to obtain new formulations to carry recombinant AnxA1 (rAnxA1) or Ac2-26 and test their efficacy in the experimental colitis model induced by dextram sodium sulfate (DSS, 0-6 days) in C57Bl/6 mice. rAnxA1 was functionalized into multiwall lipid core nanocapsules (MLNC) by Zn2+ binding, with high incorporation efficiency (92%) and administered orally, intravenously or intraperitoneally during the latent phase of the disease (6º-9º day). Only intraperitoneal treatment with MLNC-AnxA1 (12.5 µg/mL) significantly reduced the clinical signs of the disease, restored the integrity of the colonic structure and cell proliferation, as well as increased the expression of intestinal barrier cell junctions. Furthermore, MLNC-AnxA1 induced macrophage polarization to the M2 phenotype in vivo in inflamed tissue and in vitro after stimulation with bacterial lipopolysaccharides (LPS). In an attempt to obtain a therapeutic formulation with oral activity, the Ac2-26 peptide was incorporated into ordered mesoporous silica SBA-15 and coated with Eudragit® L30-D55. Peptide incorporation was effective (88%) and oral administration of Eudragit-SBA15-Ac2-26 (6º-9º day; 200 µg/mice; 8 mg/kg) significantly reduced clinical symptoms and inflammation. PET-SCAN assays showed SBA-15 remained in the intestine for up to 16 hours after administration and promoted the release of the peptide in the inflamed intestine. In epithelial cell culture (Caco-2), SBA15-Ac2-26 favored the internalization of Ac2-26. Taken together, the two experimental delivery strategies for rAnxA1 or Ac2-26 were efficient and the results obtained suggest that more studies should be carried out to confirm the treatment strategies. In order to seek tools to expand these studies, during the BEPE internship, studies were carried out in epithelial cell cultures based on adult stem cells differentiated in vitro. The results showed rAnxA1 or Ac2-26 protected epithelial integrity after challenge with LPS, by upregulating the expression of tight and adherens junctions and reducing the expression of claudin-2, responsible for increasing intercellular permeability; by negative modulation of pro-inflammatory cytokines CXCL-1 and MCP-1, and positive modulation of anti-inflammatory cytokine IL-10. In this way, we standardized a new cell culture model that has not yet been tested for AnxA1 or Ac2-26, which could be used to unravel the mechanisms of MLNC-AnxA1 and Eudragit-SBA15-Ac2-26

Interação de cepas de Lactobacillus crispatus com Streptococcus agalactiae em culturas celulares mimetizando o ambiente vaginal / Interaction between Lactobacillus crispatus and Streptococcus agalactiae strains in cell cultures mimicking the vaginal environment

A microbiota vaginal é dominada pelo gênero Lactobacillus, mantendo um pH ácido crucial para a saúde. Alterações hormonais e a menopausa podem impactar essa microbiota. Estreptococos do Grupo B (EGB) são associados a infecções neonatais e podem colonizar a microbiota vaginal. A interação entre Lactobacillus e EGB é complexa, com resultados conflitantes em estudos in vitro e in vivo. O uso de probióticos contendo Lactobacillus pode ter benefícios, como alterar a positividade para EGB em um grupo que recebeu o tratamento. Contudo a replicabilidade deste resultado é limitada, e os mecanismos envolvidos nessa interação ainda são pouco elucidados. Portanto o objetivo desse estudo foi caracterizar cepas de EGB, avaliar sua interação com L. crispatus em diversas condições. Neste estudo foram selecionadas seis cepas, pertencentes aos sorotipos Ia, II, III e V. Essas cepas foram caracterizadas de acordo com o sorotipo capsular, presença de genes de virulência (hialuronidase, ß-citolisina/hemolisina, e ilhas de pili 1, 2a e 2b), resistência a antimicrobianos (penicilina, cefepima, vancomicina, eritromicina e clindamicina), curva de crescimento, Restriction Fragment Length Polymorphism (RFLP) e Multi-Locus Sequence Typing (MLST). O resultado de MLST identificou os sequence types ST1, ST23 ST28 e ST182, e os clonal complexes CC1, CC19 e CC23. Cepas possuem a maior parte dos genes de virulência testados. Somente uma cepa apresentou alguma resistência, no caso à eritromicina e clindamicina. Resultados que estão de acordo com a literatura como os principais descritos em isolados clínicos. Os ensaios de interação realizados foram: crescimento em co-cultura, formação de biofilme, ensaios de adesão e exclusão em cultura celular e por fim, análise do perfil proteico. Alguns efeitos de L. crispatus no EGB observados foram aumento na formação de biofilme de EGB e diminuição da sua proliferação quando em co-cultura. Contudo esses resultados variaram de acordo com a cepa de EGB. Nos ensaios de competição em cultura celular, a presença de L. crispatus levou à diminuição da adesão em uma cepa pertencente ao sorotipo III, e aumento de adesão em uma cepa pertencente ao sorotipo V. Quando a cultura de células recebeu tratamento prévio de L. crispatus, nos ensaios de exclusão, houve menor adesão de EGB. Contudo todos os efeitos observados são cepa-dependente; não foram encontradas associações entre o comportamento das cepas e seu sorotipo capsular ou fenótipo MLST. Estudos futuros podem elucidar as respostas ativadas na presença de microrganismos comensais

