DEVICE FOR PREPARING A BIOLOGICAL SAMPLE FOR

VITRIFICATION, AND A METHOD FOR UTILIZING IT

FIELD OF THE INVENTION

The invention relates to the field of cryopreservation. More precisely, it relates to devices for vitrification of biological samples.

BACKGROUND OF THE INVENTION

Preservation of biological samples, for example oocytes and embryos at very low temperature is known as cryopreservation. One of the major challenges of cryopreservation is to prevent the intracellular liquid within the sample from turning into ice crystals. Two common techniques of cryopreservation are slow freezing and vitrification.

During the slow freezing process ice crystals are formed intercellularly, and as a result the remaining liquid becomes hypertonic thus allowing intracellular water to leave the cells and to pass towards an outside of the cells by exosmosis, thus preventing intracellular crystallization.

In vitrification, intercellular and intracellular water crystallization is avoided by means of a very high cooling rate. According to some vitrification protocols, the sample is plunged into a very cold cryogenic medium, e.g., liquid nitrogen (LN) or LN slush), thus resulting in very high cooling rates, which enables vitrification rather than crystallization of the intracellular and intercellular liquids.

In some protocols, vitrification may be further enabled by increasing the viscosity of the sample, for example by applying various cryoprotectants and/or other applicable additives, by reducing the volume of the sample, or by a combination thereof. For example, the publication "Vitrification of oocytes and embryos" (Amir Arav, "Embryonic development and manipulation in animal development", edited by A. Lauria and F. Gandolfi, Portland Press, London, U.K., 1992), presents a method of vitrifying cells enclosed in small drops sufficient to keep them in physiological conditions. In this publication, Arav reports that with volume of 70 nanoliter drops, good survival rates can be achieved even with low concentration of cryoprotectant.

Vitrification is further described in the following publications: "Titration of Vitrification Solution in Mouse Embryo Cryopreservation" (A. ARAV, L. GIANAROLI, AND P. SURIANO, Cryobiology 25(6), 1988) presents reducing the toxicity of the vitrification solution by decreasing the time and temperature of embryo exposure to cryoprotectant solution.

"Osmotic and cytotoxic study of vitrification of immature bovine oocytes" (A.

Arav, D. Shehu, and M. Mattioli, Journal of Reproduction and Fertility, 99: 353-358, 1993) presents experiments conducted in order to determine the composition of a solution suitable for vitrification of immature bovine oocytes.

"New trends in gamete's cryopreservation" (Amir Arav, Saar Yavin, Yoel Zeron, Dity Natan, Izik Dekel, and Haim Gacitua. Molecular and Cellular Endocrinology, 187: 77-81, 2002) presents techniques to improve freezing and vitrification of sperm, oocytes and embryos, based on 'Multi-Thermal-Gradient' (MTG) freezing.

"Measurement of essential physical properties of vitrification solutions" (S. Yavin and A. Arav. Theriogenology, 67(1): 81-9, 2007) examines the principal parameters associated with successful vitrification, and composes guidelines to aspects of the vitrification process.

"Embryo cryopreservation in the presence of low concentration of vitrification solution with sealed pulled straws in liquid nitrogen slush" (Saar Yavin, Adaya Aroyo, Zvi Roth, and Amir Arav. Human Reproduction, 24(4): 797-804, 2009) presents a vitrification method that combines LN slush and sealed pulled straws (SPS).

U.S. Patent Application 2011/0207112 (Burbank and Jones, published in 2011) discloses an automated system and method of cryopreservation and reanimation of oocytes, embryos, or blastocysts. One or more oocytes or embryos are positioned in a processing container, the processing container being configured to allow fluid to flow into and out of the processing container, where two or more fluids flow into and out of the processing container with oocytes or embryos therein.

SUMMARY OF THE INVENTION

The present invention, in some embodiments thereof, relates to the vitrification of a biological sample.

The invention relates to a device for preparing a biological sample for vitrification, the device comprising a chamber configured to contain a first liquid and to accommodate a carrier for holding the sample immersed in the first liquid; an inlet configured to add a second liquid into the chamber for modifying a composition of the first liquid; and a first drain configured to drain the first liquid from the chamber; wherein the chamber has a liquid capacity that is sufficiently large to enable a gradual modification of the composition in accordance with a modification course adequate for preparing the sample for vitrification

lln some embodiments, the device further comprises a first drain configured to drain the first liquid from the chamber; wherein the chamber and the first drain are configured together to induce a directional flow of the first liquid, wherein the directional flow forces the biological sample to reside substantially at a predetermined location

In some embodiments, the inlet is coupleable to a manual injection pump; and the manual injection pump together with the liquid capacity enables the gradual modification of the first liquid.

In some embodiments, the manual injection pump is a syringe.

In some embodiments the chamber is configured to accommodate a carrier characterized by a size adequate for placing a biological sample therewith by manual pipetting.

In some embodiments the chamber is configured to accommodate a carrier that comprises a permeable member, which is configured to enable a thru-flow of the first liquid between sides thereof, while preventing the biological sample from being carried along with the thru-flow.

In some embodiments the permeable member is located at a bottom of the carrier.

In some embodiments, the first drain is configured to enable abrupt exhaustion of substantially all the first liquid from the first container.

In some embodiments, the first drain is coupleable to a manual aspiration pump.

In some embodiments, the first drain is configured to enable abrupt exhaustion of substantially all the first liquid from the first container.

In some embodiments the first drain is configured to enable aspiration of gas therethrough;the inlet is configured to deliver gas into the chamber; and the chamber, the inlet, and the first drain are configured so that the aspiration of gas induces a flow of gas at a vicinity of the sample, thus removing a part of a residual liquid.

In some embodiments, the device further comprises a flow guiding element configured to guide a flow of the second liquid toward the carrier.

In some embodiments, the flow guiding element is configured to guide the flow to an opening of the carrier.

In some embodiments, the chamber is configured to accommodate a plurality of carriers.

In some embodiments, the chamber comprises a liquid guiding element configured to guide a flow of the second liquid toward the plurality of carriers.

In some embodiments, the device further comprises a second drain configured to enable flow of an excessive volume of the first liquid out of the chamber, wherein the excessive volume exceeds a first level limit, thus constraining the first liquid substantially below the first-liquid-limit.

In some embodiments, the first-level-limit is configurable.

In some embodiments, the chamber comprises a first container configured to contain the first liquid and to accommodate the carrier; and the device further comprises a second drain configured to enable flow of an excessive volume of the first liquid out of the first container, the excessive volume exceeds a first level limit, thus constraining the first liquid substantially below the first-liquid-limit.

In some embodiments, the first-level-limit is configurable.

In some embodiments, the device further comprises an inspection element configured to enable visual inspection of the biological sample for enabling monitoring a status thereof; and an illumination element configured to enable illuminating the sample for enabling the visual inspection.

In some embodiments, the chamber is configured to accommodate the carrier so that the sample resides substantially between the illumination element and the inspection element, thus enabling trans-illumination of the sample.

In some embodiments, the illumination element is located substantially below the carrier; and the inspection element is located substantially above the carrier.

In some embodiments, the inspection element is an internal inspection element residing inside the chamber.

In some embodiments, the internal inspection element comprises a camera. In some embodiments, the internal inspection element comprises an optic fiber.

In some embodiments, the illumination element is an internal illumination element residing inside the chamber.

In some embodiments, the internal illumination element comprises a Light

Emitting Diode (LED).

In some embodiments, the internal illumination element comprises an optic fiber.

In some embodiments, the inspection element comprises an inspection window, wherein the inspection window comprises an opening in a boundary of the chamber, and enables inspection of the sample therethrough.

In some embodiments, the inspection window is configured to be closed by an inspection-window-cover comprising a transparent member, thus enabling visual inspection while maintaining the chamber closed.

In some embodiments, the illumination element comprises an illumination window, wherein the illumination window comprises an opening in a boundary of the chamber, and enables illuminating the sample by light penetration therethrough.

In some embodiments, the illumination window is configured to be closed by an illumination-window-cover comprising a transparent member, thus enabling illumination while maintaining the chamber closed.

The invention also relates to a method for vitrifying a reproductive multi cell suspension, the method comprising immersing the multi cell suspension in a carrier in a first liquid residing in a chamber, wherein the first liquid is of a first composition; gradually modifying the first liquid from the first composition to a third composition at a first modification course, which is adequate for vitrification, by adding a second liquid of a second composition; and abruptly modifying the composition of the first liquid from the third composition to a forth composition by draining substantially all of the first liquid, and adding a third liquid of a forth composition.

In some embodiments, adding the second liquid is performed manually.

In some embodiments, adding adding the third liquid is performed manually

In some embodiments, draining liquids is performed manually.

In some embodiments, the method further comprises maintaining the multi cell suspension substantially adjacent to a permeable member of the carrier by draining part of the first liquid, thus inducing a directional flow thereof through the permeable member, and thereby forcing the multi cell suspension to reside substantially adjacent to the permeable member.

In some embodiments, the method further comprises placing the multi cell suspension at the carrier by manual pipetting.

In some embodiments, a volume of the first liquid and a volume of the second liquid occupy together at least 1 milliliter..

In some embodiments, a volume of the first liquid and a volume of the second liquid occupy together at least 5 milliliter.

In some embodiments, the method further comprises immersing two or more multi cell suspensions in two or more carriers in the first liquid residing in the chamber.

In some embodiments, the method further comprises constraining the first liquid substantially below a first-liquid-limit, by enabling out-flow of an excessive volume of the first liquid, the excessive volume exceeds a first level limit.

In some embodiments, the method further comprises visually inspecting the multi-cell suspension residing within the chamber.

In some embodiments, visually inspecting comprises trans-illuminating the multi-cell suspension.

