Avaliação da integridade das células-tronco mesenquimais derivadas do tecido adiposo humano após o bioprocesso de criopreservação

Resumo: As celulas-tronco de origem adulta surgem como promessa terapeutica na medicina regenerativa podendo ser encontradas em diversos tecidos como o adiposo. Com as perspectivas da criacao de um banco de celula-tronco para pesquisa e posterior uso terapeutico, o estudo da criopreservacao dessas celulas urge, a fim de garantir que essas celulas apos o descongelamento permanecam viaveis e funcionais. Objetivos: Comparar as celulas-tronco do tecido adiposo antes da criopreservacao e apos o seu descongelamento quanto a: a) indice de proliferacao e capacidade de formar colonias b) expressao das moleculas de superficies em especial a integrina ƒ¿4; c) capacidade de diferenciacao d) viabilidade e integridade celular. Metodos: Celulas-tronco adultas foram obtidas do tecido adiposo de 10 mulheres e 2 homens, saudaveis pela tecnica de lipoaspiracao, realizada por cirurgioes plasticos e apos o consentimento informado. O isolamento das celulas foi realizado atraves de digestao enzimatica com colagenase tipo I posterior cultivo em DMEM/F12 suplementado com 10% de SFB e 100U/mL de Penicilina/100ƒÊg/mL de Estreptomicina. Foi calculado o indice de proliferacao celular da primeira a terceira passagem para cada amostra. Realizadas analises de unidade formadora de colonia na segunda passagem, as celulas foram submetidas a diferenciacao adipogenica e osteogenica com auxilio de meios indutores. A Imunofenotipagem atraves de citometria de fluxo (FACS Calibur; Becton Dickinson, USA), utilizando-se marcadores: CD34, CD45, CD49d, CD73, CD90, CD105, anexina V e 7-AAD, seguidas do processo de criopreservacao, precedido de congelamento programavel (Nicool LM10). Vinte dias apos, as celulas foram descongeladas e os testes repetidos. Resultados: Rendimento celular: 9,09 } 4,55 X 106 celulas/mL isoladas. Indice de proliferacao antes da criopreservacao: Passagens 1= 0,24; 2= 21,33; 3= 13,03 (p=0,001), respectivamente; passagem 3=17,43 }4,11 apos descongelamento (p=0,07). Unidade formadora de colonia antes da criopreservacao: 28,08 % } 7,06%, apos o descongelamento 21,51% } 6,61% (p=0,001).Todas as amostras induzidas tiveram diferenciacao adipogenica e osteogenica, antes da criopreservacao e apos o descongelamento. A analise imunofenotipica demonstrou as seguintes expressoes antes da criopreservacao: CD34- (98,88% } 1,08%), CD45- (99,79% } 0,22%), CD49d+ (88,67% } 6,55), CD90+ (99,55 } 0,07%), CD73+ (99,57% } 0,43%) e CD105+ (99,4% } ,64%); Apos o descongelamento: CD34- (99,26% } 0,61%, p= 0,11), CD45- (99,8% } 0,14%, p= 0,79), CD49d+ (77,8% } 14,45%, p= 0,01), CD73+ (99,5% } 0,43%, p= 0,53), CD90+(99,45 } 0,7%, p= 0,62), CD105+ (98,26% } 2,07%, p= 0,05). Viabilidade celular (7-AAD-) e apoptose (Anexina V-) antes da criopreservacao: 91,39% } 5,85%, 91,34% } 4,54%; apos descongelamento das celulas: 76,31% } 13,33% (p= 0,001), 74,99% } 14,19% (p= 0,003), respectivamente. Conclusao: As celulas-tronco mesenquimais derivadas de tecido adiposo mantem suas caracteristicas imunofenotipicas, viabilidade, capacidades de proliferacao e de diferenciacao apos o descongelamento das celulas criopreservadas, a excecao da integrina ƒ¿4 (CD 49d), uma molecula de adesao que participa da integracao da celula no tecido hospedeiro

