Vitrification device and method for preparing sample

Abstract

To provide a novel vitrification device for vitrification of a biological sample which can substantially reduce a risk of sample damage and artificial error.SOLUTION: Provided is a vitrification device equipped with an input part. The input part comprises: a sample chamber which is formed in the input part, and is constituted so as to receive a sample; an input part waste reservoir which is formed in the input part; and a filtration grating which is arranged between the sample chamber and the input-part waste reservoir, and is constituted so as to allow fluid waste to flow from the sample chamber into the input-part waste reservoir via a channel formed of the filtration grating while retaining the sample in the sample chamber.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present disclosure relates generally to vitrification devices and methods for sample preparation.

[0002] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application is a continuation of U.S. Provisional Application No. 62 / 488,655, filed April 21, 2017. The benefit of and priority under 35 U.S.C. §119(e) of the present application is claimed, and is incorporated by reference in its entirety. No. 6,399,433, which is incorporated herein by reference in its entirety. [Background technology]

[0003] Human oocyte cryopreservation (oocyte cryopreservation) is a method used to preserve a woman's fertility. This is one of the techniques being developed. A woman's oocytes are extracted, frozen, and stored. Alternatively, the eggs are thawed, fertilized, and transferred to the uterus as embryos. may be frozen and stored, and later thawed and implanted into the woman's uterus.

[0004] Vitrification is a rapid freezing process in which biological specimens are frozen within seconds, for example using liquid nitrogen. Vitrification of oocytes or embryos preserves the fertility and viability of oocytes and embryos. The results are far superior to those obtained by slow freezing. As shown.

[0005] Biological specimens preserved by vitrification are typically small in size and non-vitrified. They are always delicate and susceptible to loss of activity and damage during human manipulation, such as during implantation by micropipette. Traditionally, samples are transferred to a vitrification tool where they are held and exposed to liquid nitrogen. Before being vitrified, they are pretreated and prepared for vitrification using various agents and solutions. Similarly, to thaw, frozen samples are treated with various warm agents or solutions. Each step of the procedure involves using a micropipette to transfer a sample, such as an oocyte or embryo, into a certain solution. This involves transferring the sample from one solution to another. Avoids the qualitative risks and various human errors associated with such operations It is not possible.

[0006] Thus, the risk of sample damage and human error is substantially reduced in this field. A new vitrification device for vitrification of biological specimens and related experiments A procedure is required. Summary of the Invention

[0007] One aspect of the present disclosure relates to a vitrification device. In some embodiments, the device an input having an input port; a sample chamber; and a waste container in fluid communication with the sample chamber. and, while holding a sample in the sample chamber, a fluid introduced from the input port is and a filter mechanism for selectively passing fluid from the sample chamber to the waste container. In some embodiments, the sample chamber, waste container, and filter mechanism are to draw fluid by capillary action from said filter mechanism through said filter mechanism to said waste container. It is composed.

[0008] The vitrification device further comprises at least one observation window, the observation window comprising: The sample in the sample chamber is configured to be viewable through the observation window.

[0009] In some embodiments, the vitrification device is pre-inserted to close the input port. Some embodiments further include a cap reversibly coupleable to the input portion. The cap is connected to the input portion when the cap is connected to the input portion. The input port is configured to seal the input port to prevent the flow of fluid within the chair. In some embodiments, the filter mechanism comprises a plurality of microfluidic channels to promote capillary action. A filter having a plurality of pores sized and configured to form a body channel. It is equipped with a

[0010] In some embodiments, the sample chamber comprises a material that is resistant to liquid nitrogen. In some embodiments, the sample chamber comprises a thermally conductive material. The sample chamber is made of acrylic material, polypropylene material, polycarbonate material, In some embodiments, the polyester-based material is a polyester-based material. In one embodiment, the sample chamber has walls having a thickness of 0.002 inches or less.

[0011] Another aspect of the present disclosure relates to a method for preparing a sample. In some embodiments, The method includes delivering a sample into a sample chamber adjacent a filter mechanism; A filter mechanism passes the sample through the sample chamber while retaining the sample within the sample chamber. The first fluid is then forced by a fluid force into a waste container. The step of processing the sample includes forcing the first fluid into the waste container. This initiates capillary action, which causes subsequent fluid to flow through the sample chamber into the waste container. You will be drawn into it.

[0012] In some embodiments, the method further comprises the step of: providing a filter mechanism in the sample chamber; While holding the sample, the sample is drawn by capillary action through the sample chamber and into the waste container. treating the sample with a second fluid by drawing the second fluid; In some embodiments, the method further comprises: and observing the sample in the sample chamber.

[0013] In some embodiments, the method further comprises attaching a cap to the sample chamber. In some embodiments, the fluid is introduced into the fluid passage through the input port. wherein the method further comprises vitrifying the sample in the sample chamber. In some embodiments, the sample is immersed in a liquid nitrogen bath by contacting the sample chamber with liquid nitrogen. This causes the material to be vitrified.

[0014] In some embodiments, the method comprises contacting the sample chamber with a warm solution. The method further comprises the step of thawing the sample. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 is a perspective view of a portion of a vitrification device according to some embodiments of the present disclosure.

[0016] [Figure 2A] FIG. 1 is a perspective view of a cap of a vitrification device according to some embodiments of the present disclosure.

[0017] [Figure 2B] 2B is a cross-sectional view of the cap of FIG. 2A according to some embodiments of the present disclosure.

[0018] [Figure 2C] FIG. 2B is a front view of the cap of FIG. 2A according to some embodiments of the present disclosure.

[0019] [Diagram 3] FIG. 1 is a perspective view of an input portion of a vitrification device covered by a cap according to some embodiments of the present disclosure.

[0020] [Figure 4A] FIG. 1 is a perspective view of a front portion of a vitrification device according to some embodiments of the present disclosure.

[0021] [Figure 4B] FIG. 2 is a top view of the front of a vitrification device according to some embodiments of the present disclosure.

[0022] [Figure 5A] 1A-1C are longitudinal cross-sectional views of a portion of a vitrification device according to some embodiments of the present disclosure, with a cap coupled to an input of the device.

[0023] [Figure 5B] 1A-1C are cross-sectional views of an input portion of a vitrification device according to some embodiments of the present disclosure.

[0024] [Figure 5C] FIG. 5C is a front view of the cap portion of FIG. 5B according to some embodiments of the present disclosure.

[0025] [Figure 6A] FIG. 1 is a perspective view of a portion of an input portion of a vitrification device according to some embodiments of the present disclosure.

[0026] [Figure 6B] FIG. 2 is a side view of a portion of a filtration grid according to some embodiments of the present disclosure.

[0027] [Figure 7] 7A-7C are partial views of various embodiments of an input portion of a vitrification device according to some embodiments of the present disclosure.

[0028] [Figure 8] 8A-8B are top views of various embodiments of an input portion of a vitrification device according to some embodiments of the present disclosure.

[0029] [Figure 9A] FIG. 2 is a top view of a portion of a vitrification device according to some embodiments of the present disclosure.

[0030] [Figure 9B] FIG. 9B is a cross-sectional view of the vitrification device taken along line AA of FIG. 9A.

[0031] [Figure 9C] FIG. 9C is an enlarged view of area D shown in FIG. 9B according to some embodiments of the present disclosure.

[0032] [Figure 10A] FIG. 1 is a perspective view of a portion of an input portion of a vitrification device according to some embodiments of the present disclosure.

[0033] [Figure 10B] FIG. 10B is a cross-sectional view of the vitrification device of FIG. 10A according to some embodiments of the present disclosure.

[0034] [Figure 10C] FIG. 10B is a front view of the vitrification device of FIG. 10A according to some embodiments of the present disclosure.

[0035] [Figure 11A] 1 illustrates an exemplary procedure for freezing an embryo sample using a vitrification device according to some embodiments of the present disclosure.

[0036] [Figure 11B] 1A-1D show an exemplary procedure for thawing an embryo sample using a vitrification device according to some embodiments of the present disclosure.

[0037] [Figure 12]12A-12B show an exemplary embodiment for stacking multiple units of a vitrification device according to the present disclosure.

