Toxicity and oxidative stress of canine mesenchymal stromal cells from adipose tissue in different culture passages

Pesquisa Veterinária Brasileira

Cruz, Ivana Beatrice Mânica da¹  Rosa, Matheus Pippi da²  Pippi, Ney Luis²  Sprada, Arícia Gomes²  Machado, Alencar Kolinski¹  Bayard, Paulo² 

[1] UFSM, Santa Maria, Brazil;Universidade Federal de Santa Maria, Santa Maria, Brazil

关键词:  Stem cell;    culture;    passage;    cell viability;    free radicals.;   

DOI  :  10.1590/S0100-736X2015001300004

来源: Colegio Brasileiro de Patologia Animal-CBPA

【 摘 要 】

:Stem cells in regenerative therapy have received attention from researchers in recent decades. The culture of these cells allows studies about their behavior and metabolism. Thus, cell culture is the basis for cell therapy and tissue engineering researches. A major concern regarding the use of cultivated stem cell in human or veterinary clinical routine is the risk of carcinogenesis. Cellular activities require a balanced redox state. However, when there is an imbalance in this state, oxidative stress occurs. Oxidative stress contributes to cytotoxicity, which may result in cell death or genomic alterations, favoring the development of cancer cells. The aim of this study was to determine whether there are differences in the behavior of cultured mesenchymal stem cells from canine adipose tissue according to its site of collection (omentum and subcutaneous) evaluating the rate of proliferation, viability, level of oxidative stress and cytotoxicity over six passages. For this experiment, two samples of adipose tissue from subcutaneous and omentum where taken from a female dog corpse, 13 years old, Pitbull. The results showed greater levels of oxidative stress in the first and last passages of both groups, favoring cytotoxicity and cell death.Index Terms: Stem cell; culture; passage; cell viability; free radicals.Resumo:O uso de células-tronco como terapia regenerativa tem recebido atenção de pesquisadores nas últimas décadas. A possibilidade de cultivá-las permite o estudo de seu comportamento e metabolismo. Assim, o cultivo celular representa a base para pesquisas de terapia celular e engenharia de tecidos. Uma das principais preocupações relativa ao uso de células-tronco cultivas na rotina clínica humana ou veterinária é a reprogramação dessas células em tumores benignos ou malignos. As atividades celulares necessitam de um estado redox balanceado e quando há algum desequilíbrio nessas reações ocorre o estresse oxidativo. O quadro de estresse oxidativo contribui pra a citotoxicidade podendo resultar em morte celular e até mesmo em alterações genômicas e ocorrência de células cancerígenas. O objetivo deste trabalho foi verificar se há diferenças no comportamento de células-tronco mesenquimais estromais de tecido adiposo de cão de acordo com o seu tecido de coleta (omento e subcutâneo) avaliando o cultivo dessas células quanto a sua taxa de proliferação, viabilidade, estresse oxidativo e citotoxicidade ao longo de seis passagens. Para a execução deste experimento foram utilizadas duas amostras de tecido adiposo coletas do subcutâneo e omento do cadáver de um cão, fêmea, 13 anos de idade, da raça Pitbull. O cadáver era oriundo do Hospital Veterinário Universitário e sofreu eutanásia devido a complicações no seu quadro de cardiomiopatia. As duas amostras foram encaminhadas para o isolamento e cultura celular. Os resultados mostraram que a primeira e última passagem em ambos os grupos são as passagens mais submetidas ao estresse oxidativo ficando mais sujeitas à citotoxicidade.Termos de Indexação: Células-tronco; cultura; passagem; viabilidade celular; radicais livres.IntroductionStem cells (SC) are investigated since 1960 when Ernest A. Mac Culloch and James E. Till observed for the first time a certain undifferentiated bone marrow cell with the ability of self-renewal, self-generation and differentiation (Bonventre & Yang et al. 2011). Since then, stem cells are considered a promise in the field of regenerative medicine and many studies have been developed for their better understanding (Fortier 2005). SC can be classified according to their origin: embryonic stem cells (ESC) are derived from embryos, more specifically in the blastocyst stage, and are able to differentiate themselves in all cell types; adult stem cells (ASC) are found, virtually, in any tissue, however their differentiation is limited to their germ layer origin (Tuan et al. 2003).The first studies with embryonic stem cells (EST) were performed in mice in 1981. Subsequently in 1998 a study with EST derived from a human embryo was reported and it raised ethical and religious issues (Thomson 1998). Due to the controversy involved in the use of EST, adult stem cells became an alternative to new research in cell therapy (Williams 2007). Within literature, ASC isolation has been reported in bone marrow, adipose tissue, kidney, liver, tendon, synovial membrane, amniotic fluid, placenta, umbilical cord and dental pulp (Fortier 2005, Fadel et al. 2011). Among those, adipose tissue was shown to be a reliable and easily accessible source (Kern et al. 2006).The expansion and cultivation of adult and embryonic stem cells allowed significant progress in the fields of regenerative medicine and tissue engineering, as well as in the pharmaceutical industry, in the development and evaluation of new drugs (Sareen 2009). Nevertheless, particular issues must be clarified before cultivated stem cells are widely used in clinical routine. The major concern regarding the cultivation of adult and embryonic stem cells is the development of chromosomal abnormalities, possibly leading to loss of function or potentiating the risk of carcinogenesis. These complications may prevent the therapeutic application of stem cells and should be investigated carefully (Furlani 2009, Sareen 2009).One of the main factors that could be related to chromosomal instability in cultured cells is oxidative stress (OS) (Riley et al. 2008). Some free radicals are physiologically