La vitrification en une seule étape d’embryons murins définit de nouveaux standards de cryopréservation

La cryopréservation d’embryons est un des outils les plus efficaces, économiques et utiles au niveau éthique pour préserver indéfiniment la génétique des animaux de laboratoire. Les bénéfices liés à son utilisation sont nombreux, et incluent la réduction des coûts associés à la pérennisation de lignées, la limitation de l’apparition et de la dissémination de mutations non voulues, la facilité et la sécurité pour les transferts internationaux, et la réduction du nombre d’animaux à élever en captivité. La vitrification a été démontrée comme étant plus efficace que la congélation lente en procréation médicalement assistée humaine, où elle constitue maintenant la méthode de référence. Il en va de même pour la cryopréservation des embryons de souris, où la vitrification préserve mieux l’intégrité de la chromatine, réduit la pénétration intracytoplasmique d’agents cryoprotecteurs potentiellement toxiques, et in fine permet une meilleure survie et un meilleur développement après réchauffement que la congélation lente. Cependant, pour être efficaces, les méthodes actuelles de vitrification nécessitent plusieurs étapes d’exposition des embryons à des solutions spécifiques avant le refroidissement, mais aussi au cours de leur réchauffement ultérieur. Cette relative complexité peut s’avérer difficile à gérer de manière optimale quand un grand nombre d’embryons doivent être traités au cours d’une même session. Nous avons développé et breveté une technologie de vitrification d’embryons en une étape (« one-step ») qui est aussi efficace que les meilleures méthodes de vitrification multi-étapes. De plus, nos milieux sont chimiquement définis (sans sérum ou autre composant biologique non défini), et des supports permettant la vitrification aseptique peuvent être utilisés sans perte de rendement. Notre technologie de vitrification one-step d’embryons de rongeurs répond ainsi aux problèmes d’ergonomie liés aux méthodes classiques de vitrification, et fournit ainsi aux scientifiques une solution efficace, biologiquement sûre et facile à utiliser pour la cryopréservation d’embryons de rongeurs. Elle rend ainsi l’efficacité de la vitrification plus aisément applicable aux rongeurs de laboratoire, contribuant ainsi à la réduction du nombre d’animaux nécessaires à la pérennisation et à la diffusion de lignées ou de colonies utiles.Vitricell: développement et valorisation de kits de vitrification de cellules en conditions aseptiques et chimiquement définie

VitriCell: the safest key for cryopreserving your valuable cells

VITRICELL is a future spin-off company aiming at providing standard and customized solutions for safe and efficient cryopreservation of valuable and sensible eukaryotic cells. Developed in the Embryology unit of the Faculty of Veterinary Medicine of the University of Liège, the core methodology relies on aggregation of inventor’s pioneering expertises in the fields of stem cells engineering, vitrification of gametes and embryos and in assisted reproductive technologies. VITRICELL will soon provide researchers and clinicians with vitrification kits, allowing upgrade of the current yields and safety after cryopreservation of their high-value cells. The benefits of our method are the most prominent with fragile cell lines limited in expansion ability. VITRICELL’s current product is based on a novel and user-friendly, aseptic and automatable vitrification method in sealed french straws, with bio-safe and chemically defined media. The absence of any ice crystal formation avoids excessive cell dehydration and injuries to cell membranes. As a consequence, VITRICELL’s method results in a higher efficiency (recovery rates, morphology, pluripotency…) than conventional slow freezing for cryopreserving human pluripotent stem cells (hPSCs) and other sensitive stem cell-like lines and embryos from human and non-human species. VITRICELL’s future products developments focuses on (i) vitrification of higher cells numbers in larger containers, (ii) implementation of the method to current automation processes.Vitricell: développement et valorisation de kits de vitrification de cellules en conditions aseptiques et chimiquement définie

