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

Blastocyst transfer after aseptic vitrification of zygotes: an approach to overcome an impaired uterine environment

In some IVF cycles, no fresh embryo transfer in the stimulated cycle is advisable. The cryopreservation of zygotes and the transfer of blastocysts in a cryo-embryo transfer is an option to circumvent an inadequate uterine environment due to risk of ovarian hyperstimulation syndrome, inappropriate endometrium build up, endometrial polyps or uterine myomas. For this strategy, highly secure and safe cryopreservation protocols are advisable. This study describes a protocol for aseptic vitrification of zygotes that results in high survival rates and minimizes the potential risk of contamination in liquid nitrogen during cooling and long-term storage. In mouse zygotes, there was no difference in efficiency as compared with a conventional open vitrification system. In IVF patients, aseptically vitrified zygotes showed no difference in blastocyst formation rate as compared with sibling zygotes kept in fresh culture. A clinical study comprising 173 cryo-cycles with a transfer of blastocysts originating from vitrified zygotes showed an ongoing pregnancy rate of 40.9%. The live birth rate per patient was 36.8%. A combination of good clinical results and increased safety conditions due to aseptic vitrification encourages the use of cryo-embryo transfer for patients with a suboptimal uterine environment in a fresh cycle

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

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

Closed carrier device: A reality to vitrify oocytes and embryos in aseptic conditions

Vitrification with the use of ‘‘Open’’ carrier devices (Cryoloop, cryotop, cryoleaf, Vitriplug) which allowed the contact with liquid nitrogen has become a more popular way to achieve cooling rate superior to 20.000 °C/min. Even though the question of contamination with liquid nitrogen during ultra-rapid cooling and storage remain debatable with the use of ‘‘open’’ devices, it is important to revise the carrier system in a way, which minimizes the risk of contamination. According to the EU tissues and cells directive, it is advisable that the cooling and storage should be carried out in embryo carrier devices ensuring complete separation of the embryos from liquid nitrogen in a way, which minimizes the risk of contamination. The consequence of a reduction in the cooling rate resulting from the heat-insulating barrier of aseptic devices has to be counteracted by gradually increasing intracellular concentrations of cryoprotectants without inducing a toxic effect. We developed an aseptic vitrification method of vitrification for MII oocytes and embryos at different stage of development using the ‘‘VitriSafe’’ as ‘‘closed’’ carrier device

Cryopréservation d’ovocytes et d’embryons par congélation ou vitrification dans le cadre de l’assistance médicale à la procréation

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