RESUMEN
The development of serum-/xeno-free media may help avoid the drawbacks of using serum and its components, such as probable contamination, instability of composition, or difficulty in sterilization. The objectives of this research were to investigate the use of combinations of a permeating cryoprotective agent (Me2SO) and non-permeating polymers (polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol, hydroxyethyl starch, dextran) for cryopreservation of interstitial cells (ICs) of rat testis, and to propose the mechanism of cryoprotection of such compositions. In the course of this study, the best combination was 100 mg/ml dextran (M.m. 40 kDa) (Dex40) with 0.7 M Me2SO in Ham's F12. The ICs were additionally cooled and warmed to different end temperatures (-30, -50, -50 and -196 °C) to determine which temperature intervals contributed most to the IC loss. Then, the cryoprotective action of this serum-/xeno-free medium was investigated in comparison with serum or albumin-containing media by differential scanning calorimetry (DSC) and thermomechanical analysis (TMA). The results showed that the medium based on Dex40 did not decrease the amount of ice formed. However, it could undergo other phase separation and phase transformation to form glassy states. Potential cell-damaging physical processes such as eutectic crystallization/melting, recrystallization of NaCl and/or Me2SO derivatives, found in serum-containing media and taking place in specific temperature intervals, were not observed in the Dex40 based media. This was in good correlation with indicators of cell survival. Additionally, the application of Dex40 allowed using Me2SO in lower concentrations (0.7 M) than required for serum-containing media (1.4 M), which may decrease the toxicity of serum-/xeno-free media.
Asunto(s)
Criopreservación , Dimetilsulfóxido , Animales , Criopreservación/métodos , Crioprotectores/farmacología , Medio de Cultivo Libre de Suero , Dextranos/farmacología , Dimetilsulfóxido/química , Dimetilsulfóxido/farmacología , Masculino , RatasRESUMEN
RATIONALE: Chronic alcohol exposure is associated with impaired decision making skills, cognitive deficits, and poor performance on tasks requiring behavioral flexibility. Although oral routes of alcohol administration are commonly used to examine effects of alcohol on various behaviors in rodents, only a few investigations have used intragastric exposures to evaluate ethanol's effects on behavioral flexibility in the adult rat. OBJECTIVES: The aim of the current series of experiments was to determine if behavioral flexibility impairments would be demonstrated across a variety of procedural factors, including route of administration [intraperitoneal injection (i.p.), intragastric gavage (i.g.)], ethanol dose (3-5 g/kg), number of daily exposures (once/day, twice/day), duration of exposure (2-6 weeks), or length of abstinence (5-7 days). METHODS: Adult male Sprague-Dawley rats were exposed to chronic intermittent ethanol (CIE) or vehicle and evaluated for behavioral intoxication, blood ethanol concentrations (BEC), and performance on a reversal learning odor discrimination task. RESULTS: While all rats displayed behavioral intoxication and elevated BECs, CIE i.p. rats had prolonged elevation in BECs and made the most errors during the reversal learning task. Unexpectedly, CIE i.g. exposures failed to produce deficits during reversal learning tasks regardless of ethanol dose, frequency/duration of exposure, or length of abstinence. CONCLUSIONS: Behavioral flexibility deficits resulting from CIE i.p. exposures may be due to the severity and chronicity of alcohol intoxication. Elucidating the impact of ethanol on behavioral flexibility is critical for developing a better understanding of the behavioral consequences of chronic alcohol exposure.
Asunto(s)
Conducta Animal/efectos de los fármacos , Depresores del Sistema Nervioso Central/farmacología , Etanol/farmacología , Aprendizaje Inverso/efectos de los fármacos , Intoxicación Alcohólica , Animales , Nivel de Alcohol en Sangre , Depresores del Sistema Nervioso Central/administración & dosificación , Etanol/administración & dosificación , Lavado Gástrico , Inyecciones Intraperitoneales , Masculino , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
Long-chain fatty acid amides are signaling lipids found in mammals and other organisms; however, details of the metabolic pathways for the N-acylglycines and primary fatty acid amides (PFAMs) have remained elusive. Heavy-labeled precursor and subtraction lipidomic experiments in mouse neuroblastoma N18TG2 cells, a model cell line for the study of fatty acid amide metabolism, establish the biosynthetic pathways for the N-acylglycines and the PFAMs. We provide evidence that the N-acylglycines are formed by a long-chain specific glycine-conjugating enzyme, glycine N-acyltransferase-like 3 (GLYATL3). siRNA knockdown of GLYATL3 in the N18TG2 cells resulted in a decrease in the levels of the N-acylglycines and the PFAMs. This is the first report of an enzyme responsible for long-chain N-acylglycine production in cellula. The production of the PFAMs in N18TG2 cells was reported to occur by the oxidative cleavage of the N-acylglycines, as catalyzed by peptidylglycine α-amidating monooxygenase (PAM). siRNA knockdown of PAM resulted in an accumulation of [(13)C18]N-oleoylglycine and decreased levels of [(13)C18]oleamide when the N18TG2 cells were grown in the presence of [(13)C18]oleic acid. The addition of [1-(13)C]palmitate to the N18TG2 cell growth media led to the production of a family of [1-(13)C]palmitoylated fatty acid amides, consistent with the biosynthetic pathways detailed herein.
Asunto(s)
Aciltransferasas/fisiología , Ácidos Grasos/biosíntesis , Amidas/metabolismo , Animales , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen , Lipogénesis , RatonesRESUMEN
Thawing in the water bath is often considered as a standard procedure. The thermal history of samples thawed in this way is poorly controlled, but cryopreservation and banking of cell-based products require standardization, automation and safety of all the technological stages including thawing. The programmable freezers allow implementation of the controlled cooling as well as the controlled thawing. As the cell damage occurs during the phase transformation that takes place in the cryoprotectant medium in the process of freezing-thawing, the choice of warming rates within the temperature intervals of transformations is very important. The goal of the study was to investigate the influence of warming rates within the intervals of the phase transformations in the DMSO-based cryoprotectant medium on the cell recovery and to develop a cryopreservation protocol with controlled cooling and warming rates. The temperature intervals of phase transformations such as melting of the eutectic mixture of the cryoprotectant solution (MEMCS), melting of the eutectic salt solution (MESS), melting of the main ice mass (MMIM), recrystallization before MEMCS, recrystallization before MESS and recrystallization before MMIM were determined by thermo-mechanical analysis. The biological experiments were performed on the rat testicular interstitial cells (TIC). The highest levels of the cell recovery and metabolic activity after cryopreservation were obtained using the protocol with the high (20 °C/min) warming rate in the temperature intervals of crystallization of the eutectics as well as recrystallizations and the low (1 °C/min) warming rate in the temperature intervals of melting of the eutectics as well as MMIM. The total cell recovery was 65.3 ± 2.1 %, the recovery of the 3-beta-HSD-positive (Leydig) cells was 82.9 ± 1.8 %, the MTT staining was 32.5 ± 0.9 % versus 42.1 ± 1.7 %; 57.4 ± 2.1 % and 24.0 ± 1.1 % respectively, when compared to the thawing in the water bath.