Avaliação da integridade das células-tronco mesenquimais derivadas do tecido adiposo humano após o bioprocesso de criopreservação

Resumo: As celulas-tronco de origem adulta surgem como promessa terapeutica na medicina regenerativa podendo ser encontradas em diversos tecidos como o adiposo. Com as perspectivas da criacao de um banco de celula-tronco para pesquisa e posterior uso terapeutico, o estudo da criopreservacao dessas celulas urge, a fim de garantir que essas celulas apos o descongelamento permanecam viaveis e funcionais. Objetivos: Comparar as celulas-tronco do tecido adiposo antes da criopreservacao e apos o seu descongelamento quanto a: a) indice de proliferacao e capacidade de formar colonias b) expressao das moleculas de superficies em especial a integrina ƒ¿4; c) capacidade de diferenciacao d) viabilidade e integridade celular. Metodos: Celulas-tronco adultas foram obtidas do tecido adiposo de 10 mulheres e 2 homens, saudaveis pela tecnica de lipoaspiracao, realizada por cirurgioes plasticos e apos o consentimento informado. O isolamento das celulas foi realizado atraves de digestao enzimatica com colagenase tipo I posterior cultivo em DMEM/F12 suplementado com 10% de SFB e 100U/mL de Penicilina/100ƒÊg/mL de Estreptomicina. Foi calculado o indice de proliferacao celular da primeira a terceira passagem para cada amostra. Realizadas analises de unidade formadora de colonia na segunda passagem, as celulas foram submetidas a diferenciacao adipogenica e osteogenica com auxilio de meios indutores. A Imunofenotipagem atraves de citometria de fluxo (FACS Calibur; Becton Dickinson, USA), utilizando-se marcadores: CD34, CD45, CD49d, CD73, CD90, CD105, anexina V e 7-AAD, seguidas do processo de criopreservacao, precedido de congelamento programavel (Nicool LM10). Vinte dias apos, as celulas foram descongeladas e os testes repetidos. Resultados: Rendimento celular: 9,09 } 4,55 X 106 celulas/mL isoladas. Indice de proliferacao antes da criopreservacao: Passagens 1= 0,24; 2= 21,33; 3= 13,03 (p=0,001), respectivamente; passagem 3=17,43 }4,11 apos descongelamento (p=0,07). Unidade formadora de colonia antes da criopreservacao: 28,08 % } 7,06%, apos o descongelamento 21,51% } 6,61% (p=0,001).Todas as amostras induzidas tiveram diferenciacao adipogenica e osteogenica, antes da criopreservacao e apos o descongelamento. A analise imunofenotipica demonstrou as seguintes expressoes antes da criopreservacao: CD34- (98,88% } 1,08%), CD45- (99,79% } 0,22%), CD49d+ (88,67% } 6,55), CD90+ (99,55 } 0,07%), CD73+ (99,57% } 0,43%) e CD105+ (99,4% } ,64%); Apos o descongelamento: CD34- (99,26% } 0,61%, p= 0,11), CD45- (99,8% } 0,14%, p= 0,79), CD49d+ (77,8% } 14,45%, p= 0,01), CD73+ (99,5% } 0,43%, p= 0,53), CD90+(99,45 } 0,7%, p= 0,62), CD105+ (98,26% } 2,07%, p= 0,05). Viabilidade celular (7-AAD-) e apoptose (Anexina V-) antes da criopreservacao: 91,39% } 5,85%, 91,34% } 4,54%; apos descongelamento das celulas: 76,31% } 13,33% (p= 0,001), 74,99% } 14,19% (p= 0,003), respectivamente. Conclusao: As celulas-tronco mesenquimais derivadas de tecido adiposo mantem suas caracteristicas imunofenotipicas, viabilidade, capacidades de proliferacao e de diferenciacao apos o descongelamento das celulas criopreservadas, a excecao da integrina ƒ¿4 (CD 49d), uma molecula de adesao que participa da integracao da celula no tecido hospedeiro

Why not "do simple things in a simple way": Use of the Pap test as the first step in screening genetic stability for human cultured stem cell therapy?

