WO2014057148A2 - Method for the vitrification of biological material using closed thermoplastic polymer microcapillaries introduced into cryogenic fluids - Google Patents
Method for the vitrification of biological material using closed thermoplastic polymer microcapillaries introduced into cryogenic fluids Download PDFInfo
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- WO2014057148A2 WO2014057148A2 PCT/ES2013/000228 ES2013000228W WO2014057148A2 WO 2014057148 A2 WO2014057148 A2 WO 2014057148A2 ES 2013000228 W ES2013000228 W ES 2013000228W WO 2014057148 A2 WO2014057148 A2 WO 2014057148A2
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- microcapillary
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0236—Mechanical aspects
- A01N1/0263—Non-refrigerated containers specially adapted for transporting or storing living parts whilst preserving, e.g. cool boxes, blood bags or "straws" for cryopreservation
- A01N1/0268—Carriers for immersion in cryogenic fluid, both for slow-freezing and vitrification, e.g. open or closed "straws" for embryos, oocytes or semen
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- the invention patent object of the present specification refers to a new method of cryopreservation of biological material, which achieves the vitrification of the samples with the presence of cryoprotectant.
- the procedure is based on the use of a microcapillary of thermoplastic polymers of small dimensions and heat sealing ( Figure 1 and 2), called Safespeed, introduced in liquid nitrogen to achieve the correct vitrification and avoid cross contamination.
- the patent is framed within the biotechnology sector, and within it in the area of cellular cryopreservation, that is, cell preservation at cryogenic temperatures for long periods of time keeping its functionality intact. This process has special relevance in the conservation of oocytes, embryos, stem cells, and all types of organism or biological material that can be introduced into the capillary.
- the invention patent object of the present specification is a continuation of the development of the author's own technique [15].
- the main techniques used to achieve cell preservation are based on the use of low temperatures to slow down and stop the biochemical reactions responsible for cell aging and deterioration. Within these techniques, the most widely used are the calls of slow cooling and vitrification, with a large number of variants in both cases.
- Vitrification is a process by which a liquid solidifies in a non-crystalline phase (vitreous phase) with a rapid decrease in temperature and an increase in viscosity. Vitrification completely eliminates the formation of ice crystals, which pose the greatest obstacle to cryopreservation of living cells [1]. This technique has greatly improved the results obtained by slow cooling, although it is still insufficient for the cryopreservation of certain cell types, such as oocytes [2], or stem cells [3], where sensitivity Cellular demands an improvement of the methods used today.
- vitrifying agents also called cryoprotectants
- cryoprotectants are used because of their relationship with cellular protection.
- some of them such as dimethylsulfoxide or ethylene glycol
- good results have been achieved [5,6].
- Different containers have been applied to introduce cells in liquid nitrogen, among which we find copper grids used in electron microscopy [7], OPS capillaries [8,11], (Open Pulled Straws, conventional PVC capillaries stretched to have a smaller diameter inside) or the so-called cryoloops [9,12], nylon ties in which a solution film containing the cells is suspended.
- CryoTip [14], or Cryopette are heat sealable closed systems that have also been widely used in assisted reproduction. Being heat sealable, the sample is isolated inside without any contact with the outside, thus minimizing the risk of contamination during the process of vitrification, storage in nitrogen tank, and subsequent reheating.
- the cell survival rate achieved with the present invention is much higher than that obtained with these systems.
- FIG. 1 Microcapillary geometry, main straw and outer cap in open use position. Both ends of the microcapillary and main straw are heat sealable.
- the polycarbonate microcapillary (1) heat sealed and containing the biological sample at the lower end, has a coating (2), which transfers rigidity, protection and ease of use.
- the cap or cover (3) is fixed to the main straw through an inner mechanical closure of the coating
- FIG. 1 Microcapillary geometry, main straw and outer cap in closed storage position.
- the cap or cover (3) prevents the capillary from rubbing against the walls and base of the storage system, protecting the sample.
- the container device object of the present invention (Safespeed) consists of a polycarbonate straw, optimized as a closed system for cryopreservation procedures. It consists of an ultra thin capillary (microcapillary) mounted on a straw and a protective sheath.
- the container device object of the present invention (Safespeed) prevents the nucleation and growth of ice in the process of vitrification of cell samples, obtaining cell survival rates close to 100% and allowing heat sealing and isolation of the sample, to avoid cross contamination of samples during vitrification, storage and reheating,
- the new type of cellular container (Safespeed), in which the biological sample to be cryopreserved is introduced, together with the necessary concentration of cryoprotectant and serum, is manufactured by a set of assembled thermoplastic polymers (such as polycarbonate).
- thermoplastic microcapillary In order to increase the ease in handling the thermoplastic microcapillary, it can be introduced into another straw of greater diameter and rigidity, mobile but not removable, which will in turn act as protection for the microcapillary.
- This protective straw can discover (use position) or cover (storage position) the microcapillary through a mechanical closing mechanism.
- the use position allows optimize the vitrification process, and the storage position minimizes or eliminates both the risk of capillary rupture and the risk of cell sample loss.
- the container consists of the following parts:
- a plastic capillary (smaller than 250 microns in diameter) in which the biological sample (s) are deposited.
- a main straw connected to the microcapillary that provides rigidity and allows the sample to flow to the capillary by suction through the back.
- a thin-walled rear end section that allows connection to a standard suction system and also the closing by heat (heat sealer).
- a plastic protector that slides over the outer straw and allows the capillary to be exposed for the moment of the biological sample suction and the introduction into liquid nitrogen, and which in turn allows another second storage position once the Sample is vitrified, in which it covers the capillary so that it prevents it from colliding with other objects and damaging the sample inside.