The vaginal microbiota is dominated by the genus Lactobacillus, which helps to maintain a crucial acidic pH for health. Hormonal changes and menopause can impact this microbiota. Group B Streptococcus (GBS) are associated with neonatal infections and can colonize the vaginal microbiota. The interaction between Lactobacillus and GBS is complex, with conflicting results in in vitro and in vivo studies. The use of probiotics containing Lactobacillus may have benefits, such as altering GBS positivity in a treated group. However, the replicability of this result is limited, and the mechanisms involved in this interaction are still poorly understood. The use of probiotics containing Lactobacillus may have some benefits, but further research is needed. These strains were characterized based on capsular serotype, presence of virulence genes (hyaluronidase, ß-cytolysin/hemolysin, and pili islands 1, 2a, and 2b), antimicrobial resistance (penicillin, cefepime, vancomycin, erythromycin, and clindamycin), growth curve, Restriction Fragment Length Polymorphism (RFLP), and Multi-Locus Sequence Typing (MLST) Therefore, the aim of this study was to characterize GBS strains, evaluate their interaction with L. crispatus under various conditions. Six strains were selected for this study, belonging to serotypes Ia, II, III, and V. MLST results identified sequence types ST1, ST23, ST28, and ST182, and clonal complexes CC1, CC19, and CC23. Most strains possessed the tested virulence genes, with only one strain showing resistance to erythromycin and clindamycin. These results are in line with the literature. Interaction assays included co-culture growth, biofilm formation, adhesion and exclusion assays in cell culture, and, finally, protein profile analysis. Some observed effects of L. crispatus on GBS included an increase in GBS biofilm formation and a decrease in GBS proliferation during co-culture. However, these results varied according to the GBS strain. GBS interaction with L. crispatus resulted in increased biofilm formation and decreased proliferation in co-culture. In cell culture competition assays, the presence of L. crispatus led to decreased adhesion in one GBS strain belonging to serotype III and increased adhesion in one strain belonging to serotype V. When cell culture received pre-treatment with L. crispatus, exclusion assays showed lower GBS adhesion. However, all observed effects are strain-dependent; no associations were found between strain behavior and capsular serotype or MLST phenotype. Future studies may elucidate the responses activated in the presence of commensal microorganisms

Adult human neural cells in culture following traumatic brain injury

Objective: The present study aims to evaluate the viability of adult human neural cells in culture obtained from traumatized brain tissues collected in emergency surgery procedures. Methods: Exploratory, descriptive, quantitative and cross-sectional study evaluating samples obtained from patients who underwent traumatic brain injury with extrusion of brain tissue submitted to cell culture in a standardized medium, being preserved during 168h. After observation under phase contrast microscopy and immunohistochemical processing for neuronal (MAP-2) and glial (GFAP) markers, morphometric parameters of neural cells (cell body area, dendritic field length and fractal dimension) were evaluated using ImageJ software, with data obtained after 24, 72 and 168h being compared using non-parametric Kruskal Wallis test, followed by Dunn's post hoc test. Results: The explant of the nervous tissue revealed a consolidated pattern of cell migration into the culture medium. Cell proliferation, upon reaching confluence, presented an aspect of cellular distribution juxtaposed along the culture medium at all time points analyzed. Both neurons and glial cells remained viable after 168h in culture, with their morphologies not varying significantly throughout the time points evaluated. Immunohistochemistry for MAP-2 showed a relatively well-preserved cytoskeletal organization. GFAP immunoreactivity revealed activated astrocytes especially at the later time point. Conclusions: Our results point out the viability of cell culture from traumatized human nervous tissue, opening up perspectives for the use of substances of natural origin that may contribute neuroprotectively to neuronal maintenance in culture, allowing future translational approach.

Estudio de biocompatibilidad de matrices poliméricas combinadas, responsivas al pH, con aplicación en Ingeniería de Tejido Óseo / Biocompatibility study of combined pH-responsive polymeric matrices with application in Bone Tissue Engineering

El presente trabajo muestra la obtención de un material a partir de un polímero sintético (TerP) y otro natural, mediante entrecruzamiento físico y su caracterización fisicoquímica y biológica, con el fin de emplearlos para regeneración de tejido óseo. Las membranas fueron obtenidas por la técnica de evaporación del solvente y caracterizadas por espectroscopia FTIR, ensayos de hinchamiento, medidas de ángulo de contacto y microscopia electrónica de barrido (SEM). Se encontró que la compatibilidad entre los polímeros que la constituyen es estable a pH fisiológico y que, al incorporar mayor cantidad del TerP a la matriz, esta se vuelve más hidrofóbica y porosa. Además, teniendo en cuenta la aplicación prevista para dichos materiales, se realizaron estudios de biocompatibilidad y citotoxicidad con células progenitoras de médula ósea (CPMO) y células RAW264.7, respectivamente. Se evaluó la proliferación celular, la producción y liberación de óxido nítrico (NO) al medio de cultivo durante 24 y 48 horas y la expresión de citoquinas proinflamatorias IL-1ß y TNF-α de las células crecidas sobre los biomateriales variando la cantidad del polímero sintético. Se encontró mayor proliferación celular y menor producción de NO sobre las matrices que contienen menos proporción del TerP, además de poseer una mejor biocompatibilidad. Los resultados de este estudio muestran que el terpolímero obtenido y su combinación con un polímero natural es una estrategia muy interesante para obtener un biomaterial con posibles aplicaciones en medicina regenerativa y que podría extenderse a otros sistemas estructuralmente relacionados. (AU)