[0038] [Figure 13A] FIG. 1 is a perspective view of a vitrification device according to some embodiments of the present disclosure.

[0039] [Figure 13B] FIG. 13B is a perspective view of an input portion of the vitrification device of FIG. 13A according to some embodiments of the present disclosure.

[0040] [Figure 14A] FIG. 13B is a top view of the vitrification device of FIG. 13A according to some embodiments of the present disclosure with a cap on the input.

[0041] [Figure 14B] FIG. 13B is a top view of the vitrification device of FIG. 13A with the cap removed according to some embodiments of the present disclosure.

[0042] [Figure 14C] FIG. 14C is a cross-sectional view taken along line AA in FIG. 14B.

[0043] [Figure 14D] FIG. 13B is a cross-sectional view of the vitrification device of FIG. 13A according to some embodiments of the present disclosure.

[0044] [Figure 15A] FIG. 13B is a partial cross-sectional view of the vitrification device of FIG. 13A according to some embodiments of the present disclosure.

[0045] [Figure 15B] FIG. 13B is a partial cross-sectional view of the vitrification device of FIG. 13A according to some embodiments of the present disclosure.

[0046] [Figure 16] FIG. 1 is a top perspective view of a vitrification device according to some embodiments of the present disclosure.

[0047] [Figure 17] 17 is a cross-sectional view of the vitrification device of FIG. 16 according to some embodiments of the present disclosure.

[0048] [Figure 18] FIG. 17 is a perspective view of an input portion of the vitrification device of FIG. 16 according to some embodiments of the present disclosure.

[0049] [Figure 19] 19 is a cross-sectional view of a portion of the input portion shown in FIG. 18 according to some embodiments of the present disclosure.

[0050] [Figure 20] 17 is a cross-sectional view of a portion of the vitrification device of FIG. 16 according to some embodiments of the present disclosure.

[0051] [Figure 21] 17 is a cross-sectional view of a portion of the vitrification device of FIG. 16 according to some embodiments of the present disclosure.

[0052] [Figure 22] 22A-22D are perspective views of alternative embodiments of an input portion of the vitrification device of FIG. 16 according to some embodiments of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] The present specification refers to, for example, a device for rapid freezing of samples using liquid nitrogen and In certain preferred embodiments, a method and apparatus are provided that utilizes forces such as those generated by capillary action. Low pressure is used to evaporate the device without damaging the sample(s) in the device. Fluid can be pushed or pulled through the chair.

[0054] Referring to FIG. 1, a perspective view of a vitrification device 101 according to one embodiment of the present disclosure is shown. The vitrification device 101 has an elongated shape and two ends. In the device 101, an input section is reversibly coupled to a removable cap 103. (not shown). At the other end, the device 101 comprises a handle 102. The cap 103 is coupled to the input portion and is rotatable about a longitudinal axis of the cap 103. The vitrification device 101 has a closed configuration and at least one open configuration. In some embodiments, switching between the closed and open configurations involves rotating the cap 103. In some embodiments, the coupling between the cap 103 and the input portion is It may be reversible, and the cap 103 may be removed from the input as needed by the user. This can be done.

[0055] 2A shows a perspective view of the cap 103. The cap 103 has an open end 103a and a closed end 103b. In some embodiments, the cap 103 has at least one An input port 104 and at least one observation window 105. In some embodiments In some embodiments, the cap 103 further comprises one or more openings (not shown). The closed end 103b has a grip for facilitating holding and rotating the cap 103. 106 is formed.

[0056] FIG. 2B shows a cap 103 according to one exemplary embodiment of the present disclosure. The cap 10 is generally in the shape of a hollow cylinder with a number of recesses and / or openings. 3 has an open end 103a and a closed end 103b. FIG. 2B shows the cross-sectional view taken from the closed end 103b. 2C is a front view of the cap 103; FIG. 2D is a cross-sectional view of the cap 103 taken along line AA in FIG. As shown in this figure, the cap 103 has an open end 103a, a closed end 103b and At least one connecting portion 103c is provided between the outside of the cap 103 and the cavity 103c. In this exemplary embodiment, the input ports 104 are A recess 104a in the wall of cap 103 and an input channel that forms an acute angle with the longitudinal axis of cap 103. The flow medium such as a solution delivered from the input port 104 is Directly into the sample area provided within cavity 103c. In some embodiments, The angle between the longitudinal axis of the cap 103 and the input channel 104b is about 20° to about 70°. In some embodiments, the angle is within the range of 20°, 30°, 40°, 50°, , 60° or 70°.

[0057] In some embodiments, the cap 103 also has at least one observation window 105 in a wall. In some embodiments, a user of the device may use a sump located in the cavity 103c. The observation window 105 is made of a transparent material so that the inside can be easily seen to check the condition of the filter. In various embodiments, the observation window 105 is integral with the rest of the cap 103. In some embodiments, the observation window 10 may be made of the same or different materials. The observation window 105 is formed on the wall so that the thickness of the area 5 is smaller than the average thickness of the wall. The recess may be formed by the above-mentioned method.

[0058] In some embodiments, the cap 103 further comprises one or more openings. In the exemplary embodiment shown in FIG. 1, the cap 103 has two ribs 103b connecting the outside and the cavity 103c. In some embodiments, the cap 103 is configured to receive liquid nitrogen. When the liquid nitrogen is in contact with the opening, it flows into the cavity 103c and is located there. In some embodiments, such a cap 103 may be placed in the vicinity of the sample. The number of openings is not limited to two. In various embodiments, the cap 103 may have one, two, or more openings. Or, three or more openings may be provided. The presence of multiple openings allows liquid nitrogen to enter through one opening while being closed in the cavity 103c. The trapped air can escape through the other opening, allowing liquid nitrogen to enter the sample area. This is believed to allow for rapid flow into the vicinity, thus allowing for rapid vitrification of the sample.

[0059] In the exemplary embodiment shown in FIG. 2B, the distance between the two arrows B is measured. The thickness of the grip 106 ranges from about 0.06 inches to about 0.12 inches. In one exemplary embodiment, the thickness of the grip 106 is 0.114 inches.

[0060] In some embodiments, the length of the cavity 103c measured by the distance between the two arrows D. The diameter ranges from about 0.06 inches to about 0.13 inches. In some embodiments, the cavity 103c has a diameter of 0.126 inches. The device is configured to accommodate a tapered input of a vitrification device. In an embodiment, the input portion has a draft angle of about 1°, and the cap 103 fits within the cavity 103c. When the cap 103 is coupled to the input, the taper This provides a wedging force that helps engage and / or seal the two parts. In one embodiment, the input portion and the cap 103 have matching draft angles within the range of 1° to 5°. It may have a configuration.

[0061] In some embodiments, the length of the cap 103 as measured by the distance E is about 0. The range is from 3 inches to about 0.7 inches. In one exemplary embodiment, the cap The length of the 103 is 0.64 inches.

[0062] FIG. 3 shows a perspective view of the front of the vitrification device 101, which is the input of the device. The figure shows, in particular, the connection of the part 110 with the cap 103. 101 is shown in a first open configuration. The input port 104 is in fluid communication with the sample chamber 111. The cap 103 is inserted through the input section 1 so that the observation window 105 is above the sample chamber 111. In some embodiments, the input 110 is configured to store waste materials. It further includes a waste container 112a and a waste channel 113 for disposal of the same.

[0063] With the first open configuration, the user can view the contents within the sample chamber 111 through the observation window 105. While monitoring objects and / or activities, a sample is introduced from the input port 104 into the sample chamber 111. It may also deliver a flow agent such as a solution for preparing or processing a sample. The waste is then transferred to a waste container 112a, which is in fluid communication with the sample chamber 111. a and disposed of through waste channel 113. The sample chamber 111 is configured to allow the sample to pass through it while allowing waste to pass through to a waste container 112. The device may include different mechanisms for storing the

[0064] 4A and 4B are perspective and top views of the vitrification device 101 in a second open configuration. In this configuration, one or more openings 107, 108 in the cap 103 are The cap 103 is positioned relative to the input 110 such that the input 110 is above the sample chamber 111. That is, with this configuration, a user can enter the device through at least one of the openings 107 and 108. The sample may be manipulated and / or obtained from the sample chamber 111 using the The dimensions of the openings 107, 108 in the cap 103 may be the same or different. Without being limited by theory, at least one aperture 107 may be provided through which a sample may be manipulated. It is believed that the size of the selection is large enough to make it easy to create and obtain. Generally, as long as the size of the opening(s) does not impede the flow of liquid nitrogen through the opening(s), The other openings 108 may be smaller.