In some embodiments, the method further comprises controlling the draining part of the first liquid responsive to information obtained during the visually inspecting.

In some embodiments, the method further comprises draining substantially all the first liquid; and aspiring gas, thus inducing a flow of gas at a vicinity of the multi- cell suspension, and thereby reducing a volume of a residual liquid.

In some embodiments the method further comprises inserting the multi cell suspension with a carrier into a cryogenic medium

BRIEF DESCRIPTION OF THE DRAWINGS

In order to understand the invention and to see how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which: FIG 1 illustrates a cryopreservation system for vitrification of a reproductive biological sample, according to embodiments of the invention;

FIG 2 illustrates a cryopreservation device, according to certain embodiments of the invention;

FIG 2A illustrates a cryopreservation device 2A02 comprising a flow guiding element 2A06, according to embodiments of the invention;

FIG 3 illustrates a cryopreservation device 302, further comprising a first drain 306, according to embodiments of the invention;

FIG 4 illustrates a cryopreservation device configured to enable reduction of volume of a residual liquid by aspirating gas, according to embodiments of the invention;

FIG 5 illustrates a cryopreservation device further comprising a second drain, according to embodiments of the invention;

FIG 5A illustrates a cryopreservation device, according to embodiments of the invention;

FIG 6 illustrates two cryopreservation devices comprising a second container, according to embodiments of the invention;

FIG 7 illustrates a cryopreservation device, according to embodiments of the invention;

FIG 8 illustrates a cryopreservation device configured to enable visual inspection of the reproductive biological sample residing within the chamber, according to embodiments of the invention; and

FIG 9 schematically illustrates a cryopreservation method for vitrification of a reproductive multi cell suspension, by using a cryopreservation device in accordance with embodiments of the invention.

DETAILED DESCRIPTION

In the following description components that are common to more than one figure will be referenced by the same reference numerals, unless specifically noted otherwise. In addition, unless specifically noted otherwise, embodiments described or referenced in the present description can be additional and/or alternative to any other embodiment described or referenced therein.

FIG 1 illustrates a cryopreservation system 102 for vitrification of a biological sample 104, according to embodiments of the invention. The source of the biological samples may be any animal, including but not restricted to human beings, mammals, and vertebrates. In some cases, the biological sample is a multi-cell suspension, for example an oocyte, an embryo, or another multi-cell suspension. In other cases, the biological sample is a tissue, for example a slice of an ovary tissue, etc. In some cases the invention is used for handling reproductive biological samples (such as oocytes, sperm, embryos, ovary tissues etc.). However, the invention is not limited to reproductive biological samples and may be directed to other kinds of biological samples. One non limiting example for using the invention with other (non- reproductive) kinds of biological samples is preparing a piece of tissue (or pieces of tissue) taken in a biopsy for vitrification, before the piece can be sent for analysis.

System 102 utilizes a cryopreservation device 106 which enables the preparation of a sample for vitrification. As illustrated in the present figure, preparation is initiated by placing the sample in or on a carrier 108 accommodated in device 102, so that the sample is being immersed within a first liquid 110 residing within the device. Accordingly, due to simplicity consideration, the phrase "in the carrier" is mostly used, though the reader may consider that in some cases, when applicable, the sample may also be placed "on the carrier", and therefore the phrase "in the carrier" should be understood herein as "in or on the carrier".

Understanding that every liquid has a composition, it is clear that the first liquid has a composition as well. Accordingly, subsequent to placing the sample in the carrier, the composition of the first liquid is modified, thus affecting a composition of inter-cellular and/or extra-cellular liquids residing within and around the sample. Afterwards, the carrier is removed from the cryopreservation device and is rapidly cooled by inserting it into a cryogenic medium 112, such as liquid nitrogen or liquid nitrogen slush. Accordingly, the carrier is configured to be inserted into a cryogenic medium, such as liquid nitrogen (LN) or LN slush.

Placing the biological sample in the carrier forms part of an initialization stage, while in some cases, initialization comprises placing the biological sample in the carrier, which is already accommodated in the device and immersed in the first liquid. Accordingly, in the example illustrated in FIG 1, biological sample 104 (e.g. an oocyte) is taken from a laboratory container 114 (e.g. a Petri dish) and placed in carrier 108 which is accommodated in device 106. However, this example is non- limiting, and in other cases the biological sample may be placed in the carrier before the carrier is accommodated in the device, or while it is being accommodated.

It is noted that in some embodiments, carrier 108 may be any applicable carrier, whose form is not limited by the form illustrated in the present figure. Similarly, it should be appreciated that FIG 1 is a non-limiting introductory example to the various embodiments of the invention.

FIG 2 illustrates a cryopreservation device 202, according to certain embodiments of the invention. It is noted that device 202 is an example of a cryopreservation device, such as the one generally denoted 106 in FIG 1, and additional or alternative examples may exist. It is further noted that FIGs 1 and 2 illustrate components denoted by common names in both figures. Such components constitute "common components", wherein the common component illustrated in FIG 2 is considered to be a non-limiting example of the corresponding component in FIG 1. In general, common elements may be denoted in both figures by the same reference numerals (e.g. 104 and 110) wherein the common component in FIG 2 may be a similar or a different embodiment of the corresponding component in FIG 1. In some cases, common components are denoted in FIG 2 by different reference numerals (e.g. 202 and 210) than the numerals used in FIG 1 (106 and 108, respectively), in order to indicate that the component of FIG 2 represent an example characterized by structural and/or functional aspects additional to the corresponding component of FIG 1.

Cryopreservation device 202 comprises a chamber 204 configured to contain the first liquid 110. More specifically, the chamber comprises a first container 206 confining a first space 208 configured to contain the first liquid. The term "first liquid" refers herein to a liquid contained within the first container, and more specifically within the first space thereof. It is noted that the volume and/or the composition of the first liquid may change over time, as explained below, with reference to the present figure.

Chamber 204 is further configured to accommodate a carrier 210, which is an example of the carrier generally denoted 108 in FIG 1. Carrier 210 is configured to hold the biological sample, thereby enabling immersion of the sample in the first liquid. More specifically, chamber 204 is configured to accommodate carrier 210 so that a sample held by the carrier is accordingly immersed in the first liquid.

In some embodiments, which are illustrated in FIG 2, carrier 210 comprises a permeable member 212 at a bottom thereof. In those cases carrier 210 is configured to hold the biological sample on the permeable member, and chamber 204 is configured to accommodate carrier 210 so that the sample residing in the carrier, on permeable member 212 or in proximity thereto, is immersed in the first liquid.

Permeable member 212 is configured to enable a thru-flow of the first liquid between sides thereof, while preventing the biological sample from being carried along with the thru-flow. In some embodiments, carrier 210 may comprise non- permeable side walls 214. In some embodiments, carrier 210 comprises an access opening 216 configured, e.g., to enable placing the biological sample in the carrier and taking it out therefrom via the access opening.

In some embodiments, the permeable member comprises openings characterized by a diameter larger than the molecules of the first liquid and smaller than the diameter of the biological sample. In some embodiments the permeable member comprises a mesh characterized by appropriate openings.

The characteristics of the permeable member may depend on the characteristics of the biological sample. For example, in cases where the biological sample is an oocyte, whose typical diameter is approximately 110 micrometers, the permeable member may comprise openings characterized by a diameter in the range of approximately 10 to 100 micrometers. These values are non-limiting though, and any value applicable to the case may be in use. For example, for cases where the biological sample comprises sperm or ovary slices, the diameter of the openings may be in the range of approximately 1 to 5 micrometers or approximately 0.4 to 10 millimeter, respectively. Furthermore, it should be appreciated that sometimes the biological sample may carry electric charge, hence, in such cases the size, diameter of the opening may be determined not only based on the physical, morphological form, but also based on electrical characteristics of the permeable member. Understanding this it may be further appreciated that the size of the openings of the permeable member is mostly functionally determined unlike morphological determination.

It is noted that carrier 210 is a non-limiting example of a carrier that may be utilized in device 202, and other carriers may also be used. In some embodiments, for example, the carrier comprises one or more permeable members at side-walls thereof. These examples, however, are non-limiting and other carrier, for example a capsule, or a holding pipette, or any other carrier suitable for the case may be utilized.

In some embodiments, for example, the biological sample may be coupled to the carrier. In some cases, a biological sample, for example an ovary tissue slice, may be coupled to the carrier, e.g., by suturing. In other cases, a biological sample may be coupled to the carrier by a bioadhesive or by other appropriate biocompatible adhesive.

In some embodiments, the carrier is configured to hold a single sample. In other embodiments, the carrier is configured to hold even two or more samples (i.e., the carrier is configured to hold one or more samples), thus enabling concurrent vitrification of a plurality of sample.

Moreover, in some embodiments, the cryopreservation device is configured to accommodate one carrier. In other embodiments, the cryopreservation device is configured to accommodate two or more carriers, thus enabling concurrent vitrification of samples residing in different carriers. One possible outcome of the latter feature is the possibility to reanimate samples residing in different carriers at different times. Another possible outcome is the possibility to vitrify concurrently samples originated from different entities, e.g. oocytes harvested from different sources, while keeping track on the origin of each sample. In some embodiments, the cryopreservation device comprises two or more chambers, and each one of these chambers may be configured to accommodate a single carrier per chamber, one or more carriers, two or more carriers, etc.

Further to introducing the carriers that might be used in conjunction with the present invention it should be understood that in different embodiments the carrier may be an element designed specifically for use with a device according to the invention, or a commercially available product requiring or not-requiring adaptations for the device.