- a suction device for example, a Hamilton syringe, an automatic pipette, a stripper, a mouth pipette or any other oocyte aspiration and manipulation system
- a suction device for example, a Hamilton syringe, an automatic pipette, a stripper, a mouth pipette or any other oocyte aspiration and manipulation system
- the total length of the complete device will be about 80-140 mm, which adapts to the storage standards, adjusting to the practices for assisted reproduction, allowing a comfortable manipulation and minimization of the space they occupy in the stocks.
- Example 1 Procedure for cell preservation of oocytes
- This example shows the practical application of the method for cryopreservation of mouse oocytes.
- mice C57BL / 6 x CBA / Ca virgin mice (4-8 weeks) kept under controlled conditions (12 light hours: 12 dark hours) and fed ad libitum were used. They were superovulated by intraperitoneal injection with 0.1 ml of PMSG (5 Ul). After 48-56 hours, 0.1 ml of HCG (5 Ul) was administered. They were sacrificed 14 hours later by cervical dislocation and the oviducts were removed.
- the oocytes were released from the cluster by hyaluronidase (SIGMA Ref. H4272-30 mg). After this, the oocytes were washed in M2 medium to remove the remains of hyaluronidase The previously denuded metaphase II oocytes were then transferred to an equilibrium solution, and then to the vitrification solution.
- hyaluronidase SIGMA Ref. H4272-30 mg.
- the oocytes were introduced into the microcapillary, which was heat sealed, with the biological sample at the lower end.
- the microcapillary was introduced into the liquid nitrogen container manually, in the position of use, as shown in Figure 1.
- the microcapillary was placed in storage position covered by the cap, and was transported in a plastic canister to a liquid nitrogen tank, where it remained 24 hours.
- the microcapillary was introduced into a 37.5 ° C water bath, manually (again in use position, without cap).
- mice oocytes were cryopreserved and reheated, of which 595 survived, obtaining an oocyte survival of 96%, much higher than other vitrification techniques using closed devices.
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Abstract
The invention relates to a novel method for the cryopreservation of biological material, vitrifying the samples with the presence of cryoprotector. The method is based on the use of thermoplastic polymer microcapillaries of reduced dimensions, which are heat sealed (figure 1 and 2) and introduced into liquid nitrogen in order to produce correct vitrification and prevent cross contamination.
Description
Título Title
Procedimiento de vitrificación de material biológico mediante microcapilares de polímeros termoplásticos cerrados introducidos en fluidos criogénicos Procedure of vitrification of biological material by microcapillars of closed thermoplastic polymers introduced in cryogenic fluids
Objeto de la invención Object of the invention
La patente de invención objeto de la presente memoria se refiere a un nuevo procedimiento de criopreservación de material biológico, que consigue la vitrificación de las muestras con presencia de crioprotector. El procedimiento se basa en el uso de un microcapilar de polímeros termoplásticos de reducidas dimensiones y termosellado (figura 1 y 2), llamado Safespeed, introducido en nitrógeno líquido para conseguir la correcta vitrificación y evitar la contaminación cruzada. La patente se enmarca dentro del sector de la biotecnología, y dentro de éste en el área de la criopreservación celular, es decir, conservación de células a temperaturas criogénicas durante largos períodos de tiempo manteniendo intacta su funcionalidad. Este proceso tiene especial relevancia en la conservación de ovocitos, embriones, células madre, y todo tipo de organismo o material biológico que pueda ser introducido en el capilar. La patente de invención objeto de la presente memoria es una continuación del desarrollo de la técnica del propio autor [15]. The invention patent object of the present specification refers to a new method of cryopreservation of biological material, which achieves the vitrification of the samples with the presence of cryoprotectant. The procedure is based on the use of a microcapillary of thermoplastic polymers of small dimensions and heat sealing (Figure 1 and 2), called Safespeed, introduced in liquid nitrogen to achieve the correct vitrification and avoid cross contamination. The patent is framed within the biotechnology sector, and within it in the area of cellular cryopreservation, that is, cell preservation at cryogenic temperatures for long periods of time keeping its functionality intact. This process has special relevance in the conservation of oocytes, embryos, stem cells, and all types of organism or biological material that can be introduced into the capillary. The invention patent object of the present specification is a continuation of the development of the author's own technique [15].
Estado de la técnica State of the art
Las principales técnicas empleadas para conseguir la conservación celular se basan en la utilización de bajas temperaturas para ralentizar y detener las reacciones bioquímicas responsables del envejecimiento y deterioro de la célula. Dentro de estas técnicas, las más ampliamente utilizadas son las llamadas de enfriamiento lento y las de vitrificación, con un gran número de variantes en ambos casos. The main techniques used to achieve cell preservation are based on the use of low temperatures to slow down and stop the biochemical reactions responsible for cell aging and deterioration. Within these techniques, the most widely used are the calls of slow cooling and vitrification, with a large number of variants in both cases.
El enfriamiento lento es hasta ahora el procedimiento mas usado. Se basa en el control de la tasa de enfriamiento con el objetivo de crear un delicado equilibrio entre los distintos factores que causan daño celular, entre los que se encuentran la formación de hielo, fracturas y una excesiva deshidratación de la célula. Slow cooling is so far the most used procedure. It is based on the control of the cooling rate in order to create a delicate balance between the different factors that cause cell damage, including ice formation, fractures and excessive dehydration of the cell.