[0065] FIG. 5A shows a cross-sectional view of the vitrification device 101 shown in the first open configuration of FIG. As shown in the cross-sectional view of FIG. 5A, the input portion 110 includes a waste container 112a / 112b and a fluid In some embodiments, the waste container 112b includes a sample chamber 111 that is in communication with the sample chamber 111. , which extends into the handle 102 of the device. As shown in FIG. 5A, the sample chamber 11 1 is provided at the input section 110, and the waste container 112a at the input section is fed through a waste channel 113. The waste container 112b is connected to the inside of the handle 102. The device then disposes of fluid waste into a waste container 112b in the handle 102. In some embodiments, the device is configured to collect waste from the waste container 112a / 112b into a sump. In some embodiments, the device further includes a mechanism, such as a check valve, to prevent the liquid from returning to the reservoir chamber 111. The device detects that trapped air is released as fluid enters the waste container 112b. The handheld device includes a filter or other mechanism that prevents liquid nitrogen from entering the waste container 112b. It is further provided in the module 102.

[0066] In some embodiments, the input 110 includes at least one In the exemplary embodiment shown in FIG. 5A, in particular, recess 1 14 is separated from the sample chamber 111 by a thin wall 150. In the embodiment, the cap 103 further comprises at least one opening 107, 108. In this configuration, when the cap is coupled to the input, liquid nitrogen flows through the opening into the recess. There is fluid communication between the openings 107, 108 and the recess 114 so that the fluid can enter the recess 114. In some embodiments, the device is configured to be immersed in liquid nitrogen in a closed configuration. That is, an opening on the cap allows liquid nitrogen to enter the recess 114 of the input portion 110. This allows the sample to flow into the cavity, thereby rapidly freezing the sample held therein. In some embodiments, the cap 10 can be placed in the vicinity of the sample chamber 111. 3 has at least two openings 107, 108, which allow liquid nitrogen to flow through one opening Liquid nitrogen is easier to remove because air that enters through the opening and is trapped in the cavity escapes through the other opening. It is simply allowed to flow into the recess 114 .

[0067] In some embodiments, the dimensions of the input portion 110 at different locations along its longitudinal axis are In particular, in some embodiments, the input 110 may be a tape In some embodiments, the interior space of the cap 103 is also shaped like the input portion 110. The taper shape is adapted to fit the shape of the input portion 1 when the cap 103 engages the input portion 1. In some embodiments, the input 110 and and cap 103 have matching draft angles within the range of 1° to 5°.

[0068] FIG. 5B is a cross-section of the input section 110 taken along line AA in FIG. 5C, which is a front view of the input section 110. As shown, in this exemplary embodiment, the dimensions of the input portion 110 are such that it is The input unit 110 is connected to the input terminal 102 at a position (the base of the input unit 110) from the other end of the input unit 110 (the input terminal 110) gradually becomes smaller toward the front end of the arrow D. The base dimensions of the suction cups range from about 0.1 inches to about 0.2 inches. In one embodiment, the input portion 110 measures 0.127 inches at its base. The dimension of the front end of the input section, measured as the distance between arrows B, is approximately 0.1 inches to approximately 0.2 inches. In one exemplary embodiment, the dimension at the front end is in the range of about 0.11 It is 6 inches.

[0069] In some embodiments, the length of the input portion 110, measured as the distance between the two arrows E, is , in the range of about 0.1 inches to about 0.4 inches. In one exemplary embodiment, , the length of the input portion 110 is about 0.351 inches. The length of the device including the handle 102 and the input portion 110, measured by the distance F at It ranges from about 0.5 inches to about 3 inches.

[0070] 6A and 6B show an exemplary embodiment of an input portion of a vitrification device. 6A shows a perspective view of a partial structure of the input unit 110. The waste container 112a and the opening of the waste channel 113 are shown, which are connected to the input 110. A block separating the sample chamber 111 and the waste container 112a is provided on the sample chamber 111. 115 is also provided on the input portion 110. In some embodiments, a cap (not shown) may be provided. When the input portion 110 is connected to the input portion 110, the upper end of the block 115 engages with the inner wall of the cap. In some embodiments, as shown in FIG. 6A, a cap (not shown) When the input 110 is connected to the block 115, the upper end of the block 115 is connected to the sample chamber 111 and the waste A filter mechanism, such as a filter grid 151 , is provided that maintains fluid communication with the waste container 112 .

[0071] FIG. 6B is an enlarged view showing a partial structure of the filter grid 151 shown in FIG. Also shown is a portion of the handle 102 and input portion 110 of the vice. The filter grid 151 has a plurality of alternating low portions 151a and high portions 151b. That is, when the input portion 110 is engaged with a cap (not shown), the upper end of the high portion 151b to engage the inner wall of the cap to provide fluid communication between the waste container 112a and the sample chamber 111. A channel is formed in the lower portion 151a that connects to the

[0072] In some embodiments, when the cap is attached, a fluid delivery device such as a micropipette may be used. The device is used to push waste fluid through the channel into a waste container, while the In the sample area, samples such as oocytes or embryos can be held. In this embodiment, the dimensions of the filtering grid 151 are determined based on the sample size to be used with the vitrification device. In some embodiments, the dimensions depend on the type of channel through which the waste material passes. is selected to hold the sample. For example, the sample may be a human oocyte or embryo. In an embodiment, the dimensions of each channel (such as diameter, width or length depending on the shape of the channel) are: It is in the range of 0.0005 to 0.0015 inches.

[0073] Furthermore, without intending to be bound by theory, the dimensions of the channel are The number of channels is important because it also affects the liquid pressure on the sample. Instead, the device operates on one or more channels. The multi-channel design helps reduce back pressure, which reduces the amount of sample in the sample area. This is believed to allow for less stress on the pull and quicker fluid filling.

[0074] In some embodiments, the filter grid 151 is a single filter that connects the sample chamber and the waste container. and the flow area (defined as the cross-sectional area perpendicular to the longitudinal axis of the channel) is Approximately 7×10 -7 Square inch to 1.5 x 10 -6 In other words, the area is in the square inch range. In an embodiment, the filtration grid 151 forms a plurality of channels connecting the sample chamber and the waste container. The total flow area of ​​all the channels combined is approximately 7 × 10 -7 1.5 to square inch ×10 -6 It's in the square inch range.

[0075] As will be appreciated, in some embodiments, the filtration grid 151 is disposed in the sample chamber 1 11. This facilitates the separation of the fluid waste from the specific sample held in the In this embodiment, the filter grid 151 facilitates the removal of waste from the sample chamber. In particular, in some embodiments, the fluid waste is filtered through the filter grid 151. The particles flow through the channel into the waste container 112a while the particles with a size larger than the diameter of the channel flow into the waste container 112b. A sample having a diameter of 10 mm is held in the sample chamber 111. The actuator may be actuated by a variety of mechanisms. For example, in some embodiments, the cap may be When engaged with the input port, a fluid delivery device, such as a micropipette, is The liquid pressure can be transmitted to the sample chamber to push the fluid towards the waste container. In an alternative embodiment, a suction mechanism may draw the fluid flow toward a waste container. Furthermore, in some embodiments, the microflow formed by the filter grid 151 The body channel has capillary wicking that facilitates drawing liquid waste towards a waste container. The first solution is then subjected to a fluid force by using a pipette or the like. When the fluid is introduced through the input port 104, capillary action is initiated. The fluid enters the sample chamber. 111 through the filter grid 151 and into the waste container 112. In combination with a microfluidic channel of a particular size, the presence of fluid in the waste container 112 Capillary action is used to generate a force strong enough to draw subsequent fluid through the device 101. Complete.