In some embodiments, chamber 204 further comprises a carrier-holding element 228 configured to hold the carrier. It is noted that some aspects of device 202 depend on carrier 210 being accommodated in an appropriate position within chamber 204. For example, as explained above with reference to the present figure, chamber 204 is configured to accommodate carrier 210 so that a sample residing in the carrier, on permeable member 212 or in proximity thereto, is immersed in the first liquid. Further examples of aspects depending on the position of the carrier within the chamber are presented below, for example with reference to FIG 2A, where guiding a flow of liquid towards the carrier depends on a special position of the carrier relative to a flow-guiding element. Accordingly, in some embodiment where an aspect of device 202 depends on an appropriate position of the carrier within the chamber, it should be understood that the carrier-holding element is configured to hold the carrier at the appropriate position.

In the example illustrated in FIG 2, carrier-holding element 228 comprises a carrier-holding ring 230 configured to hold the carrier which rests surrounded by the ring. However, this example is non-limiting, and any other embodiment appropriate for the case, can be implemented. For example, the structure of the device, or the chamber, may allow placing the carrier in a certain position.

Device 202 further comprises an inlet 218 configured to add a second liquid 220 into the chamber, whereby the second liquid is inserted into the first space where it is merged into the first liquid. Inlet 218 may be coupled to a first source 222 of the second liquid, wherein the first source may be a manual pump, constituting an "injection pump", (such as a syringe or a squeezable container), a container coupled to an active pump (e.g., an electrical or any other form of motorized pump), or any other applicable source configured to yield the second liquid. It should be appreciated that manual pumps are, in many cases, advantageous due to their simplicity and lower cost. Yet, in some embodiments, the first source may be configured to be controlled by an automatic controlling mechanism, such as computerized controller.

In FIG 2, inlet 218 is illustrated to comprise an inlet pipe 224 coupled to chamber 204 via a single inlet-opening 226 at a side-wall of the chamber. Inlet pipe 224 is further illustrated to be coupled to first source 222. However, the details of the example illustrated in FIG 2 are non-limiting, and other embodiments can be implemented, including, for example, embodiments where the inlet-opening is located at an upper wall or a cover of the chamber, or at a bottom thereof; embodiments where the inlet comprises two or more inlet-openings; embodiments where the inlet does not comprise a pipe and the first source is coupled directly to the inlet-opening; embodiments comprising two or more inlets; combinations of the embodiments listed above; or any other suitable embodiment.

Cryopreservation device 202 enables a gradual modification of a composition of the first liquid from a first composition into a third composition, by adding the second liquid characterized by a second composition to the first liquid, as described in the following example. According to this example a first volume V of the first liquid, of a first composition, is inserted into the first container, the carrier is accommodated in the container, and the sample is placed at the carrier. As explained above, when the carrier is accommodated in the chamber, sample 104 is immersed in the first liquid. Subsequently, a second volume V ₂ of second liquid 220, characterized by a second composition, is yielded from first source 222. The second liquid is inserted into the chamber via inlet 218 during a time period referred to herein as a "first modification period" T at a "first yielding course" Y , which represents an instantaneous yielding rate Y _\ {t) of the second liquid at time 0<ί<=Γι. It should be appreciated that the instantaneous yielding rate may be constant through T , or it may modify during T . Consequently, the composition of the first liquid, in which the sample is being immersed, is gradually modified during the first modification period from the first composition into a third composition at a first modification course C _\ , which represents the instantaneous concentration (i.e. relative portion) C _\ (t) of the second liquid within the first liquid.

It is noted that the order of operations taking place before the first modification period is non-limiting, and other orders of operations may be implemented, for example, placing the sample at the carrier prior to accommodating the carrier within the chamber, or accommodating the carrier in the chamber before inserting the first liquid to the first container, or any other order of operations appropriate for the case.

In some embodiments, a liquid of the first composition constitutes a "holding solution", a liquid of the third composition constitutes an "equilibrium solution", and the second solution is accordingly configured to modify the composition of the first liquid from the first composition into the third composition. Herein, the term holding solution refers to a solution configured to maintain the biological sample in a physiological condition, and the term equilibrium solution refers to a solution configured to bring the sample into a "first equilibrium state". The term "equilibrium state" refers herein to a state wherein (a) a portion of the water contained within the sample has been replaced by premating cryoprotectants and (b) the sample is at osmotic equilibrium with the third solution. The term "first equilibrium state" refers to an equilibrium state wherein the concentration of the premating cryoprotectants within the first liquid is equal to a first target concentration. In some embodiments, the first target concentration is within the range of 2% to 20%. For example, in cases where the biological sample comprises sperm or oocytes, the first target concentration may be about 5% or 15%, respectively. However, these first target concentration ranges, which are brought here by way of example, are non-limiting, and any other values suitable for the case may be implemented.

In some embodiments, the holding solution comprises phosphate buffer solution, HEPES solution, or culture medium. However, these compositions are brought by way of example and are therefore non-limiting, and any other suitable composition may be used when appropriate for the case.

In some embodiments the equilibrium solution comprises a combination of the holding solution and permeating cryoprotectants, wherein the permeating cryoprotectants may be, for example, DMSO (dimethylsulphoxide), PROH (1,2- propanediol), EG (ethylene glycol), GLY (glycerol), and any other permeating cryoprotectants suitable for the case. However, those examples are non-limiting, and any other suitable composition may be used if applicable.

Following is one specific example of the first, second, and third compositions, which may be applicable, for example, to cryopreservation of oocytes. In this specific example, first composition is a holding solution comprising Hepes TALP medium, the second composition comprises 16% EG and 16% DMSO, and the ratio between Vi and V ₂ is 1: 1. Therefore, in this specific example, the third composition is an equilibrium solution comprising 8% EG and 8% DMSO, and the first target concentration is 16% CPA.

Having described the gradual modification of the composition of first liquid from the first composition into the third composition by adding the second liquid thereto, it is further described that the chamber, and more specifically the first space, has a liquid capacity that is sufficiently large to enable a gradual modification in accordance for vitrification.

In addition, it should be appreciated that the modification course should be adequate for preparing the sample for vitrification, while maintaining viability and reproductive functionality thereof, as explained in the following paragraphs.

It is appreciated that successful vitrification depends on duration T of the first modification period. For example, Yavin and Arav 2006, mentioned in the background of the present invention, describes vitrification of oocytes and ovarian cortical slices, wherein the duration T _\ is within the range of 8 to 12 and 30 to 50 minutes, respectively. As explained in Arav, Shehu, and Mattioli (1993), in order to maintain viability and reproductive functionality of a reproductive biological sample, the sample should be protected from damaging osmotic stress and from a long exposure to cytotoxic substances. Therefore, T should be limited by lower and upper limits. It is further appreciated that the specific ranges of T presented in Yavin and Arav 2006 refer to specific protocols depicted therein, and other protocols may dictate other ranges. However, it is appreciated that an appropriate range, which is suitable for the case, should always be applied in order to prevent damaging osmotic stress and long exposure to cytotoxic substances, thus maintaining viability and functionality of the biological sample, such as reproductive functionality in case of a reproductive biological sample.

It is further appreciated that successful vitrification depends not only on the duration T of the modification period, but also on the first modification course C _\ as defined above. Since the premating cryoprotectants are added to the first liquid by adding the second liquid thereto, it is noted that first concentration course C _\ is determined by first volume Vi and first yielding rate Y , as can be expressed in the following equation.

Equation 1 :

It is further appreciated that due to inherent accuracies, an actual value of V , and an actual course of Y _\ might deviate from their respective value and course, and therefore an actual course of C _\ might deviate from its nominal course. Therefore, successful vitrification depends on an adequate modification course, which is determined by a maximal deviation of the actual modification course from its nominal course.

According to embodiments of the present invention, cryopreservation device 202 is configured to enable gradual modification of the composition of the first liquid from the first composition to the third composition at an adequate modification course, which is adequate for vitrification, i.e. a modification course which prevents exposing the sample to damaging osmotic stress. Furthermore, the first container of the cryopreservation device is characterized by a liquid capacity which enables the adequate modification course, as explained in the following paragraphs. It is noted that higher values of V and V ₂ implies better accuracy of first modification course C _\ . In order to appreciate this point, a general example of composing ingredient B, C, etc., into a composition A is considered, where quantities of the composition and the ingredients are denoted by X _A , ¾, X _c , etc. Due to practical limitations, actual values of ¾, X _c , etc. usually differ from their nominal values by disparity quantities A _B , A _c , etc. It can be appreciated that in this example, the accuracy of composition A is substantially determined by the ratios Δ _Β /¾, AJXc, etc. Therefore, high accuracy of composition A may be achieved by reducing the disparity quantities AB, Ac, etc., or by increasing the nominal quantities ¾, Xc, etc. Since reducing the disparity quantities typically requires higher accuracy of the equipment, increasing the nominal quantities is usually a more convenient option.

Returning now to the liquid capacity of first container 206, it is appreciated that higher liquid capacity enables higher values of Vi, V ₂ , and Y , which implies better accuracy of first modification course C _\ . Therefore, in order for device 202 to be useful for vitrification, first container 206 is configured to have a liquid capacity that is sufficiently large to enable adding second volume V ₂ of the second liquid to first volume Vi of the first liquid, wherein V _\ and V ₂ are large enough to enable adequate modification course.

Recall that in some embodiments, first source 222 comprises a manual injection pump, such as a manual syringe, configured to yield the second liquid. In such embodiments, the manual injection pump together with the liquid capacity enables the gradual modification of the composition of the first liquid in accordance with the adequate modification course. In other words, the liquid capacity is configured to be large enough to enable gradual modification of the composition of first liquid by utilizing a manual pump for yielding the second liquid, wherein the gradual modification is in accordance with the adequate modification course.

In some examples, adequate modification course can be achieved when both Vi and V ₂ are equal to or greater than 0.5 milliliter. Therefore, in those examples, first container 206 is configured to be characterized by liquid-capacity of at least 1 milliliter, which is the sum of V _\ and V ₂ . That is, the volume of the first liquid and the volume of the second liquid occupy together at least 1 milliliter.