La vitrificación es un proceso mediante el cual un líquido se solidifica en una fase no cristalina (fase vitrea) con un rápido descenso de la temperatura y un aumento en la viscosidad. La vitrificación elimina completamente la formación de cristales de hielo, los cuales suponen el mayor obstáculo para la criopreservación de células vivas [1] . Esta
técnica ha mejorado en gran medida los resultados obtenidos por el enfriamiento lento, aunque todavía es insuficiente para la criopreservación de determinados tipos celulares, como es el caso de los ovocitos [2], o de las células madre [3], en donde la sensibilidad celular exige una mejora de los métodos utilizados en la actualidad. Vitrification is a process by which a liquid solidifies in a non-crystalline phase (vitreous phase) with a rapid decrease in temperature and an increase in viscosity. Vitrification completely eliminates the formation of ice crystals, which pose the greatest obstacle to cryopreservation of living cells [1]. This technique has greatly improved the results obtained by slow cooling, although it is still insufficient for the cryopreservation of certain cell types, such as oocytes [2], or stem cells [3], where sensitivity Cellular demands an improvement of the methods used today.
Con el objetivo de incrementar la viscosidad de la muestra y evitar la formación de hielo se utilizan los llamados agentes vitrificantes, también llamados crioprotectores, por su relación con la protección celular. Con algunos de ellos, como el dimetilsulfóxido o el etilenglicol se han conseguido buenos resultados [5,6]. Diferentes contenedores han sido aplicados para introducir células en nitrógeno líquido, entre las que encontramos rejillas de cobre utilizadas en microscopía electrónica [7], capilares OPS [8,11], (Open Pulled Straws, capilares de PVC convencionales estirados para tener un menor diámetro interior) o los llamados cryoloops [9,12], lazos de nylon en los que se suspende una película de solución conteniendo las células. In order to increase the viscosity of the sample and avoid the formation of ice, the so-called vitrifying agents, also called cryoprotectants, are used because of their relationship with cellular protection. With some of them, such as dimethylsulfoxide or ethylene glycol, good results have been achieved [5,6]. Different containers have been applied to introduce cells in liquid nitrogen, among which we find copper grids used in electron microscopy [7], OPS capillaries [8,11], (Open Pulled Straws, conventional PVC capillaries stretched to have a smaller diameter inside) or the so-called cryoloops [9,12], nylon ties in which a solution film containing the cells is suspended.
CryoTip [14], o Cryopette son sistemas cerrados termosellables que han sido también ampliamente utilizados en reproducción asistida. Al ser termosellables, la muestra queda aislada en el interior sin contacto alguno con el exterior, minimizando así el riesgo de contaminación durante el proceso de vitrificación, almacenamiento en tanque de nitrógeno, y recalentamiento postetior. Sin embargo, la tasa de supervivencia celular conseguida con la presente invención es muy superior a la obtenida con estos sistemas. CryoTip [14], or Cryopette are heat sealable closed systems that have also been widely used in assisted reproduction. Being heat sealable, the sample is isolated inside without any contact with the outside, thus minimizing the risk of contamination during the process of vitrification, storage in nitrogen tank, and subsequent reheating. However, the cell survival rate achieved with the present invention is much higher than that obtained with these systems.
También en los últimos años, han aparecido otros contenedores abiertos como CryoTop [13], o Cryoleaf con buenos resultados en supervivencia ovocitaria pero con la problemática de no asegurar un potencial caso de contaminación cruzada. Also in recent years, other open containers such as CryoTop [13], or Cryoleaf have appeared with good results in ovocyte survival but with the problem of not ensuring a potential case of cross contamination.
DOCUMENTOS RELEVANTES RELEVANT DOCUMENTS
[1] Gábor Vajta, Masashige Kuwayama, Improving cryopreservation systems, [1] Gábor Vajta, Masashige Kuwayama, Improving cryopreservation systems,
Theriogenology 65 (2006), 236-244) Theriogenology 65 (2006), 236-244)
[2] David K. Gardner, In vitro fertilization, a practical approach, Taylor & Francis; Édition: 1 (29 septembre 2006) [2] David K. Gardner, In vitro fertilization, a practical approach, Taylor &Francis; Édition: 1 (September 29, 2006)
[3] S.S. Gouk, C.K.F. Tan, M.P. Hande, A. Poonepalli, G.S. Dawe and Lilia L. Kuleshova, Protein- and serum-free vitrification of neural stem cells, Cryobiology, Volume 53, Issue 3, December 2006, Page 389
[4] S. Llavin, A. Arav, Measurement of essential physical properties of vitrification solutions, Theriogenology 2007; 67; 81-89. [3] SS Gouk, CKF Tan, MP Hande, A. Poonepalli, GS Dawe and Lilia L. Kuleshova, Protein- and serum-free vitrification of neural stem cells, Cryobiology, Volume 53, Issue 3, December 2006, Page 389 [4] S. Llavin, A. Arav, Measurement of essential physical properties of vitrification solutions, Theriogenology 2007; 67; 81-89.
[5] Rail WF, Fahy GM: lce-free cryopreservation of mouse. embryos at -196 by vitrification. Nature 1985;313:573-. 575. [5] Rail WF, Fahy GM: lce-free cryopreservation of mouse. embryos at -196 by vitrification. Nature 1985; 313: 573-. 575
[6] T. Takahashi, A. Hirsh, E. F. Erbe, J. B. Bross, R. L. Steere and R. J. Williams [6] T. Takahashi, A. Hirsh, E. F. Erbe, J. B. Bross, R. L. Steere and R. J. Williams
Vitrification of human monocytes, Cryobiology, Volume 23, Issue 2, April 1986, Pages 103- 115. Vitrification of human monocytes, Cryobiology, Volume 23, Issue 2, April 1986, Pages 103-115.