[0076] 7A-7C show additional exemplary embodiments of a vitrification device. In particular, FIG. 7 is a perspective view of a partial structure of the input section 710. In this embodiment, the island section 719 is a sample chamber. 711 and waste container 712. Bypass channels on either side of island 719 718 fluidly connects the sample chamber 711 to a waste container. In some embodiments, the islands 719 are configured to capture and hold a sample therein. The capture pocket 716 is formed in the sample chamber 711. and a closed end towards a waste container 712. As shown, In some embodiments, the capture pocket 716 is "U" shaped.

[0077] In some embodiments, the top end of the island 719 may include a filter such as a plurality of filtration channels 717. The trap mechanism further comprises a trap mechanism that is connected at one end to the capture pocket 716 and at the other end to a waste receptacle. When a cap (not shown) is coupled to the input portion 710, the island The upper surface of 719 engages the inner wall of the cap, and the filtration channel 717 is in contact with the capture pocket 716. A waste container 712 is connected in fluid communication.

[0078] According to the present disclosure, the dimensions of the filtration and bypass channels may be varied. B shows that the bypass channel 718 is partially filled along the bottom, thereby 7 shows an exemplary embodiment in which the loop 718 is constrained to a smaller dimension. Without being intended to be construed as limiting the scope of the invention, the dimensions of the bypass channel 718 and the filtration channel 717 may be varied to suit the needs of the individual The flow rate of fluid over the sample located in the capture pocket 716 is affected by the liquid pressure on the sample. It is believed that the number of bypass channels 718 or filtration channels 717 is defined. It is not important that the device of the present application has at least one bypass channel 718 and and at least one filtering channel 717. where the flow area of ​​a channel is defined as the cross-sectional area perpendicular to the longitudinal axis of the channel. In some embodiments, the total flow area of ​​the bypass channel(s) is about 5×10 - 6 Square inch to about 2×10 -5 In some embodiments, the range is 100 mm. The total flow area of ​​the filtration channels is approximately 7 × 10 -7 About 1.5 to a square inch ×10 -6 It's in the square inch range.

[0079] In some embodiments, the total flow area of ​​the bypass channel and the total flow area of ​​the filtration channel The ratio of the area to the area is adjusted to achieve optimal flow and liquid pressure to the sample in the capture pocket. A high bypass to filtration ratio allows more fluid to pass through the bypass channels. (i.e., bypassing the trapping pocket) so that The sample in the sample is expected to undergo less fluid manipulation. A quantity of fluid is driven to flow through the capture pocket and filtration channel, thereby Thus, in some embodiments, the bypass The filtration ratio is in the range of about 6:1 to about 4:1. The bypass to filtration ratio is approximately 5:1.

[0080] In some embodiments, the dimensions of the filtration channel are determined by the size of the filtration channel to be used with the vitrification device. The selection is also based on the type of sample. In particular, in some embodiments, the filtration channel The dimensions of the filter are chosen so that it allows waste to pass but retains the sample within the capture pocket. For example, in an embodiment in which the sample is a human oocyte or embryo, each filtration channel The diameters range from 0.0005 to 0.0015 inches.

[0081] According to the present disclosure, the size of the capture pocket may be varied. An exemplary embodiment in which the length of the capture pocket 916 is increased compared to the embodiment shown in A. In other embodiments, the thickness and / or width of capture pocket 716 is varied. In some embodiments, the volume of the capture pocket may be large enough to accommodate several human or animal oocytes. In some embodiments, the input 910 is sufficient to hold a cell or embryo. The size of the sample chamber 711 in the sample chamber may be varied. For example, FIGS. 8A and 8B show two examples. 8A shows an exemplary embodiment, in which the sample chamber 811 is accommodated in FIG. 8A in comparison with FIG. 8B. The length of the input section 810 is increased.

[0082] 9A-9C are schematic diagrams of one embodiment of a vitrification device according to the present disclosure. The handle 902, the input section 910, and various structures provided on the input section 910 are , including a sample chamber 911, a waste container 912, an opening of a waste channel 913, and an island 919. In this embodiment, the island 919 includes a U-shaped capture pocket 916 and A filter mechanism, such as a number of filtration channels 917, connecting the U-shaped pocket with a waste container 912 FIG. 9B shows a cross-sectional view taken along line AA in FIG. The handle 902, the input unit 910, and various structures provided on the input unit 910 are as follows: Shown is a recess 914, a capture pocket 916, and a number of filtration channels 917. FIG. 9C is an enlarged view of area D shown in FIG. 9B. further includes a bypass channel 918.

[0083] In some embodiments, the U-shape measured by the distance between the two arrows D in FIG. The length of the capture pocket 916 ranges from about 0.02 inches to about 0.04 inches. In one exemplary embodiment, the length of the capture pocket 916 is 0.038 inches. .

[0084] In some embodiments, the capture point is measured by the distance between the two arrows B in FIG. The width of the socket 916 ranges from about 0.005 inches to about 0.02 inches. In the exemplary embodiment, the width of the capture pocket 916 is 0.015 inches.

[0085] In some embodiments, the island 9 is measured by the distance between the two arrows C in FIG. 9A. The length of 19 ranges from about 0.03 inches to about 0.07 inches. In one embodiment, the length of the island 919 is 0.68 inches.

[0086] In some embodiments, the capture point is measured by the distance between the two arrows E in FIG. The depth of the socket 916 ranges from about 0.01 inches to about 0.03 inches. In the exemplary embodiment, the capture pocket 916 is 0.021 inches deep.

[0087] In some embodiments, the bypass is measured by the distance between the two arrows F in FIG. The depth of the channel 918 ranges from about 0.001 inches to about 0.005 inches. In one exemplary embodiment, the bypass channel 918 has a depth of 0.005 inches. It is.

[0088] In some embodiments, when the vitrification device is in the first open configuration, the cap The input port is provided with an input section such that at least one end of the input port is in close proximity to the capture pocket. That is, the user can use, for example, a micropipette to Samples can be easily delivered into the capture pocket via the port.

[0089] In some embodiments, the filtration grid 717, 917 is located in the capture pocket 716, 916. Facilitates the separation of fluid waste from the particular sample retained. In this embodiment, the fluid waste is passed through a filter grid 717, 917 to a waste container 712, 912. The sample with a particle size larger than the diameter of the channel flows into the trap pocket. The fluid waste stream is driven by a variety of mechanisms. For example, in some embodiments, when the cap is engaged with the input, the microphone A fluid delivery device, such as a micropipette, transmits liquid pressure through an input port to the sample chamber. , which can push the fluid flow towards a waste container. A mechanism may direct the flow of fluid toward a waste container. In an embodiment, the microfluidic channel draws liquid waste towards a waste container. The device is configured to provide a capillary wicking force that facilitates the use of a pipette or the like. When the first solution is introduced into the sample chamber 711, 911 by fluid force, capillary action occurs. The fluid is discharged from the sample chamber 711, 911 through the filter grid 717, 917. The entire device is then pushed into the waste container 712, 912 and waste channel 713, 913. In combination with a microfluidic channel of a specific size through which The presence of the fluid draws subsequent fluid through the device 701, 901 using capillary action. This generates a force strong enough to penetrate the

[0090] 10A-10C show further embodiments of a vitrification device according to the present disclosure. FIG. 10A is a perspective view of a portion of the device, including a handle 1002 and an input section. The input section 1010 includes a sample chamber 1011 and a filter 1021. A filter mechanism including a sample chamber 1011 and a filter 1021, and a lamp disposed between the sample chamber 1011 and the filter 1021. FIG. 10B is a schematic diagram of the input portion 1010 and the handle 1002 of FIG. FIG 10B is a cross-sectional view of the embodiment shown in FIG 10A taken along line AA. The handle 1002 is provided with a waste channel 1013 connected to a waste container 1012. A filter 1021 is shown.

[0091] In some embodiments, the filter 1 is measured by the distance between the two arrows B in FIG. The thickness of 021 ranges from about 0.01 inches to about 0.03 inches. In the illustrated embodiment, the thickness of filter 1021 is 0.22 inches.