It is noted that according to the present invention, it is understood that larger volumes enable improved accuracy. Furthermore, larger volumes enable easier utilization of the device, e.g. by allowing shorter operation time and by relaxing the required skill level of the operator. Larger volumes also enable vitrification of large samples, and/or concurrent vitrification of plurality of samples. Accordingly, in some embodiments, the first container of the cryopreservation device is configured to be characterized by higher liquid-capacity. For example, in some embodiments the liquid capacity is about 1 to about 5 milliliter, which enables concurrent vitrification of a plurality of oocytes or embryos. For another example, in some cases the liquid capacity is about 5 to about 10 milliliter, thus enabling vitrification of ovarian cortical slices.

Further features of the cryopreservation device refer to size characteristics of the carrier accommodated thereby. In some embodiments chamber 204 of cryopreservation device 202 is configured to accommodate carrier 210 characterized by a size adequate for placing a biological sample therewith by manual pipetting. Regarding this feature, it should be appreciated that in order for device 202 to be useful for vitrification of a biological sample, carrier 210 should enable placing the sample therewith and taking it therefrom. Manual pipetting, i.e. utilization of a hand operated pipette for transferring of an object residing within a fluid, is a common practice in laboratories. Therefore, embodiments of device 202 characterized by a chamber configured to accommodate a carrier large enough to enable manual pipetting are advantageous, since they simplify handling of the biological sample.

Referring to the size of the carrier, recall that in some embodiments the carrier comprises access opening 216, as illustrated in FIG 2. In such embodiments, chamber 204 is configured to accommodate carrier 210 comprising access opening characterized by a diameter large enough to enable placing the sample at the carrier by manual pipetting. In some embodiments, for example, the diameter of the access opening is greater or equal to substantially 0.2 millimeter.

In some embodiments, the chamber of the cryopreservation device is configured to be substantially closed during the preparation of the sample for vitrification, wherein term "substantially closed" indicates that the chamber is generally closed, but may comprise some small openings, for examples the opening of the inlet, and openings of a first and/or second drain described below with reference to FIGs 3 and 5. One of the advantages of closing the chamber is, for example, protection of the sample from potential contamination. Another advantage, for example, is better thermal isolation of the first liquid from the environment. In some embodiments, the chamber comprises a cover which is configured to be opened in order to enable inserting the carrier and the sample into the chamber, and taking it thereout, and to be closed during the preparation of the sample.

As described above, the cryopreservation device is configured to enable modification of the composition of the first liquid from a first composition into a third composition by adding a second liquid into the first liquid. In this regard, it should be noted that mixing of different solutions into a homogeneous one is not necessarily instantaneous. Therefore, some embodiments of the cryopreservation device comprise elements configured to improve the mixing of the liquids. For example, some embodiments comprise elements configured to guide the second liquid to the carrier, thus enabling better mixing at the vicinity of the biological sample, i.e., in a volume affecting the biological sample, as illustrated in the next figure.

FIG 2A illustrates a cryopreservation device 2A02 comprising a flow guiding element 2A06, according to certain embodiments of the invention. It should be noted that device 2A02 is an example of the cryopreservation device, such as the one generally denoted 202 in FIG 2, and additional or alternative examples may exist. It should be further noted that FIGs 2 and 2A illustrate common components, wherein the common component illustrated in FIG 2A is considered to be a non-limiting example of the corresponding component in FIG 2. In general, common elements may be denoted in both figures by the same reference numerals (e.g. 218 and 222) wherein the common component in FIG 2A may be a similar or a different embodiment of the corresponding component in FIG 2. In some cases, common components are denoted in FIG 2A by different reference numerals (e.g. 2A04 and 2A08) than the numerals used in FIG 2 (210 and 214, respectively), in order to indicate that the component of FIG 2 represents an example characterized by structural and/or functional aspects additional to the corresponding component of FIG 2.

According to the embodiments illustrated in FIG 2A the carrier is denoted 2A04. Device 2A02 comprises flow guiding element 2A06 configured to guide flow of second liquid 220 coming from inlet 218 toward carrier 2A04. Consequently, the flow guiding element thus enables better merging of liquids at the vicinity of sample 104, which resides at carrier 2A04.

In some embodiments, the carrier comprises non-permeable side-walls 2A08, and a carrier opening 2A10 at an upper part of the carrier. In such embodiments, flow guiding element 2A06 may be configured to guide the flow of the second liquid to opening 2A10, as illustrated in the figure. In some embodiments, flow guiding element 2A06 may comprise, for example, a channel configured to guide liquid from inlet-opening 226 toward chamber 2A04, as illustrated in the figure. However, this example is non-limiting, and other flow guiding elements, for example a guiding element comprising a pipe, or any other appropriate guiding element, may be implemented.

As explained with reference to FIG 2, in some embodiments the cryopreservation device is configured to accommodate a plurality (i.e. two or more) of carriers. In such embodiments, the liquid guiding element may be configured to distribute the second liquid coming from the first inlet to the plurality of carriers substantially equally. In some embodiments, the flow guiding element may comprise, for example, plurality of channels, as illustrated in the figure. However, this example is non-limiting, and other flow guiding elements, for example a guiding element comprising a plurality of pipes, sprinklers, or any other appropriate guiding element, may be implemented

The examples described above, with reference to FIG 2A, are non-limiting though, and other embodiments may, additionally or alternatively, comprise other elements for the improvement of the mixing of the liquids. For example, some embodiments may comprise a stirring element, such a magnetic stirrer, or a sprinkler configured to generate flow, or any other element suitable for the case.

Further embodiments of the cryopreservation device may incorporate further elements and features. FIG 3 illustrates a cryopreservation device 302, further comprising a first drain 306, according to certain embodiments of the invention. It should be noted that device 302 is an example of the cryopreservation device, such as the one generally denoted 202 in FIG 2, and additional or alternative examples may exist. It should be further noted that FIGs 2 and 3 illustrate common components denoted by common names in both figures, wherein the common component illustrated in FIG 3 is considered to be a non-limiting example of the corresponding component in FIG 2. In general, common elements may be denoted in both figures by the same reference numerals (e.g. 218 and 222) wherein the common component in FIG 3 may be a similar or a different embodiment of the corresponding component in FIG 2. In some cases, common components are denoted in FIG 3 by different reference numerals (e.g. 304 and 312) than the numerals used in FIG 2 (204 and 206, respectively), in order to indicate that the component of FIG 3 represents an example characterized by structural and/or functional aspects additional to the corresponding component of FIG 2.

Appreciating that systems 2A02 (of FIG 2A) and 302 (of FIG 3) are both examples of system 202 (of Fig 2), it is noted that further examples may comprise structural and/or functional aspects from both FIG 2A and 3. For example, some embodiments may comprise a flow guiding element and a first drain resembling corresponding components illustrated in FIGs 2A and 3, respectively. This example is non-limiting, though, and any other combinations of components, which are appropriate for the case, may be implemented.

According to the embodiments illustrated in FIG 3 the first chamber is denoted

304, and the first container is denoted 312. Device 302 further comprises first drain 306, which is coupled to first space 208 and is configured to drain first liquid 110 therefrom.

In some embodiments, chamber 304 and first drain 306 are configured together to induce, by draining part of the first liquid, a directional flow of first liquid 110 through permeable member 212; and the directional produces a dragging force which forces biological sample 104 to reside at predetermined location, i.e. substantially adjacent to the permeable member, as explained below. Flow of liquid is illustrated in FIG 3 by arrows.

In the following discussion, the term "density" refers to the specific gravity of a given object, i.e. to a mass of the object divided a volume thereof. It should be appreciated that when an object A is immersed in a fluid B, wherein object A and fluid B are characterized by densities D ₀ bj _ect and Dfl _ui d, respectively, a position of object A relative to fluid B depends on the relation between the two densities D ₀ bj _ect and which determines the relationship between a buoyance of the object and a weight thereof. Accordingly, If D ₀ bj _ect > ^fiuid, the object tends to sink down to a bottom of the fluid; if D ₀ bj _ec t < the object tends to rise up to an upper surface of the fluid; and if D ₀ bj _ect = £ * fluid, the object will float within the fluid. In some vitrification protocols, the first composition is characterized by a first density which is lower than an initial density of the biological sample (i.e. prior to the preparation of the sample for vitrification). In addition, the second composition is characterized by a second density which is higher than the initial density of the sample. Therefore, during the first period the first liquid is typically characterized by a gradually increasing density. Hence, in some cases, at some moment during the first period the density of the first liquid might become equal or higher than the density of the sample, thereby causing the sample to float in the first liquid or to rise to an upper surface thereof. Accordingly, in some cases, the sample might reach an opening of the carrier and drift out of the carrier and be lost in the first container. In other cases, the sample might rise to the upper surface of the liquid and adhere to a wall of the carrier. In such cases it might be subsequently very difficult to find the sample and/or it might be damaged as well. Furthermore, in some cases the effectiveness of subsequent stages of the vitrification protocol might be deteriorated when the sample does not reside on the permeable member, as explained in the following examples. For one example, it is appreciated that in cases where the sample adheres to a non-permeable wall of the chamber rather than residing on the permeable member, it will risk dehydration. For another example, it is noted that in some cases a volume of a residual liquid which surrounds the biological sample is reduced prior to inserting the sample into the cryogenic media, as explained below with reference to FIG 4. The reduction of the residual liquid may be performed by aspiration of gas, as described below with reference to FIG 4. In those cases, the reduction of the residual liquid's volume might be less effective when the sample does not reside on the permeable member. Additionally or alternatively, the reduction of the residual liquid may be performed by placing the carrier on an element configured to suck liquids, for example an absorbing tissue. In those cases, also, the reduction of the residual liquid's volume might be less effective when the sample does not reside on the permeable member.