In the present work, the preparation of a biomaterial from a synthetic terpolymer (TerP) and a natural polymer, physically crosslinked, is shown. In order to evaluate the new material for bone tissue regeneration, physicochemical and biological characterizations were performed. The membranes were obtained by solvent casting and characterized using FTIR spectroscopy, swelling tests, contact angle measurements, and scanning electron microscopy (SEM). It was found that the compatibility between the polymers is stable at physiological pH and the incorporation of a higher amount of TerP into the matrix increases hydrophobicity and porosity.Furthermore, considering the intended application of these materials, studies of biocompatibility and cytotoxicity were conducted with Bone Marrow Progenitor Cells (BMPCs) and RAW264.7 cells, respectively. Cell proliferation, NO production and release into the culture medium for 24 and 48 hours, and proinflammatory cytokine expression of IL-1ß and TNF-α from cells grown on the biomaterials while varying the amount of the synthetic polymer were evaluated. Greater cell proliferation and lower NO production were found on matrices containing a lower proportion of TerP, in addition to better biocompatibility. The results of this study demonstrate that the obtained terpolymer and its combination with a natural polymer is a highly interesting strategy for biomaterial preparation with potential applications in regenerative medicine. This approach could be extended to other structurally related systems. (AU)

Desarrollo y validación de un método de suspensión basado en células Vero para la detección del virus Zika / Development and validation of a vero cell-based suspension method for the detection of Zika virus

Objetivo . Desarrollar y validar un método de suspensión celular utilizando células Vero 76 para el cultivo del virus Zika (ZIKV) basado en la infección de células recién sembradas no adheridas. Material y métodos . Se utilizaron tres multiplicidades de infección diferentes del ZIKV para desarrollar y comparar este novedoso método con el método estándar de monocapa de células confluentes. Además, validamos preliminarmente el método de suspensión utilizando muestras clínicas caracterizadas como positivas o negativas para el ZIKV. El método estándar de monocapa se utilizó como método de referencia, y el aislamiento viral se confirmó mediante un RT-PCR específico del ZIKV. Se estimó la sensibilidad e intervalos de confianza del 95% para el método de suspensión. Asimismo, se realizó una comparación técnica del método de suspensión contra el método de monocapa. Resultados . Nuestros hallazgos sugieren que tanto la carga viral como la replicación del ZIKV fueron comparables entre los métodos de infección en monocapa y en suspensión. Aunque ambos métodos fueron adecuados para cultivar y aislar el ZIKV, el método de suspensión se caracterizó por ser más fácil, barato y rápido, así como una técnica de aislamiento sensible. En comparación con el método de monocapa, el método de suspensión fue cuatro veces más sensible en la detección del ZIKV en casos inconclusos por RT-PCR. Conclusiones . El método de suspensión tiene el potencial de ser un método eficaz para cultivar y aislar el ZIKV y su uso es potencialmente útil tanto en la investigación como en entornos clínicos.

Objective. To develop and validate a cell suspension method using Vero 76 cells for culturing Zika virus (ZIKV) based on infection of detached freshly seeded cells. Material and methods. Three different multiplicities of infection of ZIKV were used to develop and compare this novel method to the standard confluent cell monolayer method. In addition, we preliminary validated the cell suspension method using well-characterized ZIKV positive and negative clinical samples. The standard confluent cell monolayer method was used as the reference method, and viral isolation was confirmed by a ZIKV-specific RT-PCR. The sensitivity and its 95% confidence intervals for the cell suspension method were estimated. Also, a technical comparison of the cell suspension method against the cell monolayer method was performed. Results. Our findings suggested that both the viral load and replication of ZIKV were comparable between both monolayer- and suspension-infection methods. Although both methods were suitable for culturing and isolating ZIKV, the cell suspension method was easier, cheaper, and quicker as well as a sensitive isolation technique. The cell suspension method was significantly more sensitive in detecting Zika in inconclusive cases by RT-PCR, with a fourfold increase compared to the confluent cell monolayer method. Conclusion. The cell suspension method has the potential to be an effective method for cultivating and isolating ZIKV and its application is potentially useful in both research and clinical settings.

Anti-hyperglycemic activity of aqueous extracts of cells produced in Ibervillea sonorae (S. Watson) Green cell culture on murine model / Actividad anti-hiperglucémica del extracto acuoso de células producidas en cultivo celular de Ibervillea sonorae (S. Watson) Green en un modelo murino

Ibervillea sonorae (S. Watson) Greene, is a plant native to Mexico, where its roots have been used traditionally for treating Diabetes Mellitus. The aim of this work was to establishment of cell cultures of stem explants of I. sonorae and evaluation of the anti-hyperglycemic activity of cell aqueous extract on a murine model of streptozotocin-induced diabetic rats. Cell extracts had 2.29 mg palmitic acid/g extracted, and other compounds with pharmacological activities like palmitoyl ethanolamide and palmitoyl tryptamine were also identified. Diabetic rats treated with aqueous cell extract decreased glucose levels from 350 mg/dL to 145 mg/dL, AST and ALT from 164 U/L to 49 U/L and 99 U/L to 53 U/L, respectively. Additionally, there were no changes in the cellular morphology of the pancreas, liver, kidneys, and spleen. These results revealed that the cell aqueous extract from stem explants has anti-hyperglycemic activity.