[7] Martino A, Songsasen N, Leibo S. P, Developement into blastocysts of bovine oocytes cryuopreserved by ultrarapid cooling, Biol Reprod 1996;54; 1059-1069 [7] Martino A, Songsasen N, Leibo S. P, Developing into blastocysts of bovine oocytes cryuopreserved by ultrarapid cooling, Biol Reprod 1996; 54; 1059-1069
[8] Vajta, G, Booth PJ, Holm P, Greve T, Caliesen H, Succesful vitrification of early stage bovine in vitro produced embryos with the open pulled straw (OPS) method, Cryo-Letters 1997;18;191-195 [9] Lañe M, Schoolcraft WB, Gardner DK, Vitrification of mouse and human blastocysts using a novel cryoloop container-less technique, Fértil Steril, [8] Vajta, G, Booth PJ, Holm P, Greve T, Caliesen H, Succesful vitrification of early stage bovine in vitro produced embryos with the open pulled straw (OPS) method, Cryo-Letters 1997; 18; 191-195 [ 9] Lañe M, Schoolcraft WB, Gardner DK, Vitrification of mouse and human blastocysts using a novel cryoloop container-less technique, Fertile Steril,
1999;72; 1073-1078 1999; 72; 1073-1078
[10] M.A.Nowshari and G. Bren, Effect of freezing rate and exposure time to cryoprotectant on the development of mouse pronuclear stage embryos, Human Reproduction 15 (2001), 2368-2373. [10] M.A. Nowshari and G. Bren, Effect of freezing rate and exposure time to cryoprotectant on the development of mouse pronuclear stage embryos, Human Reproduction 15 (2001), 2368-2373.
[11] G. Vajta, P. Holm, T. Greve and H. Caliesen, Vitrification of porcine embryos using the Open Pulled Straw (OPS) method, Acta Vet.Scand. 38 (1997) 349-352. [11] G. Vajta, P. Holm, T. Greve and H. Caliesen, Vitrification of porcine embryos using the Open Pulled Straw (OPS) method, Acta Vet.Scand. 38 (1997) 349-352.
[12] T. Mukaida, S. Nakamura, T. Tomiyama, S. Wada, M. Kasai and K. Takahashi, Succesful birth after transfer of vitrified human blastocysts with us of a cryoloop containerless technique, Fértil. Steril. 76 (2001) 618-620. [12] T. Mukaida, S. Nakamura, T. Tomiyama, S. Wada, M. Kasai and K. Takahashi, Succesful birth after transfer of vitrified human blastocysts with us of a cryoloop containerless technique, Fertile. Steril 76 (2001) 618-620.
[13] Kuwayama M, Vajta G, Kato O, Leibo S. P, Highly efficient vitrification method for cryopreservation of human oocytes, Reprod Biomed Online, 2005;11 ;300-308. [13] Kuwayama M, Vajta G, Kato O, Leibo S. P, Highly efficient vitrification method for cryopreservation of human oocytes, Reprod Biomed Online, 2005; 11; 300-308.
[14] Kuwayama M, Vajta G, lEda S, Kato O, Vitrification of human embryos using the CryoTip method, Reprod Biomed Online, in press RMP Online vol 11. No5.2005. [14] Kuwayama M, Vajta G, lEda S, Kato O, Vitrification of human embryos using the CryoTip method, Reprod Biomed Online, in press RMP Online vol 11. No5.2005.
[15] R. Risco, A. Olmo, J. L Sánchez, Procedimiento de criopreservación celular por vitrificación con bajas concentraciones de crioprotector mediante transferencia térmica por convección forzada. P200702565. Universidad de Sevilla, 2008 [15] R. Risco, A. Olmo, J. L Sánchez, Procedure of cell cryopreservation by vitrification with low concentrations of cryoprotectant by thermal transfer by forced convection. P200702565. University of Seville, 2008
Descripción del contenido de las figuras Description of the content of the figures
Figura 1. Geometría del microcapilar, pajuela principal y capuchón exterior en posición de uso abierta. Ambos extremos del microcapilar y pajuela principal son termosellables.
El microcapilar de policarbonato (1), termosellado y conteniendo la muestra biológica en el extremo inferior, tiene un recubrimiento (2), que le transfiere rigidez, protección y facilidad en su uso. El capuchón o cobertura (3), se fija a la pajuela principal a través de un cierre mecánico interior del recubrimiento Figure 1. Microcapillary geometry, main straw and outer cap in open use position. Both ends of the microcapillary and main straw are heat sealable. The polycarbonate microcapillary (1), heat sealed and containing the biological sample at the lower end, has a coating (2), which transfers rigidity, protection and ease of use. The cap or cover (3), is fixed to the main straw through an inner mechanical closure of the coating
Figura 2. Geometría del microcapilar, pajuela principal y capuchón exterior en posición cerrada de almacenamiento. Figure 2. Microcapillary geometry, main straw and outer cap in closed storage position.
El capuchón o cobertura (3), evita que el capilar roce con las paredes y base del sistema de almacenamiento, protegiendo la muestra. The cap or cover (3), prevents the capillary from rubbing against the walls and base of the storage system, protecting the sample.
Descripción de la invención Description of the invention
El dispositivo contenedor objeto de la presente invención (Safespeed) consiste en una pajuela de policarbonato, optimizada como sistema cerrado para los procedimientos de criopreservación. Se compone de un capilar ultra fino (microcapilar) montado sobre una pajuela y una funda protectora. The container device object of the present invention (Safespeed) consists of a polycarbonate straw, optimized as a closed system for cryopreservation procedures. It consists of an ultra thin capillary (microcapillary) mounted on a straw and a protective sheath.