One-step vitrification of murine embryos redefines cryopreservation standards

Cryopreservation of embryos is amongst the most powerful and efficient tools for indefinitely preserving the genetics of laboratory animals. The ensuing benefits are numerous and include reduction of costs associated to strain perpetuation, limitation of mutations occurrence and spreading, ease and safety of transnational shipping, and reduction of live animal husbandry. It has been demonstrated that vitrification is more efficient than slow freezing in human assisted reproduction, where it stands now as the gold standard. This is equally true for murine embryos, where vitrification has been shown to better preserve chromatin integrity, induce lower intracellular ingress of cryoprotectants and ultimately yield better embryo survival and development than slow freezing. Beside these benefits, current vitrification procedures require multiple pre-cooling and post-warming exposure steps to dedicated solutions to reach maximum effectiveness, which appears difficult to deal with when many embryos must be cryopreserved in one single session. We have developed and patented a unique one-step embryo vitrification procedure which is as efficient as the best multi-step vitrification methods. Moreover, our media are chemically defined (no serum nor undefined biological component), and aseptic vitrification carriers can be used without any yield loss. Our one-step vitrification kits and media for rodents (VitriMice™, VitriCell) address the poor ergonomy issues of classical vitrification, providing scientists with efficient, biologically safe and user-friendly solutions for embryo cryopreservation. Consequently, our one-step vitrification technology improves efficiency and applicability of cryopreservation for laboratory rodents, thereby contributing to the reduction of the number of live animals required to perpetuate and spread useful strains and colonies.Vitricell: développement et valorisation de kits de vitrification de cellules en conditions aseptiques et chimiquement définie

ONE-STEP VITRIFICATION OF MURINE EMBRYOS CHALLENGES CURRENT PARADIGMS OF CRYOBIOLOGY

peer reviewedCryopreservation of embryos is amongst the most powerful tools for preserving the genetics of laboratory animals. Vitrification is widely recognized as more efficient than slow freezing e.g. in human assisted reproduction technologies and for preserving murine embryos. Unfortunately, current vitrification procedures require multiple pre-cooling and post-warming exposure steps to dedicated solutions to reach maximum effectiveness, which appears difficult to deal with when many embryos are cryopreserved in one single session. To help solving this issue, we have developed a one-step embryo vitrification procedure. Briefly, embryos are exposed to a unique –chemically defined- vitrification solution before plunging in the liquid nitrogen. Subsequent warming is performed by immersing the vitrified embryos directly into the culture medium. Murine embryos at the zygote, two cells and morula stages have undergone our one-step procedure either under aseptic or non-aseptic conditions. After warming, direct in vitro survival, development to the blastocyst stage and in vivo development to birth were recorded as endpoints. Short exposure times to the vitrification solution (less than 60 seconds) before cooling and direct warming into culture medium led to results equivalent or better than after classical vitrification. Longer exposure times to the vitrification solution (between 90 and 150 seconds) decreased efficiency. These results demonstrate that intracellular vitrification after our one-step procedure occurs by combined effects of fast cooling (or warming) and cell dehydration, with minimal, if any, ingress of cryoprotectants. The absence of deleterious effect of warming directly into the culture medium and the relatively low sensitivity to thermal inertia (aseptic vs non-aseptic) of the carrier is a confirmation thereof. Consequently, beyond bringing a methodological simplification without any loss of efficiency, our patented one-step vitrification procedure dramatically lowers if not suppresses intracellular concentration of cryoprotectants and associated toxicity, thereby challenging some commonly accepted concepts of cryobiology.Vitricell: développement et valorisation de kits de vitrification de cellules en conditions aseptiques et chimiquement définie

Distribution of Carbohydrates in Gluten Fractions Isolated from European Wheats (Triticum aestivum l.) in a Batter System

Glucose represented 89% of the total carbohydrate of gluten isolated in a batter system from wheat cultivars exhibiting varying breadmaking qualities. Galactose, arabinose (Ara), and xylose (Xyl) were the remaining carbohydrates (ca. 10% of the total carbohydrate content), while mannose was present in minor quantities (<1%). After Pronase treatment ca. 43% of the Ara and ca. 52% of the Xyl originating from arabinoxylan (AX) could be solubilized, indicating the presence of water extractable as well as water unextractable AX. Arabinogalactan-peptide (AGP) and small amounts of beta-glucan were also present. Water extractable AX with a low Ara-to-Xyl ratio (A/X) and thus a low degree of substitution preferentially are incorporated in the gluten network probably as a result of alignment between glutenin polymers and AX in the stirring direction. In gluten from cultivars exhibiting high gluten protein recoveries (see accompanying paper; Roels et al. J. Agric. Food Chem. 1998, 46, 1344-1349), the levels of total gluten associated nonstarch polysaccharides (TOTGANSP), expressed as a percentage of total flour NSP, were also high. It was postulated that the gluten proteins of such cultivars agglomerate despite the presence of NSP since the gluten fractions with the best agglomeration properties contained the lowest amount of NSP. In the aforementioned gluten fractions the AGP/AX ratio was also unusually high, indicating that the undocumented role of AGP in wheat gluten agglomeration may well be underestimated.status: publishe