Ibervillea sonorae (S. Watson) Greene, es una planta originaria de México, donde sus raíces se han utilizado tradicionalmente para el tratamiento de la Diabetes Mellitus. El objetivo de este trabajo fue el establecimiento de cultivos celulares de explantes de tallo de I. sonorae y la evaluación de la actividad anti-hiperglucémica del extracto acuoso celular en un modelo de ratas diabéticas inducidas con estreptozotocina. El extracto celular contiene 2.29 mg de ácido palmítico/g extracto y se identificaron otros compuestos como palmitoil etanolamida y palmitoil triptamina. Las ratas diabéticas tratadas con extracto celular disminuyeron los niveles de glucosa de 350 mg/dL a 145 mg/dL, AST y ALT de 164 U/L a 49 U/L y 99 U/L a 53 U/L, respectivamente. Además, no hubo cambios en la morfología celular del páncreas, hígado, riñones y bazo. Estos resultados indican que el extracto de células de explantes de tallo de I. sonorae tiene actividad anti-hiperglucémica.

Progress on three-dimensional cell culture technology and their application / 生物医学工程学杂志

Three-dimensional (3D) cell culture model is a system that co-culture carriers with 3D structural materials and different types of cells in vitro to simulate the microenvironment in vivo. This novel cell culture model has been proved to be close to the natural system in vivo. In the process of cell attachment, migration, mitosis and apoptosis, it could produce biological reactions different from that of monolayer cell culture. Therefore, it can be used as an ideal model to evaluate the dynamic pharmacological effects of active substances and the metastasis process of cancer cells. This paper compared and analyzed the different characteristics of cell growth and development under two-dimensional (2D) and 3D model culture and introduced the establishment method of 3D cell model. The application progress of 3D cell culture technology in tumor model and intestinal absorption model was summarized. Finally, the application prospect of 3D cell model in the evaluation and screening of active substance was revealed. This review is expected to provide reference for the development and application of new 3D cell culture models.

Optimization and application of caprylic acid precipitation in the purification of monoclonal antibody / 生物工程学报

In response to the market demand for therapeutic antibodies, the upstream cell culture scale and expression titer of antibodies have been significantly improved, while the production efficiency of downstream purification process is relatively fall behind, and the downstream processing capacity has become a bottleneck limiting antibody production throughput. Using monoclonal antibody mab-X as experimental material, we optimized the caprylic acid (CA) precipitation process conditions of cell culture fluid and low pH virus inactivation pool, and studied two applications of using CA treatment to remove aggregates and to inactivate virus. Based on the lab scale study, we carried out a 500 L scale-up study, where CA was added to the low pH virus inactivation pool for precipitation, and the product quality and yield before and after precipitation were detected and compared. We found that CA precipitation significantly reduced HCP residuals and aggregates both before and after protein A affinity chromatography. In the aggregate spike study, CA precipitation removed about 15% of the aggregates. A virus reduction study showed complete clearance of a model retrovirus during CA precipitation of protein A purified antibody. In the scale-up study, the depth filtration harvesting, affinity chromatography, low pH virus inactivation, CA precipitation and depth filtration, and cation exchange chromatography successively carried out. The mixing time and stirring speed in the CA precipitation process significantly affected the CA precipitation effect. After CA precipitation, the HCP residue in the low pH virus inactivation solution decreased 895 times. After precipitation, the product purity and HCP residual meet the quality criteria of monoclonal antibodies. CA precipitation can reduce the chromatography step in the conventional purification process. In conclusion, CA precipitation in the downstream process can simplify the conventional purification process, fully meet the purification quality criterion of mab-X, and improve production efficiency and reduce production costs. The results of this study may promote the application of CA precipitation in the purification of monoclonal antibodies, and provide a reference for solving the bottleneck of the current purification process.

Advances in three-dimensional tumor models for colorectal cancer / 中华肿瘤杂志

Conventional tumor culture models include two-dimensional tumor cell cultures and xenograft models. The former has disadvantages including lack of tumor heterogeneity and poor clinical relevance, while the latter are limited by the slow growth, low engraftment successful rate, and high cost. In recent years, in vitro three-dimensional (3D) tumor models have emerged as the tool to better recapitulate the spatial structure and the in vivo environment of tumors. In addition, they preserve the pathological and genetic features of tumor cells and reflect the complex intracellular and extracellular interactions of tumors, which have become a powerful tool for investigating the tumor mechanism, drug screening, and personalized cancer treatment. 3D tumor model technologies such as spheroids, organoids, and microfluidic devices are maturing. Application of new technologies such as co-culture, 3D bioprinting, and air-liquid interface has further improved the clinical relevance of the models. Some models recapitulate the tumor microenvironment, and some can even reconstitute endogenous immune components and microvasculature. In recent years, some scholars have combined xenograft models with organoid technology to develop matched in vivo/in vitro model biobanks, giving full play to the advantages of the two technologies, and providing an ideal research platform for individualized precision therapy for specific molecular targets in certain subtypes of tumors. So far, the above technologies have been widely applied in the field of colorectal cancer research. Our research team is currently studying upon the application of patient-derived tumor cell-like clusters, a self-assembly 3D tumor model, in guiding the selection of postoperative chemotherapy regimens for colorectal cancer. A high modeling success rate and satisfactory results in the drug screening experiments have been achieved. There is no doubt that with the advancement of related technologies, 3D tumor models will play an increasingly important role in the research and clinical practice of colorectal cancer.