El dispositivo contenedor objeto de la presente invención (Safespeed) evita la nucleación y el crecimiento del hielo en el proceso de vitrificación de las muestras celulares, obteniendo tasas de supervivencia celular cercanas al 100% y posibilitando el termosellado y aislamiento de la muestra, para evitar la contaminación cruzada de las muestras durante la vitrificación, almacenamiento y recalentamiento, The container device object of the present invention (Safespeed) prevents the nucleation and growth of ice in the process of vitrification of cell samples, obtaining cell survival rates close to 100% and allowing heat sealing and isolation of the sample, to avoid cross contamination of samples during vitrification, storage and reheating,
El nuevo tipo de contenedor celular (Safespeed), en el que se introduce la muestra biológica que se quiere criopreservar, junto con la concentración necesaria de crioprotector y suero, está fabricado por un conjunto de polímeros termoplásticos ensamblados (como por ejemplo policarbonato). The new type of cellular container (Safespeed), in which the biological sample to be cryopreserved is introduced, together with the necessary concentration of cryoprotectant and serum, is manufactured by a set of assembled thermoplastic polymers (such as polycarbonate).
Con el objetivo de incrementar la facilidad en el manejo del microcapilar termoplástico, éste puede ser introducido dentro de otra pajuela de mayor diámetro y rigidez, móvil pero no desmontable, que actuará a su vez de protección para el microcapilar. Esta pajuela de protección podrá descubrir (posición de uso) o recubrir (posición de almacenamiento) el microcapilar a través de un mecanismo de cierre mecánico. La posición de uso permite
optimizar el proceso de vitrificación, y la posición de almacenamiento minimiza o elimina tanto el riesgo de rotura del capilar como el riesgo de perdida de la muestra celular. In order to increase the ease in handling the thermoplastic microcapillary, it can be introduced into another straw of greater diameter and rigidity, mobile but not removable, which will in turn act as protection for the microcapillary. This protective straw can discover (use position) or cover (storage position) the microcapillary through a mechanical closing mechanism. The use position allows optimize the vitrification process, and the storage position minimizes or eliminates both the risk of capillary rupture and the risk of cell sample loss.
El contenedor consta de las siguientes partes: The container consists of the following parts:
- Un capilar de plástico (menor de 250 mieras de diámetro) en el que se depositan la(s) muestra(s) biológica(s). - A plastic capillary (smaller than 250 microns in diameter) in which the biological sample (s) are deposited.
- Una pajuela principal conectada al microcapilar que aporta rigidez y permite que la muestra fluya hasta el capilar mediante la succión por la parte trasera. - A main straw connected to the microcapillary that provides rigidity and allows the sample to flow to the capillary by suction through the back.
- Una pajuela exterior que aporta rigidez y permite manipulación. - An external straw that provides rigidity and allows manipulation.
- Un tramo de pajuela plana que permite anotar los datos identificativos de cada paciente (nombre, código, etc.). - A section of flat straw that allows to write down the identifying data of each patient (name, code, etc.).
- Un tramo final trasero de pared delgada que permite la conexión a un sistema de aspiración estándar y también el cierre mediante calor (termoselladora). - A thin-walled rear end section that allows connection to a standard suction system and also the closing by heat (heat sealer).
- Un protector de plástico que desliza sobre la pajuela exterior y que permite dejar al descubierto el capilar para el momento de la succión de la muestra biológica y la introducción en nitrógeno líquido, y que a su vez permite otra segunda posición de almacenamiento una vez la muestra se encuentra vitrificada, en el que recubre el capilar para que evitar que éste colisione con otros objetos y se dañe la muestra del interior. - A plastic protector that slides over the outer straw and allows the capillary to be exposed for the moment of the biological sample suction and the introduction into liquid nitrogen, and which in turn allows another second storage position once the Sample is vitrified, in which it covers the capillary so that it prevents it from colliding with other objects and damaging the sample inside.
Modo de realización de la invención Embodiment of the invention
PROCEDIMIENTO DE VITRIFICACION VITRIFICATION PROCEDURE
Preparar la(s) célula(s) en el medio de enfriamiento de vitrificación recomendado (agentes crioprotectores). Prepare the cell (s) in the recommended vitrification cooling medium (cryoprotective agents).
Fijar el extremo ancho de Safespeed a un dispositivo de aspiración (por ejemplo, una jeringa Hamilton, una pipeta automática, un stripper, una pipeta de boca o cualquier otro sistema de aspiración y manipulación de ovocitos), bien directamente o bien con un conector-acoplador adecuado. Attach the wide end of Safespeed to a suction device (for example, a Hamilton syringe, an automatic pipette, a stripper, a mouth pipette or any other oocyte aspiration and manipulation system), either directly or with a connector- suitable coupler.
Cuando las muestras están listas para cargar en el Safespeed, deslizar la funda plástica protectora a lo largo de la pajuela hasta exponer el microcapilar. Con ayuda del microscopio o la lupa binocular, cargar suavemente las muestras en el Safespeed y ubicarlas entre la 2a y 3a marcas del microcapilar por aspiración, controlando la absorción
de las muestras y medio. Estas 2a y 3a marcas del microcapilar han sido calculadas al efecto de manera que la velocidad de enfriamiento y recalentamiento en dicha área esa la óptima. Este detalle es crítico. Confirmar que la/s muestra/s está situada/s entre la frontera de la marca de 2a y 3a. Precaución: El fallo en la confirmación de la ubicación de la muestra, podría dañar la muestra. When the samples are ready to load in the Safespeed, slide the protective plastic sheath along the straw to expose the microcapillary. With a microscope or stereomicroscope, gently loading samples and place them in the Safespeed between 2 and 3 marks microcapillary aspiration, controlling absorption of the samples and a half. These 2 and 3 marks microcapillary have been calculated to the effect that the speed of cooling and reheating in that area that the optimum. This detail is critical. Confirm that the / s sample / s is located / s between the border of the mark 2 and 3. Caution: Failure to confirm the location of the sample could damage the sample.