[0092] A lamp 1020 connects the filter 1021 and the sample chamber 1011. FIG. The vitrification device (without cap) is placed horizontally with the sample chamber 1021 facing upwards. As shown, the lamp 1020 is inclined relative to the surface of the sample chamber 1011. It has a corner and extends upward from one end of the sample chamber 1011. In some embodiments Use a micropipette to filter through the lamp 1020 to collect the waste. Push the fluid in the sample chamber 1011 so that it flows toward the filter 1021. While objects flow through the filter 1021 into the waste container 1012, larger sized objects It prevents the sample from flowing in, i.e. the sample is kept in the sample chamber. Theory Gravity acts on the samples, pulling them up and down without being constrained by the 20 and pull it away from the filter 1021, so that the lamp 1020 It is believed that this helps prevent the attachment of samples such as mother cells or embryos to the filter 1021. can be.

[0093] Alternatively or additionally, in some embodiments, the lamp 1020 provides at least A single microfluidic channel is formed, which connects the sample chamber 1011 to the filter A capillary suction force is provided to move the liquid waste towards 1021. When the first solution is introduced into the sample chamber 1011 by a fluid force using a nozzle or the like, The tubulation is initiated. Fluid flows from the sample chamber 1011 along the ramp to the waste container 101. 2. Combined with microfluidic channels of specific sizes running throughout the device, Thus, the presence of fluid in the waste container 1012 causes the fluid to be drawn through the device 1001 using capillary action. Alternatively or additionally, some In this embodiment, the filter 1021 is made from an absorbent material such as a sponge or filter paper. It also provides a wicking force to facilitate removal of liquid waste from the sample chamber 1011.

[0094] In some embodiments, the filter 1021 prevents the sample from passing through. , selectively passing waste to waste channel 1013. For example, in some embodiments In the filter 1021, the waste and sample are separated based on their respective sizes. In some embodiments, the mechanism allows the liquid components to pass through but not the mixture. In some embodiments, the filter 1021 retains the individual components of the oocyte or embryo. In various embodiments, the pores are small enough to prevent the passage of solid samples such as In some embodiments, the size of the pores may vary depending on the type of sample. In particular, the filter 1021 is made from a filtering material. Filter materials include, but are not limited to, sintered polyethylene beads, polymer mesh, and fiber paper. In some embodiments, the filtration material is a material that prevents adhesion of the sample to the filter 1021. Prevents wearing.

[0095] In some embodiments, the vitrification device further comprises a closing configuration (not shown). In the closed configuration, the cap is configured such that the opening on the cap is not in fluid communication with the sample chamber and the sample The cap is positioned relative to the input such that the chamber is sealed by the walls of the cap. In the closed configuration, one or more recesses or openings on the cap are in close proximity to the sample chamber. In some embodiments, in the closed configuration, one or more openings in the cap is in fluid communication with the recess on the input.

[0096] The vitrification device in a closed configuration does not directly expose the contents of the sample chamber to fluids. In some embodiments, the vitrification device may be submerged in a fluid without the need for a sample. For vitrification of the sample held in the chamber, at least the input section and the cap section are filled with liquid nitrogen. In some embodiments, the device is configured to be held in The sample may be further configured to be immersed in a warm medium to thaw the sample. In this embodiment, liquid nitrogen or a hot medium can easily access the sample through an opening on the cap or the like. However, there is no direct contact with the sample when the device is in the closed configuration. In some embodiments, at least the input portion and the cap portion of the device are liquid nitrogen resistant. In some embodiments, the rapid vitrification and thawing of samples is achieved. To achieve this, the material has thermal conductivity. Exemplary materials include, but are not limited to, acrylic-based materials, polypropylene-based materials, polycarbonate-based materials, and the like. These include carbonate-based materials, and copolyester-based materials.

[0097] In some embodiments, the switching between the open and closed configurations is performed by a carrier along its longitudinal axis. For example, about 10°, 20°, 30°, 40°, 50°, 60°, 70°, 80° , 90°, 100°, 110°, 120°, 130°, 140°, 150°, 160°, In particular, the exemplary embodiment shown in FIG. Referring again to the embodiment, the input port 104 and the openings 107, 108 are formed by the cap 1. 03. In this embodiment, the switch between the first open configuration and the closed configuration The change may be accomplished by rotating the cap 103 approximately 90 degrees along its longitudinal axis. The switching between the first and second open configurations is performed by rotating the actuator about 180° along its longitudinal axis. In some embodiments, the cap 103 may be rotated. 3 further includes a grip 106 to facilitate holding and rotating the cap.

[0098] 11A and 11B are schematic diagrams showing a method for freezing and thawing embryos using the vitrification device of the present application. As shown in FIG. 11A, the device The cap is initially positioned in a first open configuration. Culture medium enters the sample through an input port on the cap. The embryo sample is then delivered to the sample chamber through the input port. The appropriate equilibration solution is added and the embryos are allowed to soak in the equilibration solution for a predetermined period of time before waste is collected. Vitrification solution is then added through the input port and the embryos are transferred to the waste The waste is incubated in vitrification solution for a period of time before being collected. The cap is then closed. The input of the device is rotated into a chain configuration and rapidly inserted into the vitrification chamber for embryo vitrification. It is placed in a liquid nitrogen tank.

[0099] As shown in FIG. 11B, to thaw, the device frozen in the closed configuration can be quickly The cap is then quickly placed in a 37°C water bath and incubated for a specified period of time. The tube is rotated to a first open configuration. 37°C thawing solution is introduced through the input port. The embryos are then warmed for a period of time by applying a sample to the sample chamber. Solution is added and the embryos are allowed to equilibrate with the dilution solution for a period of time before waste is collected. Room temperature washing solution is then added to the sample chamber and the embryos are washed and waste collected. The incubation is allowed to equilibrate for a period of time before the sample is added to the sample chamber. The washing step may be repeated as necessary before the cap is placed in a second open configuration. The cap is rotated so that the embryos are retrieved through an opening in the cap.

[0100] The procedure for freezing and / or thawing samples using the device is The number of micropipette steps can be reduced by at least four times without compromising sample quality. In addition, a built-in waste collection mechanism is believed to be involved in the removal of carryover waste from each wetting step. The amount of solution dispensed is reduced, improving consistency from user to user. , while reducing the risk of damage associated with human handling of fragile samples. Additionally, it improves sample quality consistency by reducing human error associated with sample preparation. This includes improving sexuality.

[0101] The above freezing and thawing procedures are merely exemplary and are not intended to describe the use of the present vitrification device. The present disclosure is not intended to be limited to the specific examples given. For example, Samples that may be used are not limited to embryo samples, but may also include, for example, oocytes, somatic cells, and other suitable samples. It also includes the appropriate sample type. Additionally, the chemicals used to process the sample. The qualities are not limited to those mentioned above, the device is versatile and can be used with different gas sensors available on the market. It is believed that the device may be used with a freezing and thawing kit. Regardless of the principles of the present disclosure, those skilled in the art will be able to use the present invention to process and store samples. Further modifications and variations are possible without departing from the scope of the present invention. Positive and modified embodiments are within the scope of the present application.

[0102] 12A and 12B show that multiple units of a vitrification device according to the present disclosure, e.g. 1 illustrates exemplary embodiments that can be stacked on top of each other for convenient storage or transportation. FIG. 12A shows that each unit of the vitrification device 1201 is connected to a plug 12 at one end. 02 at the other end. The plug 1202 of one unit engages with the receptacle 1203 of the other unit, thereby The units can be stacked on top of each other. 12 shows another exemplary embodiment in which the unit has magnets 1202, 1203 at both ends. The magnets 1202, 1203 engage with each other, allowing multiple units to be stacked together. Overlap.

[0103] FIG. 13A illustrates an alternative embodiment of a vitrification device according to the present disclosure. As shown, The vitrification device 1301 includes an elongated portion having a handle portion 1302 and an input portion 1310. The input portion 1310 may be reversibly coupled to the cap 1303. In some embodiments, the input portion 1310 is cylindrical and is a cylindrical cavity within the cap 1303. In some embodiments, the input 1310 may be engaged with a proximal end of the handle 1302. and a smaller dimension at the distal end of the handle 1302. In some embodiments, the input portion 1310 has a draft of about 1° to about 5°. In some embodiments, the cavity in the cap 1303 also tapers in a conformal manner. The input portion 1310 and the cap 1303 are tapered with a draft angle that is The tapered shape of the insert creates a wedging force that helps engage and / or seal the two pieces together. In some embodiments, the input portion 1310 is configured such that the cap 1303 is The cap may further include a cap stopper 1323 to prevent further movement toward the This prevents the cap 1303 from covering the observation window 1305 or the recess 1314. In some embodiments, the cap 1303 is configured to prevent the cap 1303 from being pulled out. It further includes a grip 1306 for ease of rolling.