Overview and prospects of an in vitro cell model for studying liver fibrosis / 中华肝脏病杂志

Liver fibrosis incidence and adverse outcomes are high; however, there are no known chemical drugs or biological agents that are specific and effective for treatment. The paucity of a robust and realistic in vitro model for liver fibrosis is one of the major causes hindering anti-liver fibrosis drug development. This article summarizes the latest progress in the development of in vitro cell models for liver fibrosis, with a focus based on the analysis of induction and activation of hepatic stellate cells, cell co-culture, and 3D model co-construction, as well as concurrent potential methods based on hepatic sinusoidal endothelial cell establishment.

Spontaneous spheroids from alveolar bone-derived mesenchymal stromal cells maintain pluripotency of stem cells by regulating hypoxia-inducible factors

BACKGROUND: Spontaneous spheroid culture is a novel three-dimensional (3D) culture strategy for the rapid and efficient selection of progenitor cells. The objectives of this study are to investigate the pluripotency and differentiation capability of spontaneous spheroids from alveolar bone-derived mesenchymal stromal cells (AB-MSCs); compare the advantages of spontaneous spheroids to those of mechanical spheroids; and explore the mechanisms of stemness enhancement during spheroid formation from two-dimensional (2D) cultured cells. METHODS: AB-MSCs were isolated from the alveolar bones of C57BL/6 J mice. Spontaneous spheroids formed in low-adherence specific culture plates. The stemness, proliferation, and multi-differentiation capacities of spheroids and monolayer cultures were investigated by reverse transcription quantitative polymerase chain reaction (RT-qPCR), immunofluorescence, alkaline phosphatase (ALP) activity, and oil-red O staining. The pluripotency difference between the spontaneous and mechanical spheroids was analyzed using RT-qPCR. Hypoxia-inducible factor (HIFs) inhibition experiments were performed to explore the mechanisms of stemness maintenance in AB-MSC spheroids. RESULTS: AB-MSCs successfully formed spontaneous spheroids after 24 h. AB-MSC spheroids were positive for MSC markers and pluripotency markers (Oct4, KLF4, Sox2, and cMyc). Spheroids showed higher Ki67 expression and lower Caspase3 expression at 24 h. Under the corresponding conditions, the spheroids were successfully differentiated into osteogenic and adipogenic lineages. AB-MSC spheroids can induce neural-like cells after neurogenic differentiation. Higher expression of osteogenic markers, adipogenic markers, and neurogenic markers (NF-M, NeuN, and GFAP) was found in spheroids than in the monolayer. Spontaneous spheroids exhibited higher stemness than mechanical spheroids did. HIF-1α and HIF-2α were remarkably upregulated in spheroids. After HIF-1/2α-specific inhibition, spheroid formation was significantly reduced. Moreover, the expression of the pluripotency genes was suppressed. CONCLUSIONS: Spontaneous spheroids from AB-MSCs enhance stemness and pluripotency. HIF-1/2α plays an important role in the stemness regulation of spheroids. AB-MSC spheroids exhibit excellent multi-differentiation capability, which may be a potent therapy for craniomaxillofacial tissue regeneration.

Evaluation of different protocols for culturing mesenchymal stem cells derived from murine bone marrow

ABSTRACT Introduction: Culturing bone marrow mesenchymal stem cells (BM-MSCs) is a key point in different fields of research, including tissue engineering and regenerative medicine and studies of the bone marrow microenvironment. However, isolating and expanding murine BM-MSCs in vitro has challenged researchers due to the low purity and yield of obtained cells. In this study, we aimed to evaluate five different protocols to culture murine BM-MSCs in vitro. Methods: All protocols were based on the adhesion capacity of BM-MSCs to the tissue culture plastic surface and varied in the types of plate, culture media, serum, additional supplementation and initial cell density. Flow cytometry analysis was used to investigate lineage purity after expansion. Results: The expression of CD45 and CD11b was detected in the cultures generated according to all protocols, indicating low purity with the presence of hematopoietic cells and macrophages. The cellular growth rate and morphology varied between the cultures performed according to each protocol. Cells cultured according to protocol 5 (8 × 107cells/plate, Roswell Park Memorial Institute (RPMI) culture medium during first passage and then Iscove's Modified Delbecco's Medium (IMDM) culture medium, both supplemented with 9% fetal bovine serum, 9% horse serum, 12μM L-glutamine) presented the best performance, with a satisfactory growth rate and spindle-shape morphology. Conclusion: Our results point out that the purity and satisfactory growth rate of murine BM-MSC cultures are not easily achieved and additional approaches must be tested for a proper cell expansion.