Sellar térmicamente el microcapilar por debajo de la marca 1. A continuación la parte trasera del dispositivo se sella también con el mismo termosellador por la marca de soldadura de tubo, de manera que la muestra se encuentra aislada completamente dentro del contenedor. Thermally seal the microcapillary below the 1 mark. Then the back of the device is also sealed with the same heat sealer by the tube welding mark, so that the sample is completely isolated inside the container.
Extremadamente rápido, sumergir verticalmente de cara abajo el lado del capilar sellado de Safespeed primero, en un depósito de nitrógeno líquido o cualquier fluido líquido o gaseoso que permita una alta velocidad de enfriamiento. Deslizar la funda protectora de plástico cuidadosamente a lo largo de la pajuela hacia abajo para proteger el microcapilar (figura 3). Asegurarse de que durante este movimiento el capilar queda completamente cubierto por el nitrógeno líquido. Para el transporte, guardar la el dispositivo Safespeed en un cáliz, canister o cryocane (tubo de ensayo metálico o de plástico, que podrá adaptarse a los estándares utilizados en las clínicas u de otro tipo) y llevar al tanque de nitrógeno líquido donde será finalmente almacenado. Extremely fast, dip the side of the Safespeed sealed capillary vertically face down first, in a liquid nitrogen reservoir or any liquid or gaseous fluid that allows a high cooling rate. Slide the plastic protective sleeve carefully along the straw down to protect the microcapillary (figure 3). Ensure that during this movement the capillary is completely covered by liquid nitrogen. For transport, store the Safespeed device in a chalice, canister or cryocane (metal or plastic test tube, which can be adapted to the standards used in clinics or other) and take to the liquid nitrogen tank where it will finally be stored.
PROCEDIMIENTO DE RECALENTAMIENTO HEATING PROCEDURE
Preparar el medio de calentamiento preferido de recalentamiento (agente eliminador de crioprotector). Prepare the preferred reheating heating medium (cryoprotectant eliminating agent).
Preparar un depósito de agua (por lo menos 15 cm de profundidad) a 37 grados lo más cerca posible (máximo 15 cm) de la cryocane (cáliz o canister). Prepare a water tank (at least 15 cm deep) at 37 degrees as close as possible (maximum 15 cm) to the cryocane (calyx or canister).
Deslizar cuidadosamente la funda protectora de plástico a lo largo y hacía arriba de la pajuela asegurando que en todo momento el capilar está completamente sumergido en el nitrógeno líquido, quedando el microcapilar al descubierto.
Extremadamente rápido (menos de 1 segundo), sumergir verticalmente cara abajo el capilar sellado de Safespeed, en el recipiente de agua, agitando a mano durante dos segundos para una mejor transferencia de calor. Carefully slide the plastic protective sheath along and up the straw ensuring that at all times the capillary is completely submerged in liquid nitrogen, leaving the microcapillary exposed. Extremely fast (less than 1 second), soak the Safespeed sealed capillary vertically face down in the water container, shaking by hand for two seconds for better heat transfer.
Inmediatamente después, sacar Safespeed del agua y secar subvente su exterior (con papel absorbente, por ejemplo). Colocar el Safespeed en una plataforma de corte, posteriormente cortar el Safespeed justo por encima del termosellado en el extremo más ancho. Immediately afterwards, remove Safespeed from the water and dry its underside (with absorbent paper, for example). Place the Safespeed on a cutting platform, then cut the Safespeed just above the heat seal at the widest end.
Conecte el dispositivos de succión (convenientemente acoplado) al extremo ancho de Safespeed. Coloque el Safespeed en una plataforma de corte y posteriormente corte el Safespeed justo debajo de la 1a marca en el extremo final del microcapilar. Descargar cuidadosamente y expulsar la muestra dentro del medio de calentamiento por vitrificación recomendado. Connect the suction devices (conveniently attached) to the wide end of Safespeed. Place the Safespeed in a cutting and then cut just below the Safespeed 1 to mark the end of the microcapillary end. Carefully discharge and expel the sample into the recommended vitrification heating medium.
La longitud total del dispositivo completo será de unos 80-140 mm, lo que se adapta a los estándares de almacenamiento, ajustándose a las prácticas al uso en reproducción asistida, permitiendo una cómoda manipulación y minimización del espacio que ocupan en los stocks. The total length of the complete device will be about 80-140 mm, which adapts to the storage standards, adjusting to the practices for assisted reproduction, allowing a comfortable manipulation and minimization of the space they occupy in the stocks.
Ejemplo 1. Procedimiento de conservación celular de ovocitos Example 1. Procedure for cell preservation of oocytes
Este ejemplo muestra la aplicación práctica del método para la criopreservación de ovocitos de ratona. This example shows the practical application of the method for cryopreservation of mouse oocytes.
Se emplearon ratonas vírgenes C57BL/6 x CBA/Ca (4-8 semanas) mantenidas bajo condiciones controladas (12 horas luz: 12 horas oscuridad) y alimentadas ad libitum. Fueron superovuladas por inyección intraperitoneal con 0.1 mi de PMSG (5 Ul). Tras 48- 56 horas, se les administró 0.1 mi de HCG (5 Ul). Se sacrificaron 14 horas después por dislocación cervical y se extrajeron los oviductos. C57BL / 6 x CBA / Ca virgin mice (4-8 weeks) kept under controlled conditions (12 light hours: 12 dark hours) and fed ad libitum were used. They were superovulated by intraperitoneal injection with 0.1 ml of PMSG (5 Ul). After 48-56 hours, 0.1 ml of HCG (5 Ul) was administered. They were sacrificed 14 hours later by cervical dislocation and the oviducts were removed.