Returning to FIG 3, device 302 is configured to overcome the difficulty described above. According to embodiments illustrated in the figure, chamber 304 and first drain 306 are configured so that when part of first liquid 110 is drained by the first drain, a directive flow of liquid through permeable member 212 is consequently being induced. This induced flow produces a dragging force on sample 104, thereby forcing it to reside substantially adjacent to the permeable member 212. Flow of liquid is illustrated in FIG 3 by arrows. It should be noted that the sample is subject to two forces, an elevation force determined by the difference between the buoyancy and weight of the sample, and the dragging force produced determined by the induced directional flow. It is further noted that since those forces may fluctuate over time, a position of the sample within the first liquid may also change over time. However, as long as the sample does reach a level of an opening of the carrier, and does not reach the upper surface of the first liquid, the sample will not be drifted out of the carrier, and will not adhere to the wall of the carrier, and will eventually reside at the permeable member. Accordingly, as long as the sample remains below the upper level of the first liquid, and does not reach a level of an opening of the carrier, the sample is considered to reside substantially adjacent to the permeable member.

In the example illustrated in FIG 3, the first drain is configured to drain the first liquid out of the chamber. However, this example is non-limiting, and in other embodiments the first drain may comprise an element, e.g. a pump, configured to transfer part of the first liquid from a first location to a second location within the chamber. For example, in some embodiments the first drain may be configured to drain liquid from a first location lower than the permeable member, to a second location higher than the permeable member.

In some embodiments, which are illustrated in FIG 3, first drain 306 comprises a first drain opening 308 in the first container. In some embodiments, also illustrated in FIG 3, first drain 306 is coupleable to an aspiration device 310 configured to aspire the first liquid. In some embodiments, the aspiration device comprises an aspiration pump, for example a syringe, as illustrated in FIG 3. However, the details illustrated in FIG 3 are non-limiting, and other embodiments can also be implemented. In some embodiments, for example, the first drain may comprise a proximal opening coupled to the first space and a distal opening that is located lower than the proximal opening, thereby enabling passive drainage of the first liquid by gravitation.

As explained above, with reference to the current figure, in some embodiments first drain 306 is configured to enable minor drainage, i.e. drainage of a small portion of the first liquid configured to enable flow through the permeable member. It may be considered that in some cases, e.g., wherein the sample is a piece of tissue whose size is large enough (for example, above 0.25 millimeter in radius), or when the sample is a bulk of cells comprising, for example, at least 0.5 milliliter of cells (net volume), or a cell suspension having a net volume of 0.5 milliliter, it is less important to maintain the sample residing substantially adjacent to the permeable member and hence, in such cases, minor drainage may be skipped.

Additionally or alternatively to what has been described so far, in some embodiments, the first drain is configured to enable "major drainage", which may even exhaust substantially all of the first liquid from the first container. In this regard, it should be noted that due to practical limitations, some small portion of the first liquid might remain within the first container even after substantially all the first liquid is exhausted therefrom. It is appreciated that it is advantageous to evacuate almost all the liquid from the chamber. However, as long as an upper level of the first liquid is reduced below a level of the permeable member, the major drainage may be considered to exhaust substantially all the first liquid from the first container.

In some embodiments, device 302 may comprises two first drains 306, one configured for minor drainage and the other configured for major drainage. In other embodiments, device 302 may comprise first drain 306 configured for both minor and major drainage. Likewise, in some embodiments, two aspiration devices may be coupled to the first drain, one for minor and the other for major drainage, and in some embodiments the same aspiration device may be used for both minor and major drainage.

Major drainage capability may be utilized for abrupt modification of the composition of the first liquid. In some vitrification protocols, after the biological sample reaches the first equilibrium state, the composition of first liquid is abruptly modified from the third composition into a fourth composition. In some protocols, the fourth composition is a final-vitrification-solution configured to surround and protect the sample, and to prevent formation of extra-cellular ice crystals. In some protocols, the final-vitrification solution is further configured for partially dehydrating the sample. It is noted that the modification of the composition from the third into the fourth solution is abrupt (e.g. no longer than 60 seconds), in order to avoid overloading the sample with further permeable cryoprotectants, exposing it to damaging osmotic stress. In some protocols the vitrification solution comprises 15% DMSO (dimethyl sulfoxide), 15% ethandiol, 0.5 moVliter sucrose, 10% serum in medium or 16% DMSO, 16% propanediol, 0.5 mol/liter trehalose, and 10% HAS (human serum albumin) in PBS (phosphate buffered saline). In some protocols, the final-vitrification-solution may further comprise some non-permeating additives, for example sucrose or trehalose, or any other non-permeating additive suitable for the case. However, those compositions are brought by way of example, and are therefore non-limiting, and any other combination of the above ingredients, or any other composition appropriate for the case, may be utilized.

According to some embodiments of the invention, abrupt modification of the composition of the first liquid is enabled by inserting liquids to the first container through the inlet and exhausting liquids thereof through the first drain. For example, abrupt modification may be performed by exhausting the first liquid from the first container, followed by inserting liquids. Inserting and exhausting can be performed either concurrently or sequentially. It should be appreciated that any sequence of insertion and exhaustion operations may be implemented, as appropriate for the case.

Still referring to FIG 3, alternative or additional unitizations of the major drainage capability are now described. In some embodiments, for example, major drainage is utilized for reducing a volume of a "residual liquid" which surrounds the biological sample, as explained in the following paragraph.

In order to understand the term "residual liquid" and to appreciate the advantage of reducing the volume thereof, it should be recalled that the carrier holding the sample is taken out of the cryopreservation device and is inserted into the cryogenic medium. It should be further recalled that according to some vitrification protocols, the carrier is immersed in liquid residing within the cryopreservation device. Consequently, when the carrier is taken out of the device, the sample may reside within a residual volume of liquid, referred herein as the volume of the residual liquid. As demonstrated in Arav 1992, reduction of volume of the residual liquid is advantageous for vitrification, since the probability of successful vitrification is inversely proportional to the residual liquid's volume.

Returning to FIG 3, chamber 304 and first drainage 306 are configured so that when the first liquid is drained out of the first container via the first drain, the volume of the residual liquid is reduced. Accordingly, device 302 is configured to reduce the volume of the residual liquid by draining substantially all the first liquid out of the first container via the first drain.

In some embodiments, inlet 218 is further configured to enable flow of gas into the chamber. In some embodiments, device 302 comprises two inlets 218, one configured to deliver liquids and the other configured to enable flow of gas. In other embodiments, the same inlet 218 is configured for both. For example, in some embodiments, when coupled to the first source 212 the inlet delivers liquids, and when detached from the first source, the same inlet enables flow of gas. In some embodiments, inlet 218 may be coupleable to two first sources 212, one configurable to yield first liquid 220 and the other configured to yield gas. In some embodiments, first source 212 that is configured to yield gas may be a gas yielding pump, for example a syringe containing gas. In some embodiments, inlet 218 may be coupleable to first source 212 which is configured to yield liquid at some stages and to yield gas at other stages. It should be recalled that in some embodiments, device 302 is configured to enable major drainage of the first liquid from the first container. In some embodiments, inlet 218 is configured to enable the major drainage by allowing gas to enter into the first container and replace the first liquid being drained out. It should be noted that without entrance of gas into the chamber, drainage of liquids from the chamber might produce a low pressure therein, thus hampering further drainage and possibly damaging the sample.

In some embodiments of device 302, first drain 306 is further configured to enable aspiration of gas therethrough from the chamber. In some embodiments, device 302 comprises two first drains 306, one configured to drain liquid and the other configured to aspirate gas. In other embodiments, a single first drain is configured for both.

Recall that in some embodiments first drain 306 is coupleable to aspirating device 310. In some embodiments, first drain 306 which is configured to drain liquid is coupleable to aspiration device 310 configured to aspirate liquids. In some embodiments, first drain 306 which is configured to aspirate gas is coupleable to aspiration device 310 configured to aspirate gas. In some cases, the same aspiration device 310 is configured to aspirate both liquid and gas.

It should be recalled that, in some embodiments, device 302 is configured to reduce the volume of the residual liquid by draining the first liquid out of the first container. It should also be recalled that reducing the volume of the residual liquid result in higher cooling rate, which improves the likelihood of successful vitrification with no formation, or with minimal formation, of harmful ice crystals. In some embodiments, device 302 is configured to enable further reduction of the volume of the residual liquid, after the drainage of the first liquid from the chamber. The further reduction is achieved by aspiration of gas from the drained chamber, as explained with reference to the next figure.

FIG 4 illustrates a cryopreservation device configured to enable reduction of volume of a residual liquid by aspirating gas, according to certain embodiments of the invention. As illustrated in FIG 4, gas is aspired from a chamber 404 through a first drain 408. It should be noted that device 402 is an example of the cryopreservation device, such as the one generally denoted 302 in FIG 3, and additional or alternative examples may exist. It should be further noted that FIGs 3 and 4 illustrate components denoted by common names in both figures, wherein the common component illustrated in FIG 4 is considered to be a non-limiting example of the corresponding component in FIG 3. In general, common elements may be denoted in both figures by the same reference numerals (e.g. 210 and 310) wherein the common component in FIG 4 may be a similar or a different embodiment of the corresponding component in FIG 3. In some cases, common components are denoted in FIG 4 by different reference numerals (e.g. 404 and 408) than the numerals used in FIG 2 (304 and 308, respectively), in order to indicate that the component of FIG 4 represents an example characterized by structural and/or functional aspects additional to the corresponding component of FIG 3.