The aim of this study was to analyze adipose tissue-derived mesenchymal stem cells (AT-MSCs) using the Pap test as a first screening step to evaluate genetic stability. Human adipose tissue from six healthy female donors was obtained from elective liposuction procedures. The cells were isolated, cultivated at P2/P3, characterized by flow cytometric analysis, and differentiation induced. The AT-MSCs were stained by Papanicolaou staining and analyzed according to the Bethesda classification, and viability-apoptosis relationships were evaluated. The results of the Pap test for Sample I indicated high-grade alterations consistent with genetic instability; for Samples II-V, atypical cells of undetermined significance; and for Sample VI, normal cells. These results demonstrate the potential of using the Pap test as an initial screening step to evaluate the genetic stability of cultured AT-MSCs and also suggest its use for other adherent cells such as embryonic stem cells or induced pluripotent stem cells

Avaliação da integridade das células-tronco mesenquimais derivadas do tecido adiposo humano após o bioprocesso de criopreservação

Resumo: As celulas-tronco de origem adulta surgem como promessa terapeutica na medicina regenerativa podendo ser encontradas em diversos tecidos como o adiposo. Com as perspectivas da criacao de um banco de celula-tronco para pesquisa e posterior uso terapeutico, o estudo da criopreservacao dessas celulas urge, a fim de garantir que essas celulas apos o descongelamento permanecam viaveis e funcionais. Objetivos: Comparar as celulas-tronco do tecido adiposo antes da criopreservacao e apos o seu descongelamento quanto a: a) indice de proliferacao e capacidade de formar colonias b) expressao das moleculas de superficies em especial a integrina ƒ¿4; c) capacidade de diferenciacao d) viabilidade e integridade celular. Metodos: Celulas-tronco adultas foram obtidas do tecido adiposo de 10 mulheres e 2 homens, saudaveis pela tecnica de lipoaspiracao, realizada por cirurgioes plasticos e apos o consentimento informado. O isolamento das celulas foi realizado atraves de digestao enzimatica com colagenase tipo I posterior cultivo em DMEM/F12 suplementado com 10% de SFB e 100U/mL de Penicilina/100ƒÊg/mL de Estreptomicina. Foi calculado o indice de proliferacao celular da primeira a terceira passagem para cada amostra. Realizadas analises de unidade formadora de colonia na segunda passagem, as celulas foram submetidas a diferenciacao adipogenica e osteogenica com auxilio de meios indutores. A Imunofenotipagem atraves de citometria de fluxo (FACS Calibur; Becton Dickinson, USA), utilizando-se marcadores: CD34, CD45, CD49d, CD73, CD90, CD105, anexina V e 7-AAD, seguidas do processo de criopreservacao, precedido de congelamento programavel (Nicool LM10). Vinte dias apos, as celulas foram descongeladas e os testes repetidos. Resultados: Rendimento celular: 9,09 } 4,55 X 106 celulas/mL isoladas. Indice de proliferacao antes da criopreservacao: Passagens 1= 0,24; 2= 21,33; 3= 13,03 (p=0,001), respectivamente; passagem 3=17,43 }4,11 apos descongelamento (p=0,07). Unidade formadora de colonia antes da criopreservacao: 28,08 % } 7,06%, apos o descongelamento 21,51% } 6,61% (p=0,001).Todas as amostras induzidas tiveram diferenciacao adipogenica e osteogenica, antes da criopreservacao e apos o descongelamento. A analise imunofenotipica demonstrou as seguintes expressoes antes da criopreservacao: CD34- (98,88% } 1,08%), CD45- (99,79% } 0,22%), CD49d+ (88,67% } 6,55), CD90+ (99,55 } 0,07%), CD73+ (99,57% } 0,43%) e CD105+ (99,4% } ,64%); Apos o descongelamento: CD34- (99,26% } 0,61%, p= 0,11), CD45- (99,8% } 0,14%, p= 0,79), CD49d+ (77,8% } 14,45%, p= 0,01), CD73+ (99,5% } 0,43%, p= 0,53), CD90+(99,45 } 0,7%, p= 0,62), CD105+ (98,26% } 2,07%, p= 0,05). Viabilidade celular (7-AAD-) e apoptose (Anexina V-) antes da criopreservacao: 91,39% } 5,85%, 91,34% } 4,54%; apos descongelamento das celulas: 76,31% } 13,33% (p= 0,001), 74,99% } 14,19% (p= 0,003), respectivamente. Conclusao: As celulas-tronco mesenquimais derivadas de tecido adiposo mantem suas caracteristicas imunofenotipicas, viabilidade, capacidades de proliferacao e de diferenciacao apos o descongelamento das celulas criopreservadas, a excecao da integrina ƒ¿4 (CD 49d), uma molecula de adesao que participa da integracao da celula no tecido hospedeiro