Los ovocitos fueron liberados del cúmulo por acción de la hialuronidasa (SIGMA Ref. H4272-30 mg). Tras esto, los ovocitos se lavaron en medio M2 para eliminar los restos de
hialuronidasa. Los ovocitos en metafase II previamente denudados se transfirieron entonces a una solución de equilibrio, y después a la solución de vitrificación. The oocytes were released from the cluster by hyaluronidase (SIGMA Ref. H4272-30 mg). After this, the oocytes were washed in M2 medium to remove the remains of hyaluronidase The previously denuded metaphase II oocytes were then transferred to an equilibrium solution, and then to the vitrification solution.
Los ovocitos se introdujeron en el microcapilar, que fue termosellado, con la muestra biológica en el extremo inferior. El microcapilar se introdujo en el recipiente de nitrógeno líquido de forma manual, en la posición de uso, como muestra la figura 1. The oocytes were introduced into the microcapillary, which was heat sealed, with the biological sample at the lower end. The microcapillary was introduced into the liquid nitrogen container manually, in the position of use, as shown in Figure 1.
Una vez la muestra con el capilar alcanzó temperaturas criogénicas, el microcapilar fue puesto en posición de almacenamiento recubierto por el capuchón, y fue transportado en un canister de plástico hasta un tanque de nitrógeno líquido, donde permaneció 24 horas. Once the sample with the capillary reached cryogenic temperatures, the microcapillary was placed in storage position covered by the cap, and was transported in a plastic canister to a liquid nitrogen tank, where it remained 24 hours.
En el proceso de recalentamiento, el microcapilar fue introducido en un baño de agua a 37.5 °C, de forma manual, (de nuevo en posición de uso, sin capuchón). In the reheating process, the microcapillary was introduced into a 37.5 ° C water bath, manually (again in use position, without cap).
Después de unos minutos, se estudió la viabilidad de los ovocitos, mediante una gama amplia de valoraciones morfológicas y la técnicas de tinción para ver compromiso de la membrana (trypan blue). After a few minutes, the viability of the oocytes was studied, using a wide range of morphological assessments and staining techniques to see membrane involvement (trypan blue).
Siguiendo dicho procedimiento se criopreservaron y recalentaron 620 ovocitos de ratona, de los cuales sobrevivieron 595, obteniéndose una supervivencia de ovocitos de un 96%, mucho mayor a otras técnicas de vitrificación que utilizan dispositivos cerrados. Following this procedure, 620 mouse oocytes were cryopreserved and reheated, of which 595 survived, obtaining an oocyte survival of 96%, much higher than other vitrification techniques using closed devices.
Descrita la naturaleza de la Invención, y la manera de llevarla a la práctica, se hace constar que las disposiciones anteriormente indicadas son susceptibles de modificaciones de detalle, en tanto no alteren su principio fundamental, siendo lo que constituye la esencia de la referida Invención, reservándose los Peticionarios el derecho a obtener los correspondientes certificados de adición por las mejoras o perfeccionamientos que en lo sucesivo pudiera aconsejar la práctica, reivindicándose a título privativo las siguientes particularidades sobre las cuales ha de recaer la concesión del privilegio de la Patente de invención que se solicita.
Describing the nature of the Invention, and the way of putting it into practice, it is stated that the provisions indicated above are subject to modifications in detail, as long as they do not alter its fundamental principle, being what constitutes the essence of said Invention, the Petitioners reserving the right to obtain the corresponding certificates of addition for the improvements or refinements that the practice may advise on from now on, claiming privately the following particularities on which the granting of the privilege of the Patent of invention to be granted must fall request.
Claims
1. Procedimiento de criopreservación de material biológico mediante vitrificación, caracterizado por el uso de microcapilares poliméricos termoplásticos como contenedores del material biológico, introducidos en fluidos criogénicos para conseguir la vitrificación de la muestra biológica. 1. Cryopreservation procedure of biological material by vitrification, characterized by the use of thermoplastic polymer microcapillars as containers of biological material, introduced into cryogenic fluids to achieve vitrification of the biological sample.
2. Procedimiento de criopreservación celular según la reivindicación 1 , caracterizado por la utilización de microcapilares poliméricos termoplásticos fabricados con policarbonato, o con cualquier otro material de similares características. 2. A cell cryopreservation method according to claim 1, characterized by the use of thermoplastic polymer microcapillars made of polycarbonate, or any other material with similar characteristics.
3. Procedimiento de criopreservación celular según la reivindicación 1, caracterizado por la utilización de microcapilares poliméricos termoplásticos termosellables, para evitar la contaminación cruzada de la muestra y que éste quede completamente aislada del exterior. 3. Method of cellular cryopreservation according to claim 1, characterized by the use of thermo-plastic thermoplastic polymer microcapillaries, to avoid cross contamination of the sample and that it is completely isolated from the outside.
4. Procedimiento de criopreservación celular según la reivindicación 1 , caracterizado porque la muestra celular se introduce previamente bañada en agentes crioprotectores y suero en el interior del microcapilar, a distintas concentraciones dependiendo del tipo celular a criopreservar. 4. A cell cryopreservation method according to claim 1, characterized in that the cell sample is previously introduced in cryoprotective agents and serum inside the microcapillary, at different concentrations depending on the cell type to be cryopreserved.
5. Procedimiento de criopreservación celular según la reivindicación 1 , en el que el microcapilar polimérico termoplástico está ensamblado sobre un conjunto de pajuelas que le proporcionan rigidez y facilidad en el uso, no desmontables 5. The cell cryopreservation method according to claim 1, wherein the thermoplastic polymer microcapillary is assembled on a set of straws that provide stiffness and ease of use, not removable
6. Procedimiento de criopreservación celular según la reivindicación 1 y 5, en el que una de las pajuelas actúa de protector del capilar en su posición de almacenamiento, móvil pero no desmontable. 6. A cell cryopreservation method according to claim 1 and 5, wherein one of the straws acts as a capillary protector in its storage position, mobile but not removable.