Efeito do alendronato, da dexametasona e da associação de ambos sobre a osteoclastogênese: um estudo in vitro / Effect of alendronate, dexamethasone and the association of both on osteoclastogenesis: an in vitro study

O carcinoma secretório em glândula salivar é uma neoplasia recentemente descrita que tem os mesmos aspectos morfológicos, imuno-histoquímicos e genéticos do carcinoma secretório de origem mamária. O carcinoma secretório tem características celulares reminiscentes de uma célula secretora lactacional, isto é, um citoplasma vacuolado repleto de gotas lipídicas e um material secretado, por vezes de forma apócrina, que pode lembrar o leite. Mais recentemente, algum nível de diferenciação lactacional foi sugerida no carcinoma secretório de origem salivar. O objetivo do estudo foi verificar se existe uma diferenciação do tipo lactacional em carcinomas secretórios de origem salivar, comparando a outros tipos de carcinomas salivares mais comuns. Foram realizadas reações imuno-histoquímicas para as seguintes proteínas: receptores hormonais (receptor de prolactina e receptor do hormônio do crescimento), proteínas associadas ao produto de secreção da glândula mamária lactacional (mucina-1 (MUC-1), MUC4, globulina de gordura 1 do leite humano, lactoferrina) e proteínas associadas à via Akt-mTOR (PTEN, p-Akt, p-mTOR, p4EBP1, eIF4E, pS6). A maioria dos casos de carcinoma secretório foi negativa para receptor de prolactina e de hormônio do crescimento. Lactoferrina foi positiva em todos os grupos tumorais, porém somente em carcinoma secretório observou-se um padrão de marcação intensa, difuso tanto em célula como em secreção. Todos os casos de carcinoma secretório foram positivos para globulina de gordura do tipo 1, porém o mesmo padrão de marcação foi observado em outros tumores. A maioria dos casos de carcinoma secretório foram positivos para MUC1 e MUC4. Nenhum caso de carcinoma secretório foi positivo para Akt, mas PTEN foi difusamente expresso em 57,1% dos casos. mTOR foi expresso em mais da metade dos casos de carcinoma secretório e dos outros tumores salivares. Entre as proteínas à jusante de mTOR, somente eIF4E demonstrou alta expressão no grupo de estudo. A expressão de marcadores lactacionais não é exclusiva do carcinoma secretório, porém a expressão de lactoferrina é distinta neste grupo de tumores quando comparado aos demais tumores salivares estudados.

Mitochondrial energy metabolism in baby hamster kidney (BHK-21/C13) cells treated with karnozin extra® / Metabolismo energético mitocondrial en células de riñón de hámster bebé (BHK-21 / C13) tratadas con karnozin extra®

SUMMARY: Carnosine is known as a natural dipeptide, which inhibits the proliferation of tumor cells throughout its action on mitochondrial respiration and cell glycolysis. However, not much is known about its effects on the metabolism of healthy cells. We explored the effects of Karnozin EXTRA® capsule with different concentrations of L-carnosine, on the cell viability and the expressions of intermediate filament vimentin (VIM) and superoxide dismutase (SOD2) in normal fibroblasts BHK-21/C13. Furthermore, we investigated its action on the energy production of these cells. Cell viability was quantified by the MTT assay. The Clark oxygen electrode (Oxygraph, Hansatech Instruments, England) was used to measure the "intact cell respiration rate", state 3 of ADP-stimulated oxidation, maximum oxidation capacity and the activities of complexes I, II and IV. Results showed that Karnozin EXTRA® capsule in concentrations of 2 and 5 mM of L-carnosine did not induce toxic effects and morphological changes in treated cells. Our data revealed a dose-dependent immunofluorescent signal amplification of VIM and SOD2 in the BHK-21/C13 cell line. This supplement substantially increased the recorded mitochondrial respiration rates in the examined cell line. Due to the stimulation of mitochondrial energy production in normal fibroblasts, our results suggested that Karnozin EXTRA® is a potentially protective dietary supplement in the prevention of diseases with altered mitochondrial function.

RESUMEN: La carnosina se conoce como dipéptido natural, que inhibe la proliferación de células tumorales a través de su acción sobre la respiración mitocondrial y la glucólisis celular. Sin embargo, no se sabe mucho de sus efectos sobre el metabolismo de las células sanas. Exploramos los efectos de la cápsula Karnozin EXTRA® con diferentes concentraciones de L-carnosina, sobre la viabilidad celular y las expresiones de vimentina de filamento intermedio (VIM) y superóxido dismutasa (SOD2) en fibroblastos normales BHK-21 / C13. Además, estudiamos su acción sobre la producción de energía de estas células. La viabilidad celular se cuantificó mediante el ensayo MTT. Se utilizó el electrodo de oxígeno Clark (Oxygraph, Hansatech Instruments, Inglaterra) para medir la "tasa de respiración de células intactas", el estado 3 de oxidación estimulada por ADP, la capacidad máxima de oxidación y las actividades de los complejos I, II y IV. Los resultados mostraron que la cápsula de Karnozin EXTRA® en concentraciones de 2 y 5 mM de L- carnosina no indujo efectos tóxicos ni cambios morfológicos en las células tratadas. Nuestros datos revelaron una amplificación de señal inmunofluorescente dependiente de la dosis de VIM y SOD2 en la línea celular BHK-21 / C13. Este suplemento aumentó sustancialmente las tasas de respiración mitocondrial registradas en la línea celular examinada. Debido a la estimulación de la producción de energía mitocondrial en fibroblastos normales, nuestros resultados sugirieron que Karnozin EXTRA® es un suplemento dietético potencialmente protector en la prevención de enfermedades con función mitocondrial alterada.