According to the embodiments illustrated in FIG 4, the chamber, inlet, and first drain are denoted by 404, 406, and 408, 410, respectively. Chamber 404, inlet 406, and first drain 408 are configured so that aspiration of gas via the first drain induces a flow of gas at a vicinity of biological sample 104, thus removing a part of the residual liquid, by causing it to evaporate. The induced gas flow is illustrated in FIG 4 by arrows. It should be noted that gas aspiration, as illustrated in FIG 4, is usually performed when the chamber is substantially free of liquids, for example after major drainage, as described with reference to FIG 3.

Further embodiments of the cryopreservation device incorporating further elements and features are described in the next figures. FIG 5 illustrates a cryopreservation device 502, further comprising a second drain 508, according to certain embodiments of the invention. It should be noted that device 502 is an example of the cryopreservation device, such as the one generally denoted 202 in FIG 2. It should be further noted that FIGs 2 and 5 illustrate common components denoted by common names in both figures, wherein the common component illustrated in FIG 5 is considered to be a non-limiting example of the corresponding component in FIG 2. In general, common elements may be denoted in both figures by the same reference numerals (e.g. 218 and 222) wherein the common component in FIG 5 may be a similar or a different embodiment of the corresponding component in FIG 2. In some cases, common components are denoted in FIG 5 by different reference numerals (e.g. 504 and 506) than the numerals used in FIG 2 (204 and 206, respectively), in order to indicate that the component of FIG 5 represents an example characterized by structural and/or functional aspects additional to the corresponding component of FIG 2. Appreciating that systems 2A02, 302, 402, and 502 (of FIGs 2A, 3, 4, and 5, respectively) are all examples of system 202 (of Fig 2), it is noted that further examples may comprise structural and/or functional aspects from FIGs 2A, 3, 4, and 5. For example, some embodiments may comprise a flow guiding element, a first drain, and a second drain resembling the corresponding components illustrated in FIG 2A, 3, and 5, respectively. These examples are non-limiting, though, and any other combinations appropriate for the case may be implemented.

According to the embodiments illustrated in FIG 5, the chamber is denoted 504, and the first container is denoted 506. Device 502 further comprises second drain 508, which is configured to enable flow of an excessive volume of the first liquid exceeding a first-level- limit 510 out of the first container, thus constraining a level of the first liquid substantially below the first-liquid-limit.

It is noted that the level of the term "below" is considered in the last sentence to mean "below or equal". It is further noted that in some cases the level of the first liquid may temporarily exceed the first-liquid-level, due to hydrodynamic effects, while the excessive liquid flows out of the first container. Additionally or alternatively, the level of the first liquid may slightly exceed the first-liquid-level due to a surface tension of the first liquid. Accordingly, "substantially below the first- liquid-limit" is considered herein to mean below or equal, and to further allow for some transient exceeding due to hydrodynamic effects, and for some slight steady- state exceeding due to liquid tension.

The second drain may be implemented in various configurations, some of which are brought, by way of example, in FIGs 5, 5A, and 6. FIG 5A illustrates a cryopreservation device 5A02 which enables in-field configuration of the first-liquid- level-limit, and FIG 6 illustrates two cryopreservation devices 602a and 602b comprising a second container 612. In some embodiments the second drain is configured to enable flow of the excessive volume of the first liquid out of the first chamber. For example, in embodiments illustrated in FIGs 5 and 5A, the excessive liquid flows out of the chamber and is collected in an auxiliary container 512. However, this is non-limiting, and in other embodiments the excessive liquid may still remain within the chamber. In embodiments illustrated in FIG 6, for example, the chamber further comprises a second container 612, configured to collect the excessive liquid flowing out of the first container. In some embodiments the second drain comprises a pipe coupled to a space confined by the first container. In the embodiments illustrated in FIG 5 and 5A, for example, the second drain comprises a pipe comprising a proximal and distal second- drain-pipe openings, located inside and outside the first container, respectively. However, this is non-limiting, and other embodiments may be implemented. In one embodiment illustrated in FIG 6, for example, second drain 608a comprises a second- drain opening in a container side-wall 614 of the first container. In another embodiment illustrated in FIG 6, for example, second drain 608b comprises a second- drain opening residing above an upper edge of container side-wall 614.

In some embodiments, the first-liquid-limit is a fixed predetermined level. In the embodiments illustrated in FIG 6, for example, first-liquid-limit 610 is determined by the vertical location of the second-drain openings 608a and 608b. In other embodiments, the first-liquid-level may be configured at the laboratory as appropriate for the case. In the embodiment illustrated in FIG 5A, for example, the first-liquid- level 5A12 can be varied by varying the vertical level of the second-drain-pipe opening 5A14.

It should be noted in this regard that the examples of the second drain, as illustrated in FIGs 5, 5 A, and 6, are non-limiting. Accordingly, any appropriate combination of structural and functional features illustrated by the figures and/or described with reference thereto, or any other embodiment appropriated for case, may be implemented.

In some embodiments, the flow of the excessive liquid out of the first container may enable improved merging of the second liquid into the first liquid. Recall that, as described with reference to FIG 2A, in some embodiments the cryopreservation device comprises flow guiding element 2A06 configured to guide flow of second liquid 220 coming from inlet 218 toward carrier 2A04, in order to improve merging of liquids at the vicinity of sample 104, which resides at carrier 2A04.

FIG 7 illustrates a cryopreservation device 702 configured to improve merging of liquids by utilizing flow of excessive liquid, according to certain embodiments of the invention. It should be noted that device 702 is an example of the cryopreservation device, such as the one generally denoted 202 in FIG 2. It should be further noted that FIGs 2 and 7 illustrate common components denoted by common names in both figures, wherein the common component illustrated in FIG 7 is considered to be a non-limiting example of the corresponding component in FIG 2. In general, common elements may be denoted in both figures by the same reference numerals (e.g. 218 and 222) wherein the common component in FIG 7 may be a similar or a different embodiment of the corresponding component in FIG 2. In some cases, common components are denoted in FIG 7 by different reference numerals (e.g. 704 and 706) than the numerals used in FIG 2 (204 and 206, respectively), in order to indicate that the component of FIG 7 represents an example characterized by structural and/or functional aspects additional to the corresponding component of FIG 2.

Appreciating that systems 2A02, 302, 402, 502, 5A02, 602a, and 602b (of FIGs 2A, 3, 4, 5, 5A, and 6, respectively) are all examples of system 202 (of Fig 2), it is noted that further examples may comprise structural and/or functional aspects from FIGs 2A, 3, 4, 5, 5A, and 6. For example, some embodiments may comprise a flow guiding element, a first drain, a second drain, and a second container resembling the corresponding components illustrated in FIG 2A, 3, and 5, 5A, and 6, respectively. These examples are non-limiting, though, and any other combinations appropriate for the case may be implemented.

According to embodiments illustrated in FIG 7, the chamber is denoted 704, the first container is denoted 706, the flow guiding element is denoted 708, the second drain is denoted 710, the first-level- limit is denoted 712, and the carrier is denoted 714. The carrier comprises non-permeable walls 716, a permeable member 718, and a carrier opening 718. In the example illustrated in the figure non-permeable walls 716 are side-walls, permeable member 718 is located at a bottom of the carrier, and carrier opening 718 is located at an upper part thereof. However, this example is non- limiting, and in other carriers the carrier may be configured with the walls, the permeable member, and the carrier opening, located at other relative positions.

As illustrated in the figure, chamber 704 is configured to accommodate carrier 714 so that permeable member 718 and carrier opening 718 reside below and above first-level-limit 712, respectively. Furthermore, flow guiding element 708 is configured to guide second liquid 220 to flow into carrier opening 718.

As further illustrated in the figure, the arrangement described above results in a flow of the second liquid into the carrier, where it is merged with the first liquid residing therein, and a flow of the first liquid out of the carrier, via permeable member 718, thus enabling further flow of second liquid into the carrier. Consequently, this arrangement enables improved merging of the second liquid into the first liquids at the vicinity of sample 104, which resides at the carrier. The flow of the liquids is illustrated in the figure by arrows.

FIG 8 illustrates a cryopreservation device 802 configured to enable visual inspection of the biological sample residing within the chamber, according to certain embodiments of the invention. It should be noted that device 802 is an example of the cryopreservation device, such as the one generally denoted 202 in FIG 2. It should be further noted that FIGs 2 and 8 illustrate common components denoted by common names in both figures, wherein the common component illustrated in FIG 8 is considered to be a non-limiting example of the corresponding component in FIG 2. In general, common elements may be denoted in both figures by the same reference numerals (e.g. 212 and 222) wherein the common component in FIG 8 may be a similar or a different embodiment of the corresponding component in FIG 2. In some cases, common components are denoted in FIG 8 by different reference numerals (e.g. 804 and 806) than the numerals used in FIG 2 (204 and 206, respectively), in order to indicate that the component of FIG 8 represents an example characterized by structural and/or functional aspects additional to the corresponding component of FIG 2.

Appreciating that systems 2A02, 302, 402, 502, 5A02, 602 (a and b), and 702 (of FIGs 2A, 3, 4, 5, 5A, 6, and 7, respectively) are all examples of system 202 (of Fig 2), it is noted that further examples may comprise structural and/or functional aspects from FIGs 2A, 3, 4, 5, 5A, 6, and 7. For example, some embodiments may comprise a flow guiding element, a first drain, a second drain, and a second container resembling the corresponding components illustrated in FIG 2A, 3, and 5, 5A, and 6, respectively. These examples are non-limiting, though, and any other combinations appropriate for the case may be implemented.

According to embodiments illustrated in FIG 8, the chamber is denoted 804, and the first container is denoted 806.