7. Procedimiento de criopreservación celular según la reivindicación 1 y 5, en el que una de las pajuelas es plana para facilitar la escritura e identificación de los datos del paciente.
7. A cell cryopreservation method according to claim 1 and 5, wherein one of the straws is flat to facilitate the writing and identification of patient data.
8. Procedimiento de criopreservacion celular según la reivindicación 1 , en el que el microcapilar es introducido en fluidos criogénicos con la parte inferior del microcapilar conteniendo la muestra biológica sin el protector del capilar exterior, para optimizar el proceso de enfriamiento. 8. The cell cryopreservation method according to claim 1, wherein the microcapillary is introduced into cryogenic fluids with the lower part of the microcapillary containing the biological sample without the outer capillary protector, to optimize the cooling process.
9. Procedimiento de criopreservacion celular según la reivindicación 1 , caracterizado por la utilización de un baño de recalentamiento para el proceso de recalentamiento, en el que el microcapilar polimérico termoplástico termosellado, es introducido con la parte inferior conteniendo la muestra biológica, sin el protector del capilar exterior, para optimizar el proceso de recalentamiento. 9. Method of cellular cryopreservation according to claim 1, characterized by the use of a reheating bath for the reheating process, in which the thermo-sealed thermoplastic polymer microcapillary is introduced with the lower part containing the biological sample, without the protector of the outer capillary, to optimize the reheating process.
10. Procedimiento de criopreservacion celular según la reivindicación 1, caracterizado por la utilización de agua a 37° o de un fluido similar para el baño de recalentamiento.
10. A cell cryopreservation method according to claim 1, characterized by the use of water at 37 ° or a similar fluid for the reheating bath.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ESP201201020 | 2012-10-10 | ||
| ES201201020A ES2459893B2 (en) | 2012-10-10 | 2012-10-10 | Procedure and device for vitrification of biological material using microcapillars of closed thermoplastic polymers using ultra-fast reheating |
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| Publication Number | Publication Date |
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| WO2014057148A2 true WO2014057148A2 (en) | 2014-04-17 |
| WO2014057148A3 WO2014057148A3 (en) | 2014-06-12 |
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| PCT/ES2013/000228 WO2014057148A2 (en) | 2012-10-10 | 2013-10-10 | Method for the vitrification of biological material using closed thermoplastic polymer microcapillaries introduced into cryogenic fluids |
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| WO (1) | WO2014057148A2 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113143584A (en) * | 2021-03-09 | 2021-07-23 | 河南省立眼科医院 | Handheld device for collecting and cryogenically preserving animal corneal epithelial cells and collecting method |
| US11116206B2 (en) | 2018-10-01 | 2021-09-14 | Cook Medical Technologies Llc | Cryocontainer |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2737681A1 (en) * | 2018-07-11 | 2020-01-15 | Univ Sevilla | DEVICE AND PROCEDURE FOR PREPARING CELLS FOR ULTRA-FAST VITRIFICATION BY DEHYDRATION (Machine-translation by Google Translate, not legally binding) |
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|---|---|---|---|---|
| EP1353383A2 (en) * | 2002-04-10 | 2003-10-15 | Victor Company Of Japan, Ltd. | Image-sensing device having a plurality of output channels |
| US20060046243A1 (en) * | 2004-08-27 | 2006-03-02 | Tyho-Galileo Research Laboratories | Method for vitrification of mammalian cells |
| US20080233633A1 (en) * | 2005-09-28 | 2008-09-25 | Philippe Clairaz | Sheathing for Packaging a Predetermined Volume of a Biological Substance Designed to be Immersed in a Liquid Cryogenic Agent |
| US20090123996A1 (en) * | 2007-11-12 | 2009-05-14 | Milton Chin | Vitrification Device with Shape Memory Seal |
| EP2156735A1 (en) * | 2008-08-13 | 2010-02-24 | Dr. H. Zech GmbH | Method and instrument for vitrification and storing of biological specimen |
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2012
- 2012-10-10 ES ES201201020A patent/ES2459893B2/en active Active
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- 2013-10-10 WO PCT/ES2013/000228 patent/WO2014057148A2/en active Application Filing
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1353383A2 (en) * | 2002-04-10 | 2003-10-15 | Victor Company Of Japan, Ltd. | Image-sensing device having a plurality of output channels |
| US20060046243A1 (en) * | 2004-08-27 | 2006-03-02 | Tyho-Galileo Research Laboratories | Method for vitrification of mammalian cells |
| US20080233633A1 (en) * | 2005-09-28 | 2008-09-25 | Philippe Clairaz | Sheathing for Packaging a Predetermined Volume of a Biological Substance Designed to be Immersed in a Liquid Cryogenic Agent |
| US20090123996A1 (en) * | 2007-11-12 | 2009-05-14 | Milton Chin | Vitrification Device with Shape Memory Seal |
| EP2156735A1 (en) * | 2008-08-13 | 2010-02-24 | Dr. H. Zech GmbH | Method and instrument for vitrification and storing of biological specimen |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11116206B2 (en) | 2018-10-01 | 2021-09-14 | Cook Medical Technologies Llc | Cryocontainer |
| CN113143584A (en) * | 2021-03-09 | 2021-07-23 | 河南省立眼科医院 | Handheld device for collecting and cryogenically preserving animal corneal epithelial cells and collecting method |
Also Published As
| Publication number | Publication date |
|---|---|
| ES2459893A2 (en) | 2014-05-12 |
| ES2459893R2 (en) | 2014-06-26 |
| ES2459893B2 (en) | 2015-12-28 |
| WO2014057148A3 (en) | 2014-06-12 |
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