Cytotoxic effect of hydroalcoholic extract of Cota tinctoria (L) J. Gay on AGS and Hep-G2 cancer cell lines / Efecto citotóxico del extracto hidroalcohólico de Cota tinctoria (L) J. Gay sobre líneas celulares de cáncer AGS y Hep-G2

Cota tinctoria is a medicinal plant which has been used for management of cancer in folk medicine of various regions. The aim of present study is to investigate cytotoxic activity of different concentrations of hydroalcoholic extract of C. tinctoria flowers on gastric (AGS) and liver (Hep-G2) cancer cell lines as well as Human Natural GUM fibroblast (HUGU) cells. Cell mortality rates were examined after 24, 48 and 72 h incubations using the MTT assay. IC50of extract on AGS cells after 24, 48 and 72h was 1.46, 1.29 and 1.14 µg/mL respectively. The extract demonstrated IC50 of 5.15, 3.92 and 2.89 µg/mL on Hep-G2 cells after 24, 48 and 72 h respectively. No cytotoxic effect was detected on HUGU (Human Natural GUM fibroblast) cells. C. tinctoria seems to have a promising potential to be considered as a source for anticancer drug discovery. However, more experimental and clinical studies are required.

Cota tinctoria es una planta medicinal que se ha utilizado para el tratamiento del cáncer en la medicina popular de varias regiones. El objetivo del presente estudio es investigar la actividad citotóxica de diferentes concentraciones de extracto hidroalcohólico de flores de C. tinctoria en líneas celulares de cáncer gástrico (AGS) e hígado (Hep-G2), así como en células de fibroblasto GUM humano natural (HUGU). Se examinaron las tasas de mortalidad celular después de incubaciones de 24, 48 y 72 h utilizando el ensayo MTT. La CI50 del extracto en células AGS después de 24, 48 y 72 h fue de 1,46; 1,29 y 1,14 µg respectivamente. El extracto demostró una CI50 de 5,15, 3,92 y 2,89 µg/mL en células Hep-G2 después de 24, 48 y 72 h, respectivamente. No se detectó ningún efecto citotóxico en las células HUGU (fibroblasto GUM humano natural). C. tinctoria parece tener un potencial prometedor para ser considerada como una fuente de descubrimiento de fármacos contra el cáncer. Sin embargo, se requieren más estudios experimentales y clínicos.

Progress in the application of three-dimensional cell culture model in toxicity tests of xenobiotic / 中华预防医学杂志

In the process of xenobiotic toxicity prediction and risk assessment, in vitro cell culture models possess high practical application value. With the rapid development of biological technologies such as three-dimensional (3D) bio-printing, organoid culture and organ-on-a-chip systems, in vitro cell culture models have made great progress. Sharing the similarities in structure, function and the physiological environment with tissues or organs in vivo, hazard identification and dose-response analysis based on 3D cell culture models provide access to more accurate toxicity data as a theoretical basis for risk assessment and risk management of chemicals. This review summarizes the establishment of three typical 3D cell culture models, i.e., human cell line-based co-culture model, 3D-printed scaffold-based cell culture model and organoids, and their application in toxicity tests of xenobiotics.

Development and future promise of salivary gland organoids and salivary gland tumor organoids / 中华口腔医学杂志

Salivary glands are important organs in the oral and maxillofacial region. Environment and genetic factors may cause salivary gland tumors or non-neoplastic diseases, but the mechanisms of those diseases are still unclear. One of the important reasons is the short of researching media and model. As a new technique and research model, organoids have been widely used in the research of various diseases. Organoid culture plays a bridging role between two-dimensional cell culture and living animal models, and it is also the most promising translational research model that could connect the clinical research to basic research. This review will discuss the recent development of organoid techniques in the culture of normal salivary glands and salivary gland tumors, also their applications and challenges in tissue engineering, etiological research, and tumor therapy.

Continuous purification and culture of rat type 1 and type 2 alveolar epithelial cells by magnetic cell sorting / 中华创伤杂志(英文版)

PURPOSE@#The incidence of acute lung injury (ALI) in severe trauma patients is 48% and the mortality rate following acute respiratory distress syndrome evolved from ALI is up to 68.5%. Alveolar epithelial type 1 cells (AEC1s) and type 2 cells (AEC2s) are the key cells in the repair of injured lungs as well as fetal lung development. Therefore, the purification and culture of AEC1s and AEC2s play an important role in the research of repair and regeneration of lung tissue.@*METHODS@#Sprague-Dawley rats (3-4 weeks, 120-150 g) were purchased for experiment. Dispase and DNase I were jointly used to digest lung tissue to obtain a single-cell suspension of whole lung cells, and then magnetic bead cell sorting was performed to isolate T1α positive cells as AEC1s from the single-cell suspension by using polyclonal rabbit anti-T1a (a specific AEC1s membrane protein) antibodies combined with anti-rabbit IgG microbeads. Afterwards, alveolar epithelial cell membrane marker protein EpCAM was designed as a key label to sort AEC2s from the remaining T1α-neg cells by another positive immunomagnetic selection using monoclonal mouse anti-EpCAM antibodies and anti-mouse IgG microbeads. Cell purity was identified by immunofluorescence staining and flow cytometry.@*RESULTS@#The purity of AEC1s and AEC2s was 88.3% ± 3.8% and 92.6% ± 2.7%, respectively. The cell growth was observed as follows: AEC1s stretched within the 12-16 h, but the cells proliferated slowly; while AEC2s began to stretch after 24 h and proliferated rapidly from the 2nd day and began to differentiate after 3 days.@*CONCLUSION@#AEC1s and AEC2s sorted by this method have high purity and good viability. Therefore, our method provides a new approach for the isolation and culture of AEC1s and AEC2s as well as a new strategy for the research of lung repair and regeneration.