Distribution and Structural Variation of Arabinoxylans in Common Wheat Mill Streams

In 19 wheat-milling fractions total pentosan content, calculated as 0.88 x (% L-arabinose + % D-xylose), varied between 1.44 and 30.66% on dry matter (dm). It increased with ash content once the latter exceeded 0.6% (dm basis). Water-extractable arabinoxylans were recovered by saturating water extracts to 65% ethanol. Their contents in the milling fractions varied between 0.35 and 1.38%, and above 0.6% ash content also increased with this parameter. Their L-arabinose-to-D-xylose ratios ranged between 0.65 and 0.39, with the lowest values found for the fractions with highest ash content, indicating that the ash-rich tissues contain more arabinoxylans that are less branched. (1)H NMR spectroscopy revealed that the decrease in L-arabinose-to-D-xylose ratio was accompanied by an increase in unsubstituted xylose residues and a decrease in disubstituted xylose residues, while the contents of monosubstituted xyloses were virtually constant.status: publishe

Contents and Structural Features of Water-Extractable Arabinogalactan in Wheat Flour Fractions

In Camp Remy Buhler MLU-202 roller mill flour fractions, the levels of water-extractable arabinoxylan (WE-AX) (0.30-0.41%, dry basis) were comparable to those of water-extractable arabinogalactan (WE-AG) (0.29-0.38%, dry basis). Minaret had more WE-AX (0.49-0.68%, dry basis) than WE-AG (0.27-0.36%, dry basis). The ratio of WE-AG to WE-AX for the different flour fractions varied between 0.83 and 1.03 for Camp Remy and between 0.46 and 0.57 for Minaret. For both wheat varieties, the percentage of WE-AX and WE-AG was higher for the second and third reduction roll fractions than for the three break and the first reduction roll fractions. There was little structural variation in WE-AG of different flour fractions. The arabinose to galactose (A/G) ratio (w/w) varied between 0.63 and 0.69 for Camp Remy WE-AG and between 0.66 and 0.72 for Minaret WE-AG. The molecular weight range of the water-extractable arabinogalactan-peptide (WE-AGP) isolated from the different flour fractions of Camp Remy and Minaret was 5 x 10(4)-10 x 10(4). The 1H NMR spectra of the WE-AGP isolated from the different flour fractions were comparable and display the following diagnostic peaks (300 MHZ, D2O, 85 degrees C): delta 5.26, anomeric protons of alpha-lined arabinofuranosyl residues; and delta 4.47-4.54, anomeric protons of beta-linked galactose residues.status: publishe

Growth hormone in meat production

peer reviewedaudience: researcher, professional, studentThe somatotrope axis has been described in relation with the physiological effects associated with growth horone and somatomedins secretion. Animal productions were descried when growth hormone was administred to livestock.Les différents composants de l'axe somatotrope ont été décrits dans le care des effets physiologiques associés à la libération de l'hormone de croissance et des somatomédines. Les effets zootechniques de l'administration d'hormone de croissance chez les animaux de rente ont également été étudiés

Single step vitrification metrhod

publication date: 2018-07-05; filing date: 2017-12-15Ce brevet rapporte la description et les résultats d'une nouvelle méthode de vitrification cellulaire dont le conditionnement repose sur une seule et brève étape d'exposition des cellules à un milieu vitrifiant avant le refroidissement brutal dans l'azote liquide. Il en résulte une (quasi-)absence de pénétration intracellulaire de cryoprotecteurs, le conditionnement reposant essentiellement sur une déshydratation. Le réchauffement est effectué lui aussi en une seule étape, sans nécessité de passer par des étapes d'équilibration dans des solutions hypertoniques. Cette méthode remet en cause les paradigmes de la vitrification, qui postulent la nécessité d'une équilibration progressive des milieux extra et intracellulaires préalables au refroidissement et durant le réchauffement, et où des cryoprotecteurs pénètrent dans la cellule.BEST Stem cells, VitriCel