In some embodiments, the chamber of the cryopreservation device may be constructed of transparent materials, whereby enabling visual inspection of its content. However, in many other cases the chamber is non-transparent. Therefore, in some embodiments, which are illustrated in FIG 8, cryopreservation device 802 is configured to enable visual inspection of the biological sample according to embodiments described in the following examples, or a combination thereof, or any other embodiments appropriate for the case. In some embodiments, the chamber of the cryopreservation device comprises an inspection element configure to enable visual inspection of the sample. In some embodiments, the inspection element may be an internal inspection element residing inside the chamber. In some embodiments the internal inspection element comprises a CCD camera. However, this example is non-limiting, and other internal inspection elements, for example an optic fiber, or any other inspection element suitable for the case, may be utilized. In other embodiments, as illustrated in FIG 8, the inspection element comprises an inspection window 808, which constitutes an opening in a boundary (e.g. side-wall, cover, floor, etc.) of the chamber, wherein the inspection window enables inspecting the sample therethrough.

In some embodiments the chamber of the cryopreservation device comprises an illumination element configure to enable illuminating of sample 104 while inspecting thereof. In some embodiments, the illumination element may be an internal illumination element residing inside the chamber. In some embodiments the internal inspection element is a LED (liquid emitting diode). However, this example is non- limiting, and other internal illumination elements, for example an optic fiber, or any other illumination element suitable for the case, may be utilized. In other embodiments, as illustrated in FIG 8, the illumination element comprises an illumination window 810, which constitutes an opening in a boundary of the chamber, wherein the illumination window enables illuminating the sample by light penetration thereby.

In some embodiment of the cryopreservation device, the chamber is configured to accommodate the carrier so that the sample resides substantially between the illumination element and the inspection element, thus enabling trans- illumination of the sample, i.e. illuminating the inspected sample by light passing therethrough. It is noted that since oocytes and embryos are substantially transparent, trans-illumination is more convenient for visual inspection thereof than reflected illumination.

In some embodiments illustrated in FIG 8, chamber 804 is configured to accommodate carrier 220 so that sample 104 resides substantially between illumination window 810 and inspection window 808, thus enabling transillumination of the sample.

In the example illustrated in FIG 8, trans-illumination is enabled by locating illumination window 808 substantially below carrier 220, e.g. at a bottom of first container 806, and locating inspection window 810 substantially above the carrier, e.g. at a cover of chamber 804. However, the embodiment illustrated in the figure is non-limiting, and other embodiments may be implemented, for example embodiment wherein trans-illumination is further enable by a reflective surface (e.g. a mirror) located within the carrier, or embodiments which enable reflected-illumination rather than trans-illumination, or any other embodiment appropriate for the case.

In some embodiments, the inspection window and/or the illumination window are configured to be closed by an inspection-window-cover and or by an illumination- window-cover comprising a transparent member, thus enabling visual inspection while maintaining the chamber closed. In some embodiments, chamber 804 further comprises one or more fastening mechanisms configured to fasten the inspection- window-cover and or the illumination-window-cover to the boundary of the chamber, thus securing the closure of the chamber. Fastening mechanism may be implemented in according to various methods known in the art, including for example, seal supported threading or any other method appropriated for the case.

In some embodiments, the one or more window covers may be disposable elements. In other embodiments the one or more window covers may be elements configured to be sterilized. In some embodiments, the one or more window covers may be Petri dishes, which enable convenient cleaning and sterilization of the device.

In some embodiments, the inspection window comprises one or more optical lenses.

It is noted that the capability of the cryopreservation device to enable visual inspection of the biological samples residing therein enable better control on the preparation of the biological sample for vitrification, thereby resulting in higher probability for successful vitrification. One example for the benefit of the visual inspection is related to the minor drainage of the first liquid. As explained with reference to FIG 3, in some embodiments a minor part of the first liquid is drained through the first drain, in order to force the biological sample to reside substantially adjacent to the permeable member. Visual inspection enables preforming the minor drainage responsive to the distance of the sample from the permeable member, thus maintaining a desired location of the sample at minimal drainage.

Further example for the benefit of the visual inspection is related to the gradual modification of the composition of the first liquid. As explained with reference to FIG 2, successful vitrification depends on the modification course of the first liquid, which is determined by the first yielding course of the second liquid. Visual inspection enables controlling the first yielding course responsive to information obtained during visual indication about a status of the sample. In some cases, the first yielding course may be controlled responsive to changes in a volume of the sample. In some cases, for example, an excessive reduction of the volume of the sample indicates an excessive osmotic stress, and is therefore responded by a reduction of the yielding rate. Alternatively or additionally, the first yielding course may be controlled responsive to changes in a position of the sample. In some cases, for example, rising of the sample indicates an excessive difference in concentration between the sample and the first liquid, and is therefore responded by a reduction of the yielding rate. In this regard it is noted that the yielding course should not be reduced below a minimal level, in order not to increase toxicity by excessive exposure to the vitrification solution.

The above examples for utilization of the visual inspection are, however, non- limiting, and other utilizations, for example asserting the viability of the sample, or any other utilization appropriate for the case, may be implemented.

Following the description of various embodiments of the cryopreservation device, methods for utilization thereof are presented below. FIG 9 schematically illustrates a cryopreservation method 902 for vitrification of a reproductive multi cell suspension, by using a cryopreservation device in accordance with embodiments of the invention. As explained above, with reference to FIG 1, the multi cell suspension (cells that are in suspension) may comprise an oocyte, an embryo, sperm, or another reproductive multi-cell suspension. According to embodiments illustrated in the figure, the cryopreservation method comprises initialization 904, gradual modification 906, abrupt modification 908, and rapid cooling 912. In some embodiments, method 902 may also comprise residual liquid reduction.

Initialization comprises immersing the multi cell suspension in a first liquid residing in a chamber of the device. The multi-cell suspension is associated with a carrier, and the carrier is accommodated within the chamber, thereby immersing the multi-cell suspension in the first liquid. Volume and composition of the first liquid during the initialization are referred to as the first volume and the first composition, respectively. The initialization was described, in depth, with reference to FIGs 1 and 2. Gradual modification comprises modifying the first liquid from the first composition to a third composition by adding a second liquid thereto. Composition and volume of the second liquid are referred to as the second composition and the second volume, respectively. Gradual modification is performed at a first modification course, which is adequate for vitrification. The gradual modification was described, in depth, with reference to the previous figures, and mainly with reference to FIG 2.

Abrupt modification comprises modifying the first liquid from the third composition to a forth composition. Abrupt modification is performed by draining substantially all the first liquid, and adding a third liquid into the chamber through the inlet. A composition of the third liquid is referred to as the forth composition. Abrupt modification was described, in depth, with reference to the previous figures, and mainly with reference to FIG 3.

The rapid cooling comprises taking the carrier, which the multi-cell suspension associated with it, out of the chamber, and inserting the carrier into a cryogenic medium. The rapid cooling was described, in depth, with reference to the previous figures, and mainly with reference to FIG 1.

In some embodiments adding liquid for gradual modification and/or for abrupt modification is performed manually, e.g. by a manual injection pump, such as a syringe. The utilization of a manual injection pump was described, in depth, with reference to the previous figures, and mainly with reference to FIG 2.

In some embodiments minor and/or draining of liquids is performed manually, e.g. by a manual aspiration pump, such as a syringe. The utilization of a manual aspiration pump was described, in depth, with reference to the previous figures, and mainly with reference to FIG 3.

In some embodiments the vitrification method further comprises maintaining the multi cell suspension substantially adjacent to a permeable member of the carrier. Maintaining adjacent to a permeable member is performed by draining part of the first liquid, which is referred to as minor drainage. The minor drainage induces a directional flow of the first liquid through the permeable member, and the directional flow applies a dragging force on the multi-cell suspension, thereby forcing the multi- cell suspension to reside substantially adjacent to the permeable member. In some embodiments, minor drainage is performed during the gradual modifying. However, this is non-limiting, and minor drainage may be performed, additionally or alternatively, at other times too. The minor drainage and its utilization for maintaining the multi cell suspension substantially adjacent to the permeable member were described, in depth, with reference to the previous figures, and mainly with reference to FIG 3.

In some embodiments, associating the multi cell suspension with the carrier is performed by manual pipetting, for example by placing the multi cell suspension in the carrier by manual pipetting. Utilization of a manual pipette was described, in depth, with reference to the previous figures, and mainly with reference to FIG 2.

In some embodiments, the sum of the first volume and the second volume is at least 1 milliliter. In other embodiments, the sum of the first volume and the second volume is at least 5 milliliter. The volumes of the first and the second liquids are described, in depth, with reference to the previous figures, and mainly with reference to FIG 2.

In some embodiments, the chamber is configured to accommodate two or more carriers. Accordingly, in some embodiments the method comprises concurrent vitrification of multi-cell suspensions associated with a plurality of carriers. In such embodiments, two or more multi cell suspensions, which association with two or more carriers, are immersed concurrently immersed in the first liquid residing in the chamber, and concurrently prepared for vitrification. Concurrent vitrification is described, in depth, with reference to the previous figures, and mainly with reference to FIGs 2 and 2A.

In some embodiment, the method further comprises constraining a level of the first liquid substantially below a first-liquid-limit. Constraining is performed by enabling out-flow of an excessive volume of the first liquid, wherein the term "excessive volume" refers to a volume of the first liquid which exceeds the first level limit. In some embodiments, the excessive volume flows out of the chamber. In other embodiments, the excessive volume flows out of a first container of the chamber, and is collected in a second container. Constraining a level of the first liquid is described, in depth, with reference to the previous figures, and mainly with reference to FIGs 5, 5A, and 6.

In some embodiments, the vitrification method further comprises visual inspection of the multi-cell suspension residing within the chamber. In some embodiments, visual inspection comprises trans-illumination of the multi-cell suspension. Visual inspection is described, in depth, with reference to the previous figures, and mainly with reference to FIG 8.