Estudo do composto STX140, um análogo do estradiol, em linhagens de melanoma humano / STX140 compound study in human melanoma

O câncer de pele pode ser classificado como não melanoma e melanoma. O melanoma apresenta baixa incidência entre os cânceres de pele, porém é a forma mais letal e é considerado um dos tipos mais resistentes ao tratamento. Devido à infiltração de células malignas nos tecidos, vasos linfáticos e vasos sanguíneos, o melanoma invade e se espalha rapidamente. Suas metástases são frequentemente localizadas em linfonodos, cérebro, fígado e outros órgãos. Melanomas metastáticos abrigam múltiplas mutações gênicas e muitos tumores apresentam resistência aos tratamentos, como por exemplo com inibidores BRAF, devido à mutações e ativação de vias paralelas. Ou seja, existe uma necessidade clara da busca de novas opções de tratamento. Em trabalho realizado por nosso grupo, Massaro et al mostraram que o derivado de estradiol 2- Metoxiestradiol induz apoptose em células de melanoma e senescência. Neste sentido, o composto STX140, (um análogo do estradiol com biodisponibilidade superior), que já se mostrou eficaz no combate ao câncer de mama em diversos estudos in vitro e in vivo, será então avaliado para sua ação no melanoma de forma inédita. Este trabalho teve como principal objetivo explorar a ação antitumoral em células de melanoma do composto STX140, especialmente a indução de senescência. Utilizando a cultura de células de melanoma foram realizados os ensaios de: viabilidade celular - IC50, formação de colônias, análise do ciclo celular e caracterização de morte celular por citometria de fluxo, ensaio In vitro scratch, coloração para ß-galactosidase, PCR quantitativo e ELISA. Os resultados mostraram que o composto STX140: diminui a viabilidade celular, inibe a proliferação, formação de colônias e migração em linhagens de melanoma (não resistentes e resistentes ao vemurafenibe, inibidor de BRAF). Além do mais, o composto atuou diminuindo a secreção da interleucina pró-tumoral IL-8 em células resistentes. O STX140 induziu senescência nas células de melanoma que foram positivas para ß-galactosidase, também havendo aumento da expressão de genes chave de vias de senescência (CDKN1A e GADD45A) nas células de melanoma resistentes tratadas com o composto. Em conclusão, o STX140 mostrou ter um potencial antitumoral contra o melanoma, diminuindo sua viabilidade celular, inibindo sua proliferação e migração, induzindo senescência, diminuindo a secreção de interleucina pró- tumoral, com efeito mais acentuado nas linhagens de melanoma resistente

Skin cancer can be classified as non-melanoma and melanoma. Melanoma has a low incidence among skin cancers, but it is the most lethal form and is considered one of the most resistant to treatment. Due to the infiltration of malignant cells into tissues, lymphatic vessels and blood vessels, melanoma invades and spreads rapidly. Its metastases are often located in lymph nodes, brain, liver and other organs. Metastatic melanomas presents multiple gene mutations and many tumors are resistant to treatments, such as with BRAF inhibitors, due to mutations and activation of parallel pathways. In other words, there is a clear need to search for new treatment options. In work carried out by our group, Massaro et al showed that the estradiol derivative 2- Methoxyestradiol induces apoptosis in melanoma cells and senescence. In this sense, the compound STX140, (an estradiol analogue with superior bioavailability), which has already been shown to be effective against breast cancer in vitro and in vivo studies will be then evaluated for its action on melanoma. The main objective of this work is to explore the antitumor action of the compound STX140 in melanoma cells, especially the induction of senescence. Using the melanoma cell culture the following assays were performed: cell viability - IC50, clonogenic, cell cycle analysis and cell death characterization by flow cytometry, wound assay, staining for ß-galactosidase, quantitative PCR and ELISA. Preliminary data from this work showed that the compound STX140: decreases cell viability, inhibits proliferation, colony formation and migration in melanoma cell lines (non-resistant and resistant to vemurafenib, BRAF inhibitor). It also decreased the secretion of pro-tumor interleukin IL-8 in resistant cells. STX140 induced senescence in melanoma cells, that were positive for ß-galactosidase, and there was also increased expression of key genes of senescence pathways (CDKN1A and GADD45A) in resistant melanoma cells treated with the compound. In conclusion, STX140 has been shown to have antitumor potential against melanoma, decreasing its cell viability, inhibiting its proliferation and migration, inducing senescence, decreasing pro-tumor interleukin secretion, with a more pronounced effect on resistant melanoma cell lines