[0104] 13B is a perspective view of an input portion 1310 of a vitrification device. In the embodiment, the input 1310 is connected to a waste container (not shown) contained within the handle 1302. The input port 1304 is connected to the outside and the sample chamber (not shown). The input section further includes an observation window 1305, and connects at least one of the sample chambers. A portion may be visible from the outside through an observation window 1305. In some embodiments, the input The portion 1310 further comprises one or more recesses 1314 in close proximity to the sample chamber. In some embodiments, when in contact with liquid nitrogen, the recess 1314 allows Liquid nitrogen is in close proximity to the sample chamber to ensure good vitrification of samples held in the In some embodiments, the input unit 1310 is a The micropipette further includes a recess 1315 adjacent to the opening. This allows sample delivery tools such as a sample delivery device to more easily access the opening of the input port 1304. The recess 1315 is vacant when the cap 1303 is pressed onto the input portion 1310. It also prevents air from entering the sample chamber and causing bubbles.

[0105] 14A-14D show the handle 1302, input portion 1310 and cap 1303. 14A shows a vitrification device 1301. As shown in the top view of FIG. The portion of the input portion 1310 distal to the barrel portion 1302 is inserted into the cap 1303. In some embodiments, the cap stopper 1323 prevents the cap 1303 from reaching the observation window 130. In particular, in some embodiments, the cap stopper 1 303 is provided distal to the handle 1302 than the observation window 1305. In some embodiments, the cap may include a cap 1303 for ease of holding and handling. FIG. 14B is a top view of the vitrification device 1301. In this view, the cap 1303 has been removed from the input portion 1310, Port 1304 is exposed.

[0106] FIG. 14C shows a cross section taken along line AA in FIG. 14B. As shown in the figure, the input section 1 310 includes a sample chamber 1311 that is connected to the outside via an input port 1304. The sample chamber 1311 has at least one surface 1321 on which a sample can be placed. The activity and / or contents within the sample chamber 1311 can be viewed from the outside. The observation window 1305 is placed over the sample chamber 1311 as shown in FIG. In this exemplary embodiment, the sample chamber 1311 and the recess The location 1314 may be, for example, a sample in the sample chamber 1311 and liquid nitrogen in the recess 1314. In some embodiments, the inputs are separated by a thin wall that allows for fast heat transfer between them. The portion 1310 further includes a recess 1315 adjacent the opening of the input port 1304 .

[0107] The sample chamber 1311 is connected to a waste container 1312 that extends into the handle 1302 of the device. A filter mechanism 1320 is in fluid communication with the sample chamber 1311 and the waste container 1312. In some embodiments, the filter mechanism 1320 is disposed between the sample The sample is retained within the chamber 1311 while waste is selectively passed to a waste container 1312. In some embodiments, the filter mechanism 1320 separates the waste and sample from each other. In some embodiments, the filter mechanism 1320 separates the liquid based on its size. In some embodiments, the filter allows bodily components to pass through but retains solid components of the mixture. The mechanism 1320 is small enough to prevent the passage of an individual sample, such as an oocyte or embryo. In various embodiments, the size of the pores varies depending on the type of sample. You may do so.

[0108] In some embodiments, the filter mechanism 1320 is a mesh. In this state, holes through the mesh allow the wicking force to draw in fluid by capillary action. In some embodiments, the holes are small enough to allow for passage of the mesh during use. Fluid pressure is first provided to the sample chamber 1311 to push the liquid in so that the waste volume The presence of fluid in the reservoir 1312 drives subsequent fluid flow through the device 1301 using capillary action. It also creates a force strong enough to draw in the water, which then flows through the mesh and wets the holes. The liquid is arranged to pass the liquid and / or further liquid through the mesh without the need for fluid pressure. According to the present disclosure, suitable materials for the filter mechanism 1320 include Materials include, but are not limited to, sintered polyethylene beads, polymer mesh, and fiber paper. In some embodiments, the material has a smooth surface to prevent sample sticking or adhesion. ingredient.

[0109] FIG. 14D shows a cross-sectional view of the entire device. The cap 1303 is connected to the input section 131. In some embodiments, the cap 10 has an internal cavity 1324 for receiving the cap 10. When cap 1303 is coupled to input 1310, input port 1304 is sealed. When 303 is pressed onto input 1310, an air pocket forms in recess 1315. That is, when the cap 1303 is coupled to the input portion 1310, the recess 1315 is vacant. This prevents air from being forced into the input port 1304 and causing air bubbles in the sample chamber 1311. In some embodiments, the input portion 1310 is configured to be inserted into the input port 1310 when the cap 1303 is blocking the observation window 1305. In some embodiments, the device further includes a cap stopper 1323 to prevent The chair further includes a mechanism to prevent waste from returning from the waste container 1312 to the sample chamber 1311. In some embodiments, the device is configured to collect waste in the waste container 1312. A filter 1322 or the like is provided to allow air to escape and prevent liquid nitrogen from entering the waste container 1312. In some embodiments, the filter 1322 is configured to filter liquid waste from the waste stream. It also prevents leakage from the container 1312. In some embodiments, the filter 1322 is Pores small enough to prevent liquid from passing through, but large enough to allow air to pass through In some embodiments, the filter 1322 has a pore size of about 20 microns. In some embodiments, the filter 1322 is made of PTFE, polyethylene, It is made from polyethylene or polypropylene.

[0110] 15A and 15B show perspective views of an alternative embodiment of a vitrification device 1301. A cross section of the device along its longitudinal axis is also shown. In particular, FIG. 15A shows the handle 130. 2 (partially shown) and an input portion 1310 (partially shown) of the vitrification device 130 The partial structure of the input port 1310 is shown. The input port 1310 includes a sample chamber 1311. The port 1304 connects the sample chamber to the outside. In this exemplary embodiment, a waste container 1312 is provided that extends from the A filter mechanism 1320 is disposed between the sample chamber 1311 and the waste container 1312. A user views the activity and / or contents within the sample chamber 1311 through an observation window 1305. An observation window 1305 is placed above the sample chamber 1311 so that the input section 13 The sample chamber 1311 further comprises at least two recesses 1314 adjacent to the sample chamber 1311 . Upon contact with liquid nitrogen, the recess 1314 allows the liquid nitrogen to flow through the glass to improve the vitrification effect. A cap (not shown) is attached to the input 1311. 310, the input port 1304 is sealed. The portion 1310 further includes a recess 1315 near the opening of the input port 1304. 315 allows sample delivery tools such as micropipettes to be easily inserted into the input port 13 The recess 1315 allows the cap to access the opening of input portion 1310. It also prevents air from entering the sample chamber 1311 and forming bubbles when the sample is pressed into the sample chamber 1311 .

[0111] As shown in FIG. 15A, the sample chamber 1311 is a small space in which a sample can be placed. The sample receiving surface 1321 has at least one surface 1321. The sample receiving surface 1321 receives the sample on the surface 132. 1 may be at an angle to the input port 1304 to facilitate delivery to In some embodiments, a gap between the input port 1304 and the sample receiving surface 1321 may be provided. The angle is in the range of about 20° to about 70°. The dimensions of the sample receiving surface 1321 and the input port 1304 are not critical, so long as they can be FIG 15B shows an alternative embodiment in which the area of ​​the sample receiving surface 1321 is reduced. In this exemplary embodiment, the input port 1304 has two segments. The first segment adjacent to the opening of the input port 1304 is adjacent to the sample chamber. In another embodiment, the input port has a larger diameter than the second segment. 1304 has a large diameter at the opening and a smaller diameter toward the sample chamber 1311. The recess may be tapered.