Quercetin-Rich Extracts from Onions (<i>Allium cepa</i>) Play Potent Cytotoxicity on Adrenocortical Carcinoma Cell Lines, and Quercetin Induces Important Anticancer Properties

Adrenocortical carcinoma (ACC) is a rare subtype of cancer, with a poor prognosis in children and adults. Mitotane is the only approved adrenolytic drug for the treatment of ACC, which has controversies regarding its efficacy and side effects on patients. Onion (Allium cepa), a worldwide consumed food, is associated with many health benefits. Along with its glycosides, the flavonoid quercetin is abundant in onions. After evaluating the cytotoxicity of A. cepa extracts on adrenocortical carcinoma cell line (H295R), the rich quercetin fractions had better results. Then, we aimed to compare the quercetin vs. mitotane effectiveness, using adrenocortical carcinoma cell lines H295R and SW-13. Quercetin showed a higher cytotoxicity response on both cancerous cell lines after 10 µM concentration, while mitotane only after 30 µM. Cell cycle dynamics were altered upon quercetin treatments, with G2 phase increase with 30 µM of quercetin on H295R cell line and G1 arrest on SW-13 cell line with 15 µM. Early and late apoptosis, alongside intracellular calcium, were increased on SW-13 treated with 30 µM of quercetin, and ROS rates were reduced by quercetin on H295R. Therefore, quercetin-rich onions have the potential to be a natural source of anticancer agents for adrenocortical carcinoma

Anti-Inflammatory Effect of Malva sylvestris, Sida cordifolia, and Pelargonium graveolens Is Related to Inhibition of Prostanoid Production

The ability of plant extracts and preparations to reduce inflammation has been proven by different means in experimental models. Since inflammation enhances the release of specific mediators, inhibition of their production can be used to investigate the anti-inflammatory effect of plants widely used in folk medicine for this purpose. The study was performed for leaves and flowers of Malva sylvestris, and leaves of Sida cordifolia and Pelargonium graveolens. These are three plant species known in Brazil as Malva. The anti-inflammatory activity of extracts and fractions (hexane, chloroform, ethyl acetate, and residual) was evaluated by quantitation of prostaglandins (PG) PGE2, PGD2, PGF2α, and thromboxane B2 (the stable nonenzymatic product of TXA2) concentration in the supernatant of lipopolysaccharide (LPS)- induced RAW 264.7 cells. Inhibition of anti-inflammatory mediator release was observed for plants mainly in the crude extract, ethyl acetate fraction, and residual fraction. The results suggest superior activity of S. cordifolia, leading to significantly lower values of all mediators after treatment with its residual fraction, even at the lower concentration tested (10 μg/mL). M. sylvestris and P. graveolens showed similar results, such as the reduction of all mediators after treatment, with leaf crude extracts (50 μg/mL). These results suggest that the three species known as Malva have anti-inflammatory properties, S. cordifolia being the most potent

Adipose-Derived Stromal Cells and Mineralized Extracellular Matrix Delivery by a Human Decellularized Amniotic Membrane in Periodontal Tissue Engineering