In some embodiments, visual inspection is utilized for controlling the preparation of the multi-cells for vitrification. In some embodiments, adding the second liquid is controlled responsive to information obtained during visual inspection of the multi-cell suspension. A yielding rate, at which the second liquid may be added to the first liquid, may be controlled responsive to visual indications of a status of the multi-cell suspension, thereby resulting in an adequate modification course. Additionally or alternatively, in some embodiments, the minor drain is controlled respective to information obtained during the visual inspection, thus enabling maintaining the multi cell suspension substantially adjacent to a permeable member of the carrier.

In some embodiments, the vitrification method further comprises a residual liquid reducing, wherein a volume of a residual liquid surrounding the multi-cell suspension is reduced. In some embodiments, residual liquid reducing is performed by draining substantially all the first liquid and aspiring gas. The aspiration induces a flow of gas at a vicinity of the multi-cell suspension, thereby removing a part of the residual liquid. Gas aspiration and its utilization for reducing the volume of the residual liquid is inspection is described, in depth, with reference to the previous figures, and mainly with reference to FIG 4.

The advantages of the cryopreservation device and the method of utilization thereof, in accordance with embodiments of the invention, have been examined in experiments, some examples thereof presented in Appendices A and B. The very high survival rates (90%) obtained in the experiments asserts the advantageous of the invention in preserving the viability of the vitrified oocytes and ovarian tissues.

APPENDIX A

The "devices" mentioned with reference to this appendix are devices described above, as embodiments of the invention.

An example of a bovine oocyte vitrification protocol:

Media preparation

Vitrification solutions:

1. ES (equilibration solution): 16% ethylene glycol (EG)

16% dimethyl sulfoxide (DMSO)

in - HEPES-TALP medium + 3% BSA

2. VS (vitrification solution): 16% ethylene glycol (EG)

16% dimethyl sulfoxide (DMSO)

1M sucrose

in - HEPES-TALP medium + 3% BSA

Materials Equipment

HEPES-TALP (pre-warmed) (5.5 ml) Filter

ES (6.5 ml) 10 ml syringe (3)

VS (2.5 ml) Pipette (10 ml)

LN (liquid nitrogen) Box for LN

Stripper + tips

Procedure 1. Add 5.5 ml HEPES-TALP into a device, e.g., a device described in the

embodiments above.

2. Put a filter into the device

3. Transfer COCs into the filter Start injecting ES from syringe A and at the same time remove the HEPES- TALP with syringe B.

Wait for 16 min. until the syringe with ES is empty.

Evacuate the liquid from the device with syringe D.

Inject 2.5 ml of VS into the filter above the COCs, using syringe C.

Evacuate the liquid from the device, using syringe D.

Aspirate air using syringe D.

Transfer the filter (containing the COCs) into LN.

An example of an oocyte warming protocol:

Media preparation

Warming solutions:

WS - 1M sucrose in HEPES-TALP medium

WS - 0.5M sucrose in HEPES-TALP medium

WS - 0.25M sucrose in HEPES-TALP medium

Materials Equipment

LN (liquid nitrogen) Box for LN

WS 1M (pre-warmed) (2.5 ml) 10 ml syringe (3)

WS 0.5M (pre-warmed) (0.5 ml) Filter

WS 0.25M (pre-warmed) (0.5 ml) Pipette (10 ml)

Culture medium (pre-warmed) Stripper + tips

Mineral Oil (pre-warmed) (6ml per dish) Petri dish (2; 60 mm)

Pipettor + tips

4-well dish

Procedure Remove the filter from the LN and transfer it into a Petri dish with WS 1 M pre-warmed at 37 C

Collect the COCs from the filter and transfer them into a 4-well dish: WS 1 M,

WS 0.5 and WS 0.25 M, for 2.5 min in each solution.

Wash the COCs 3-4 times and transfer them to the culture media. An example of a bovine ovarian tissue vitrification protocol:

Media preparation

Vitrification solutions:

1. ES (equilibration solution): 16% ethylene glycol (EG)

16% dimethyl sulfoxide (DMSO)

in - HEPES-TALP medium + 3% BSA

2. VS (vitrification solution): 20% ethylene glycol (EG)

20% dimethyl sulfoxide (DMSO)

1M sucrose

in - HEPES-TALP medium + 3% BSA

Materials Equipment

HEPES-TALP (pre-warmed) (-20 ml) 10 ml syringe (4)

Filter Pipette (10 ml)

ES (10 ml) Box for LN

VS (15 ml) Stripper + tips

LN (liquid nitrogen) Scalpel handle

Disposable sterile scalpel blades (size 20 or 21)

Petri dish (100x20 mm)

Procedure

1. Put the ovary in 100x20 mm Petri dish with 15 ml HEPES-TALP

2. Cut with scalpel through the center of each ovary (through the hilus)

3. Remove the medulla using scissors until the cortical zone appears translucent

4. Cut the cortical zone into lxl cm pieces

5. Add 5.5 ml HEPES-TALP into the device (a device described above as an embodiment of the invention)

6. Put a filter into the device (pay attention that no air bubble created)

7. Transfer one piece of the ovarian tissue into the filter

8. Inject ES (10 ml) from syringe A for 35 min (until the syringe with ES is empty). Inject VS (10 ml) from syringe B for 15 min (until the syringe with VS is empty).

Remove the liquid from the device with syringe C.

Inject 5 ml of VS into the filter above the ovarian tissue, using syringe D. Remove the liquid from the device with syringe C.

Transfer the filter (containing the ovarian tissue) into LN.

An example of an ovarian tissue warming protocol:

Media preparation

Warming solutions:

WS - 1M sucrose in HEPES-TALP medium

Materials Equipment

LN (liquid nitrogen) Box for LN

Filter

WS 1M (pre- warmed) (40 ml) 10 ml syringe (3)

Pipette (10 ml)

Petri dish (100x20

Pipettor + tips

Procedure Remove the filter from the LN and immediately put it into a Petri dish with 1M WS.

Wash the ovarian tissue with 1M WS using a syringe.

Transfer the filter into a device (a device described above as an embodiment of the invention) and immediately add 5.5 ml 1M WS into the filter above the ovarian tissue, using syringe C.

Inject 10 ml HEPES-TALP from syringe A for 10 min. Following are data obtained during several experiments performed in order to test the effects of the invention.

EXPERIMENT 1: Effect on oocyte maturation

COCs were divided into 3 groups:

1. CONTROL GROUP

2. SOLUTION EFFECT GROUP

3. VITRIFICATIONAV ARMING GROUP

EXPERIMENT 2: Ovarian tissue vitrification

Ovarian tissue divided into 4 groups: 1. CONTROL GROUP 1

2. CONTROL GROUP 2

3. AUTOMATIC VITRIFICATIONAVARMING GROUP (PINK DEVICE)

4. AUTOMATIC VITRIFICATIONAVARMING GROUP (GREY DEVICE)

The tissue pieces preserved in PFA

EXPERIMENT 3: Effect on oocyte developmental competence, fertilization and cleavage

COCs were divided into 3 groups:

1. CONTROL GROUP

2. SOLUTION EFFECT GROUP

3. VITRIFICATIONAVARMING GROUP

Two different devices were used:

"PINK DEVICE" (manual)

"GREY DEVICE" (automatic)

(The devices have been described above as an embodiment of the invention)

RESULTS:

5

APPENDIX B

The "devices" mentioned with reference to this appendix are devices described above, as embodiments of the invention.

Example of an experiment on human ovarian slices Vitrified ovaries were donated in this experiment. The ovaries were warmed in the conventional warming procedure and histological evaluation was done by microscopy.

The ovaries were then placed in the device and processed according to the following protocol:

Ovarian Tissue Vitrification protocol

Media preparation

Vitrification solutions:

1. ES (equilibration solution): 16% ethylene glycol (EG)

16% dimethyl sulfoxide (DMSO)

in - HEPES-TALP medium + 3% BSA

2. VS (vitrification solution): 20% ethylene glycol (EG)

20% dimethyl sulfoxide (DMSO)

1M sucrose

in - HEPES-TALP medium + 3% BSA

Materials Equipment

HEPES-TALP (pre-warmed) (-20 ml) Filter

ES (10 ml) 10 ml syringe (4)

VS (15 ml) Pipette (10 ml)

LN (liquid nitrogen) Box for LN

Stripper + tips

Scalpel handle

Disposable sterile scalpel blades (size 20 or 21)

Petri dish (100x20 mm)

Scissors

Procedure

14. Add 5.5 ml HEPES-TALP into the device

15. Put a filter into the device (pay attention that no air bubble created)

16. Transfer one piece of the ovarian tissue into the filter

17. Inject ES (10 ml) from syringe A for 35 min (until the syringe with ES is empty).

18. Inject VS (10 ml) from syringe B for 15 min (until the syringe with VS is empty).

19. Evacuate the liquid from the chamber with syringe C 20. Inject 5 ml of VS into the filter above the ovarian tissue, using syringe D.

21. Evacuate the liquid from the chamber with syringe C.

22. Transfer the filter (containing the ovarian tissue) into LN.

Ovarian Tissue Warming protocol

Media preparation

Warming solutions:

WS - 1M sucrose in HEPES-TALP medium

Materials Equipment

LN (liquid nitrogen) Box for LN

Filter

WS 1M (pre-warmed) (40 ml) 10 ml syringe (3)

Pipette (10 ml)

Petri dish (100x20 mm)

Pipettor + tips

Procedure

5. Remove the filter from the LN and immediately put it into a Petri dish with 1M WS.

6. Wash the ovarian tissue with 1M WS using a syringe.

7. Transfer the filter into the device and immediately add 5.5 ml 1M WS into the filter above the ovarian tissue, using syringe C.

8. Inject 10 ml HEPES-TALP from syringe A for 10 min.

Results

No differences were seen in the histology sections between conventional vitrification and double vitrification using a device in accordance with the invention (in the second time).