generated during cellular metabolism and often play an important role as messengers and regulators in proliferation, differentiation and apoptosis processes (Dröge 2002, Halliwell 2007). However, when free radical and reactive oxygen species are not regulated by a cascade of antioxidants systems, oxidative stress occurs. OS is detrimental to cells causing ruptures of membranes, protein degradation and DNA damage, which may lead to cell death or genomic abnormality, also favoring the occurrence of carcinogenesis (Pujalté et al. 2011).The aim of this study was to evaluate the rate of proliferation, viability, oxidative stress and damage of mesenchymal stem cell from canine subcutaneous and omental adipose tissue over six passages.Materials and MethodsFor this study was used the corpse of a 13-year-old female Pitbull from the Veterinary Hospital routine. The patient was euthanized due to complications of dilated cardiomyopathy and its death had no relation with this experiment. The owners donated the corpse for research. At about 30 minutes after euthanasia, the samples of adipose tissue were removed from subcutaneous (SUB) and omentum (OM). For the transport of the material previous to cells' isolation, the adipose tissue was separately in falcon tubes containing Hanks' balanced solution (1% streptomycin, 1% amphotericin B) to avoid contamination.Inside the laminar flow, the fragments of adipose tissue from SUB and OM were transferred to two Petri plates. Making use of two scalpel blades each fat was sectioned into small fragments. These fragments were placed separately in 50mL falcon tube along with 2mg/mL collagenase type I in order to promote tissue degradation. The tubes remained in water bath at a temperature of 37°C for 40 minutes being manually shaken every 10 minutes. After this process, the tubes were referred to laminar flow again and complete DMEN medium (1% penicillin, 1% amphotericin B, 10% fetal bovine serum) was added to the solution at a ratio of two parts of medium to one part of collagenase to neutralize the collagenase, given its citotoxicity. The tubes were centrifuged at 600 G for ten minutes. The supernatant was discarded and a new complete medium was added to the cells pellet. The latter solution was transferred to a cell culture flask identified with SUB and OM according to the tissue origin. The flasks were maintained in a CO2 incubator at 37º and 5% concentration of carbon dioxide. The first medium exchange was performed every 24 hours, the following exchanges were realized at every 72 hours. The cells were submitted to six passages and each passage length varied according to the cell growth. When the flask reached 90% of cell confluence, a new passage was made.Each passage was performed after complete removal of the medium by adding 2.5mg/mL trypsin in the flask to remove cells from the plastic. The bottles were subsequently placed in the co2 incubator for five minutes. At the end of this process a new complete medium was placed to inactivate trypsin. The solution from the bottle was then transferred to falcon tubes and centrifuged for five minutes at a speed of 1900 rpm. The precipitate obtained was allocated in the cell culture flask along with complete medium. For every passage the rates of viability, proliferation, cytotoxicity and oxidative stress were assessed.The viability and proliferation rates were estimated by counting viable and non-viable cells in a Neubauer chamber. For this 20 μl of the cell solution was placed in a 1mL micro tube and it was added 20 μl of trypan blue dye at 0.4%. Cells were counted in a Neubauer chamber and the estimation was performed using the following formula: Number of cell per mL = Number of cells counted x dilution coefficient x 104. The number of cell counted was divided by the number of quadrants counted. The dilution coefficient was defined by the rate of cell suspension and the amont of dye (i.e., 1:1) which results in two.Cytotoxicity was measured by means of the DNA fragmentation by fluorimetric Picogreen analyses. A small amount of DNA picogreen was added to the samples, which remained at rest in the dark for five minutes. Soon after, the free-DNA concentration was measured by the fluorometer excited at 485 nm wavelength and fluorescence intensity at 520 nm. Samples were prepared in quadruplicate.The production of oxidative stress was evaluated using a non-fluorescent cell-permeating compound 2-70-dichlorofluorescein diacetate (DCFH-DA assay). DCFH-DA is hydrolysed by intracellular esterases to dichlorofluorescein (DCFH), which is trapped within the cell. This non-fluorescent molecule is then oxidized to fluorescent dichlorofluorescein (DCF) by cellular oxidants. The samples of each culture passage were treated with DCFH-DA for 60 minutes at 37°C. The fluorescence was measured at en excitation wavelength of 485nm and emission of 520 nm. The calibration curve was performed with standard DCF and the level of reactive oxide species production was calculated as nmol DCF formed/mg protein.The lipid peroxidation was determined by measuring thiobarbituric acid reactive species (TBARS). The cell solutions were centrifuged for 10 minutes at 2000rpm, the supernatant was discarded and saline solution (0.9% NaCl) was added, followed by two additional centrifugations at 2000rpm for 10 minutes. After that, the supernatant was discarded and 100ml Butylated hydroxytoluene (BHT 100 mM) and 500μl of trichloroacetic acid (TCA 20%) were added to the sample, followed by final centrifugation at 2000rpm for 5 minutes. Immediately after centrifugation, two samples with 900μl of the supernatant were mixed with a reaction medium containing thiobarbituric acid (TBA 0.8%). Then, the samples were incubated at 95°C for one hour. The absorbance was measured at a wavelength of 532nm in a spectrophotometer. The results were expressed in nmol MDA/106 cells.The results of this experiment were analyzed with one-way analysis of variance followed by Tu

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