Periodontitis is a prevalent disease characterized by the loss of periodontal supporting tissues, bone, periodontal ligament, and cementum. The application of a bone tissue engineering strategy with Decellularized Human Amniotic Membrane (DAM) with adipose-derived stromal cells (ASCs) has shown to be convenient and valuable. This study aims to investigate the treatments of a rat periodontal furcation defect model with DAM, ASCs, and a mineralized extracellular matrix (ECM). Rat ASCs were expanded, cultivated on DAM, and with a bone differentiation medium for four weeks, deposited ECM on DAM. Periodontal healing for four weeks was evaluated by micro-computed tomography and histological analysis after treatments with DAM, ASCs, and ECM and compared to untreated defects on five consecutive horizontal levels, from gingival to apical. The results demonstrate that DAM preserves its structure during cultivation and healing periods, supporting cell attachment, permeation, bone deposition on DAM, and periodontal regeneration. DAM and DAM+ASCs enhance bone healing compared to the control on the gingival level. In conclusion, DAM with ASC or without cells and the ECM ensures bone tissue healing. The membrane supported neovascularization and promoted osteoconduction

Assessment of Neurotoxic Effects of Oxycodone and Naloxone in SH-SY5Y Cell Line

Opioid drugs have analgesic properties used to treat chronic and post-surgical pain due to descending pain modulation. The use of opioids is often associated with adverse effects or clinical issues. This study aimed to evaluate the toxicity of opioids by exposing the neuroblastoma cell line (SH-SY5Y) to 0, 1, 10, and 100 µM oxycodone and naloxone for 24 h. Analyses were carried out to evaluate cell cytotoxicity, identification of cell death, DNA damage, superoxide dismutase (SOD), glutathione S-transferase (GST), and acetylcholinesterase (AChE) activities, in addition to molecular docking. Oxycodone and naloxone exposure did not alter the SH-SY5Y cell viability. The exposure to 100 µM oxycodone and naloxone significantly increased the cells’ DNA damage score compared to the control group. Naloxone exposure significantly inhibited AChE, GST, and SOD activities, while oxycodone did not alter these enzymes’ activities. Molecular docking showed that naloxone and oxycodone interact with different amino acids in the studied enzymes, which may explain the differences in enzymatic inhibition. Naloxone altered the antioxidant defenses of SH-SY5Y cells, which may have caused DNA damage 24 h after the exposure. On the other hand, more studies are necessary to explain how oxycodone causes DNA damage

Laser Ablated Albumin Functionalized Spherical Gold Nanoparticles Indicated for Stem Cell Tracking

Cell tracking in cell-based therapy applications helps distinguish cell participation among paracrine effect, neovascularization, and matrix deposition. This preliminary study examined the cellular uptake of gold nanoparticles (AuNPs), observing cytotoxicity and uptake of different sizes and AuNPs concentrations in Adipose-derived stromal cells (ASCs). ASCs were incubated for 24 h with Laser ablated Albumin functionalized spherical AuNPs (LA-AuNPs), with average sizes of 2 nm and 53 nm in diameter, in four concentrations, 127 &micro;M, 84 &micro;M, 42 &micro;M, and 23 &micro;M. Cytotoxicity was examined by Live/Dead assay, and erythrocyte hemolysis, and the effect on the cytoskeleton was investigated by immunocytochemistry for &beta;-actin. The LA-AuNPs were internalized by the ASCs in a size and concentration-dependent manner. Clusters were observed as dispersed small ones in the cytosol, and as a sizeable perinuclear cluster, without significant harmful effects on the cells for up to 2 weeks. The Live/Dead and hemolysis percentage results complemented the observations that the larger 53 nm LA-AuNPs in the highest concentrated solution significantly lowered cell viability. The demonstrated safety, cellular uptake, and labelling persistency with LA-AuNPs, synthesized without the combination of chemical solutions, support their use for cell tracking in tissue engineering applications