[0112] 16-21 show alternative embodiments of a vitrification device according to the present disclosure. 1 is a perspective view of a vitrification device 1601. The vitrification device 1601 has two ends. At one end, the device 1601 includes a removable cap. An input (not shown) is provided that can be reversibly coupled to the cap 1603. In this embodiment, the input portion 1610 is cylindrical and is connected to a cylindrical cavity in the cap 1603. At the second end, the device has a handle portion 1602 and a cap 163. 0. The device 1601 also includes at least one observation window 1605. Glass The device may be configured in a closed configuration (shown in FIG. 16) and an open configuration (shown in FIGS. 18-19). Switching to the closed and open configurations involves the input 1610 and the cap In the illustrated embodiment, the cap 1603 is The cap 1603 has a plurality of caps 1604 that form grips for easy holding and handling. Has a protuberance.

[0113] FIG. 17 illustrates a vitrified disk in a closed configuration with a cap 1603 coupled to an input 1610. In this configuration, the input port 1604 and the sample The chamber 1611 is sealed by the walls of the cap 1603. Vitrification in a Closed Configuration The device 1601 can be configured to directly expose the contents of the sample chamber 1611 to a fluid as described above. The device may be submerged in the fluid without any need for a

[0114] When in the open configuration, the input port 1604 allows the sample to be delivered to the sample chamber 1611. The input port 1604 is configured to receive a sample chamber 1611 and a flow agent. The sample chamber is in fluid communication with a waste container via a filter mechanism including a filter 1621. The observation window 1605 is in fluid communication with the sample chamber 1612. The opening 1607 is provided in the handle portion 160 diametrically opposite the observation window 1605. It is formed into 2.

[0115] FIG. 18 shows a part of the handle 1602 and the input part 1610 of the vitrification device 1601. The vitrification device 1601 is opened so that the input port 1604 is exposed. The input port 1604 is shown in a 32-bit configuration (with cap 1603 removed). A trough-like recess 1604a in the wall of the input section 1610 and an input leading to the sample chamber 1611 The recess 1604a allows a sample such as a micropipette to be inserted into the recess 1604a. This allows a delivery tool to more easily access the opening of input channel 1604b. The flow agent delivered by the input channel 1604a is introduced into the sample chamber via the input channel 1604b. Flow directly to the sample at 1611.

[0116] The observation window 1605 allows a user of the device to view the condition of a sample located in the sample chamber 1611. In some embodiments, the observation window 1605 allows the operator to observe and inspect the state of the device. The outer wall of the sample chamber 1611 may be formed by an opening in the wall so that the outer wall is exposed. In other embodiments, a user of the device may determine the state of a sample located in the sample chamber 1611. The observation window 1605 is made of a transparent material so that one can easily see inside to inspect the state of the device. In various embodiments, the observation window 1605 is made to be transparent to the remainder of the device 1601. The sample chamber 1611 may be made of the same or different material as the sample in the chamber. In some embodiments, the sensor is surrounded by a transparent material to allow the sensor to be viewed. The walls of the sample chamber 1611 are less than 0.005 inches thick, and preferably less than 0.002 inches thick. less than 1 mm thick, thereby allowing the sample to thaw at a desired rate.

[0117] FIG. 19 is a cross-sectional view of a portion of the handle 1602 and the input portion 1610 shown in FIG. In this view, aperture 1607 is shown diametrically opposite observation window 1605. 1607 exposes the space surrounding the sample chamber 1611. The input 1610 is filled with liquid nitrogen. When the sample is submerged in water, for example, liquid nitrogen can flow through opening 1607 into sample chamber 1611. In some embodiments, the number of such openings is one. In various embodiments, the observation window 1605 also functions as a similar opening. In some embodiments, having at least two openings means that liquid nitrogen is introduced through one opening. Air that enters through the mouth and is trapped in the space surrounding the sample chamber 1611 exits through the other opening. Since the liquid nitrogen can escape, it flows quickly to the vicinity of the sample area, i.e. It is believed to allow rapid vitrification of the samples.

[0118] A user may monitor the contents and / or activity within the sample chamber 1611 through an observation window 1605. While observing the sample, a sample or a sample is introduced into the sample chamber 1611 from the input port 1604. A flow agent, such as a solution for preparation or processing, may be delivered. The waste is then delivered to a waste container. In various embodiments, the device 1601 includes a waste container 1612. A filter machine to store samples while allowing waste to pass through to reach 612. For example, in this embodiment, the filter 1621 is connected to the waste container 1612. A sample chamber 1611 is separated from the

[0119] A detail of the filter 1621 is shown in perspective cross-sectional view in Figure 20. Some embodiments In the example, the filter 1621 prevents the sample from passing through while directing waste to a waste container 1622. 612. For example, in some embodiments, the filter 162 1 includes a filtering mechanism that separates waste and sample based on their respective sizes. In some embodiments, the filtration mechanism allows liquid components to pass but retains solid components of the mixture. That is, the sample is held in the sample chamber 1611. In some embodiments, The filter 1621 is sufficiently porous to prevent the passage of individual samples such as oocytes or embryos. In various embodiments, the size of the pores depends on the type of sample. In some embodiments, the filter 1621 may have multiple pores, e.g. , having a laser drilled hole provided in the injection molded component; The number and / or arrangement are selected to promote capillary action (described below). In some embodiments, the filter 1621 components have pores with a center-to-center spacing of 0.003. In another embodiment, the pores are spaced apart by about 0.002 inches in diameter in a 7x9 array. The filter 1621 may have different numbers of pores in an array, different diameters of pores, and / or differences between the pores. The interval may be any interval.

[0120] In other embodiments, the filter 1621 is made from a filtering material. Suitable filter media that may be used include, but are not limited to, sintered polyethylene beads, polymer meshes, etc. In some embodiments, the filtration material includes a filter 1621. This prevents the sample from sticking.

[0121] The vitrification device 1601 is connected to an input port 1604 via capillary action and a filter mechanism. The nozzle is sized and configured to draw fluid through the nozzle structure into a waste container 1612. In this way, the sample can be shaped without the aid of, or even against, an external force. The sample flows from the input port 1604 through the sample chamber 1611 to the waste container 1612. In some embodiments, the input port 1604, the sample chamber 1611, and the filter 1621 The microfluidic channel is formed by a small hole in the microfluidic channel 1612 and / or a small passage through the waste container 1612. A filter is formed to direct the liquid waste from the sample chamber 1611 towards the filter 1621. Capillary suction force is provided to move the liquid by using a pipette, etc. When the first solution is introduced through the input port 1604, capillary action is initiated. The liquid is pushed from the sample chamber 1611 through the pores of the filter 1621 into the waste container 1612. Combined with microfluidic channels of specific sizes running through the entire device, the waste The presence of fluid in the waste container 1612 induces subsequent transport through the device 1621 using capillary action. It generates a force strong enough to draw in the fluid. Additionally, in some embodiments, The filter 1621 has a wicking mechanism that moistens and facilitates removal of liquid waste from the sample chamber 1611. They are made from absorbent materials such as sponge, filter paper or mesh that provide a lifting force. In an embodiment, as liquid flows through the material and wets the holes, capillary action is initiated and suction occurs. The lifting force allows the liquid and / or subsequent additional liquid to pass through the material without the need for fluid pressure. Support.

[0122] Liquid waste is collected in a waste container 1612 carried in the handle portion 1602. In some embodiments, the waste container is used during pre-processing and preparation of the sample to be vitrified. It is sized to have the capacity to hold all waste fluids that will be used, and so that the waste container 1612 never reaches maximum capacity and never needs to be emptied. A waste container 1612 is located at the opposite end of the input 1610 as shown in FIG. 16, which is connected to a cap 1630 that is adapted to hold the fluid in place when the fluid is drawn from the sample chamber 1611. Additionally, the cap 1630 is adapted to hold liquid nitrogen (for freezing) and (for hot water to pass around the waste container 1612) To provide a method for disposing of a waste container 1612, the position of the waste container 1612 must be maintained on the axial centerline of the device 1601. In some embodiments, the cap 1630 can be molded in multiple colors to aid in color matching. This allows users to have their own unique color discrimination and allows them to easily identify the different sizes, uses, etc. of the device 1621. Be unique.

[0123] The device 1621 is adapted to seal the sample chamber 1611 for submersion in liquid nitrogen. In particular, the cap 1603 is disposed at the end of the input portion 1610 of the device 1621. The other end is sealed with a freezing solution in a waste container 1612 acting as a plug. It will be sealed.

[0124] 22A-22D show alternative embodiments of the input portion 1610 of the vitrification device 1601. As shown in these figures, the input 1610 is a device for inputting the freezing or thawing of a sample. Various design features are included to prevent or limit the passage of air bubbles into the sample chamber 1611 during freezing. The device 1 includes an input port 1604 having a cap 1603 after the solution is introduced. 621, the cap 16 As a result, the air bubble is directed toward the opening of input channel 1604b. Air bubbles can damage the sample in the sample chamber 1611. 22A-22D help limit the possibility of sample damage.

[0125] In FIG. 22A, the input port 1604 is recessed from the input recess 1604a by a raised portion 1622. The cap (not shown) is connected to an input channel 1604b, which is separated from the end of the cap. When placed on input 1610, any bubbles that form will flow into the input above ridge 1622. It remains above channel 1604b. When frozen, the contraction of the frozen liquid causes air bubbles to form. There is no chance that air will be sucked into the room. Therefore, when it is thawed, the room will be free of air bubbles. is maintained.

[0126] In FIG. 22B, the input port 1604 is different relative to the opening of the input channel 1604b. The second recess 1624 is lower than the input port recess 1604a but is The opening of the force channel 1604b is at a higher elevation. In this way, the liquid level is A small amount of liquid will cause the liquid to flow above the second recess 1624 (and from the opening to the input channel 1604b). The lands separating the input port recess 1604a and the second recess 1624 are The cap helps to keep the liquid level above the opening of input channel 1604b. In the case where the depths of the recesses 1604a that require a large amount of fluid for thawing are all the same, The thawing process may be faster than the time required for thawing. If thawing takes longer, the viability of the sample may be compromised. may be put at risk.

[0127] 22C-22D, the input port recess 1604a includes a narrow portion 1626. A bubble with a width equal to the recess 1604a of the port cannot be forced through the narrow portion 1626. Therefore, air bubbles are kept away from the opening of the input channel 1604b. In FIG. 22C, the input recess 1604a is inclined, and the minimum amount of Allows the fluid to be used while not allowing the fluid level to drop too low (i.e., input channel This also ensures that the liquid is kept above the opening to channel 1604b. This allows the hot solution to reach the sample more quickly, thereby improving sample quality. Promote survival capabilities.

[0128] In Figure 22D, there is a trough 1628 in the input recess 1604a. The trough 1628 allows liquid to flow even if air bubbles are trapped near the opening in 1604b. The trough 1628 is intended to be constantly filled with liquid. Therefore, by having a passage for the liquid to flow, bubbles are formed. However, there is a liquid movement that is very important when thawing a sample. Air bubbles can obstruct the flow path. , the viability of the sample is compromised.

[0129] In some embodiments, at least the input portion and the cap portion of the device are immersed in liquid nitrogen. In some embodiments, the rapid vitrification and The material is thermally conductive so that thawing can be achieved. Exemplary materials that may be used include, but are not limited to, acrylic-based materials, polypropylene ... These include recarbonate-based materials, and copolyester-based materials.

[0130] The above vitrification devices according to the various disclosed embodiments include similar features and The present invention operates according to the principle of Details may be omitted from the description of certain embodiments, and similar elements may be found in other aspects of the vitrification device. may perform the same functions or include the same features as those previously described in connection with the embodiments of It should be understood.

[0131] (Equivalent) The technology described in this application is intended as an example of one particular aspect of the current technology. The present invention is not limited to the specific embodiments illustrated herein without departing from its spirit and scope. Many modifications and variations of the techniques are possible and will be apparent to those skilled in the art. In addition to those enumerated herein, functionally equivalent methods and apparatuses within the scope of the present technology are also contemplated. Those skilled in the art will be aware from the foregoing description. Such modifications and variations are within the scope of the present technology. The present technology is intended to encompass specific methodologies, reagents, compound compositions or methods which may of course vary. It should be understood that the term "biological system" as used herein is not limited to biological systems in which it can be used. It is understood that the terminology is for the purpose of describing particular embodiments only and is not intended to be limiting. It should be understood.

[0132] As will be appreciated by those skilled in the art, for any and all purposes, the disclosed subject matter may be modified in any manner in accordance with the teachings set forth herein. In providing clarity, all scopes disclosed herein are to be construed as including any and all possible Any range recited herein is intended to be construed as including any subrange or combination of subranges. is divided at least equally into halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, the present invention Each range discussed in the subsection can be easily divided into a lower third, a middle third, and an upper third, etc. As will be understood by those skilled in the art, the terms "up to," "at least," "greater than," "better ..." and "better than" can be used interchangeably. All terms such as "less than" and "not more than" are inclusive of the recited numbers and are subsequently divided into subranges as described above. Finally, as will be appreciated by those of ordinary skill in the art, each range also refers to a separate Contains elements.

[0133] All numerical designations, e.g., pH, temperature, time, concentration, amount, and molecular weight, including ranges, are It is an approximation that varies by (+) or (-) 10%, 1% or 0.1%, as appropriate. Although not necessarily implied, it is understood that all numerical designations may be preceded by the word "about." It should be understood that, although not always explicitly stated, the reagents described herein may It is to be understood that these are merely examples and that equivalents thereof are known in the art. It is.

Claims

1. an input section having an input port; A sample chamber; a waste container in fluid communication with the sample chamber; A fluid is introduced through the input port while retaining a sample within the sample chamber. a filter mechanism that selectively allows the passage of the sample from the sample chamber into the waste container; Structure Equipped with The sample chamber, the waste container and the filter mechanism are arranged to a glass capillary configured to draw fluid through the glass capillary into the waste container. Device.

2. The sample chamber further includes at least one observation window, the observation window being configured to allow the sample in the sample chamber to be observed. The vitrified device of claim 1 , configured to be viewable through the observation window. vinegar.

3. a cap reversibly connectable to said input portion to close said input port; The vitrification device of claim 1 further comprising:

4. The cap is adapted to couple the vitrification device to the input when the cap is coupled to the input.

4. The method of claim 3, further comprising the steps of: sealing the input port to prevent the flow of fluid therethrough; The vitrification device described herein.

5. The filter mechanism forms a plurality of microfluidic channels to promote capillary action. a filter having a plurality of pores sized and configured to Item 2. The vitrification device according to item 1.

6. 10. The vitrification device of claim 1, wherein the sample chamber comprises a material that is resistant to liquid nitrogen. Vice.

7. The vitrification device of claim 1 , wherein the sample chamber comprises a thermally conductive material.

8. The sample chamber is made of an acrylic material, a polypropylene material, or a polycarbonate material. and a copolyester-based material. Device.

9. 10. The glass of claim 1, wherein the sample chamber has walls having a thickness of 0.002 inches or less. Intelligent device.

10. 1. A method for preparing a sample, comprising: delivering the sample into a sample chamber adjacent to a filter mechanism; The filter mechanism holds the sample within the sample chamber while the sample chamber a first flow path by forcing the first fluid through the first flow path and into a waste container by a hydrodynamic force; treating the sample with a body; Including, Capillary action is initiated by forcing the first fluid into the waste container, and subsequent fluid is drawn through the sample chamber and into the waste container.

11. The filter mechanism holds the sample within the sample chamber while the sample chamber by drawing a second fluid by capillary action through said waste container The method of claim 10 further comprising treating the sample with a second fluid.

12. The sample in the sample chamber is observed through an observation window in the sample chamber. The method of claim 10 further comprising the step of:

13. Attaching a cap seals an input port for fluid flow into the sample chamber. The method of claim 10 further comprising the step of:

14. The method of claim 10 further comprising the step of vitrifying the sample in the sample chamber. The method described.

15. The sample is vitrified by contacting the sample chamber with liquid nitrogen. The method of claim 14.

16. thawing the sample by contacting the sample chamber with a warm solution. The method of claim 14, further comprising:

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