WO2024155998A1

WO2024155998A1 - Hanging drop-based microfluidic system, device, and method for liquid exchange for vitrification of a specimen

Info

Publication number: WO2024155998A1
Application number: PCT/US2024/012451
Authority: WO
Inventors: Linda Griffith; Scott Manalis; Georgios KATSIKIS; Haidong FENG; Joseph Doyle
Original assignee: Cryotech LLC; Massachusetts Institute of Technology
Current assignee: Cryotech LLC; Massachusetts Institute of Technology
Priority date: 2023-01-20
Filing date: 2024-01-22
Publication date: 2024-07-25
Anticipated expiration: 2025-07-20

Abstract

A hanging drop device for vitrification of a specimen includes a housing, a plurality of microfluidic channels disposed in the housing and a plurality of inlets disposed in the housing. Each inlet is connected to and in fluid communication with at last one of the plurality of microfluidic channels. The hanging drop device further includes an outlet disposed in the housing and connected to and in fluid communication with at least one of the plurality of microfluidic channels, and an opening disposed in the housing and connected to and in fluid communication with each of the plurality of microfluidic channels. The opening is configured to form a hanging drop chamber from a liquid loaded into the opening and the hanging drop chamber is configured to receive the specimen.

Description

HANGING DROP-BASED MICROFLUIDIC SYSTEM, DEVICE, AND METHOD FOR LIQUID EXCHANGE FOR VITRIFICATION OF A SPECIMEN

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is based on, claims priority to, and incorporates herein by reference in its entirety U.S. Serial No. 63/440,180 fded January 20, 2023, and entitled “Liquid Exchange And Positioning Of Cellular Entities In A Liquid- Air Interface.”

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] N/A

BACKGROUND

[0003] Egg vitrification is a process by which a patient’s eggs can be stored indefinitely in an ultra-cooled state, and represents a powerful tool in fertility preservation. Vitrification can be used for the cryopreservation of an unfertilized egg or a fertilized egg (i.e., an embryo). Historically, the egg vitrification procedure was designated an experimental treatment reserved for women possessing medical and genetic disorders impacting ovarian function, or for women requiring fertility impairing medical interventions. Recent refinement of the egg freezing process has led fertility clinics nationwide to offer it as a standard treatment, resulting in explosive growth in the number of cycles performed. However, egg freezing is a technically sensitive process due to the manual nature of the procedure, achieving consistent high fidelity outcomes only by highly-trained embryologists in high complexity and volume embryology labs. This translates into significant out-of-pocket costs to patients, geographic inaccessibility, and high variability in clinical outcomes. Automation across multiple industries has shown that, relative to human operators, automated platforms can perform defined, repetitive tasks with improved accuracy and precision, often at a fraction of the cost and scale, while generating knowledge about the given process. The few prior attempts to automate the egg freezing process have not been successful, due to the complexity of steps and different facets of technology that must be integrated. As a result, there currently exists no widely adopted, automated commercial platform for egg (or embryo) freezing. SUMMARY OF THE DISCLOSURE

[0004] In accordance with an embodiment, a hanging drop device for liquid exchange for use in vitrification of a specimen includes a housing, a plurality of microfluidic channels disposed in the housing and configured to facilitate liquid exchange around the specimen, and a plurality of inlets disposed in the housing. Each inlet is connected to and in fluid communication with at last one of the plurality of microfluidic channels. The hanging drop device further includes an outlet disposed in the housing and connected to and in fluid communication with at least one of the plurality of microfluidic channels, and an opening disposed in the housing and connected to and in fluid communication with each of the plurality of microfluidic channels. The opening is configured to form a hanging drop chamber from a liquid loaded into the opening and the hanging drop chamber is configured to receive the specimen.

[0005] In accordance with another embodiment, a method for vitrification of a specimen using a hanging drop device for liquid exchange around the specimen includes receiving a basic solution in at least one microfluidic channel of a hanging drop device to form a hanging drop chamber in an opening of the hanging drop device. The hanging drop chamber is defined by a liquid-air interface. The method further includes receiving the specimen in the hanging drop chamber where the specimen is supported by the liquid-air interface of the hanging drop chamber, and loading, using the at least one microfluidic channel, a plurality of liquids into the hanging drop chamber to facilitate liquid exchange around the specimen. At least one liquid of the plurality of liquids is a cryoprotectant.

[0006] In accordance with another embodiment, a hanging drop device for liquid exchange for use in vitrification of a specimen includes a housing, a plurality of microfluidic channels disposed in the housing and configured to facilitate liquid exchange around the specimen, and a plurality of inlets disposed in the housing. Each inlet is connected to and in fluid communication with at least one of the plurality of microfluidic channels. The hanging drop device further includes an outlet disposed in the housing and connected to and in fluid communication with at least one of the plurality of microfluidic channels, and a plurality of openings disposed in the housing. Each opening is connected to and in fluid communication with at least one other opening in the plurality of openings using at least one of the plurality of microfluidic channels. Each opening is also connected to and in fluid communication with at least one inlet in the plurality of inlets and the outlet, wherein each opening is configured to form a hanging drop chamber from a liquid loaded into the opening, the hanging drop chamber configured to receive one specimen.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] The present invention will hereafter be described with reference to the accompanying drawings, wherein like reference numerals denote like elements.

[0008] FIG. l is a schematic diagram of a hanging drop device for liquid exchange for use in vitrification of a specimen in accordance with an embodiment;

[0009] FIG. 2a is a schematic diagram of a hanging drop device for liquid exchange for use in vitrification of a specimen and a vitrification carrier for retrieval of the specimen in accordance with an embodiment;

[0010] FIG. 2b is a schematic diagram of a hanging drop device for liquid exchange for use in vitrification of a specimen and a vitrification carrier and robotic arm for retrieval of the specimen in accordance with an embodiment;

[0011] FIG. 3 a shows a perspective view of a hanging drop device for liquid exchange for use in vitrification of a specimen in accordance with an embodiment;

[0012] FIG. 3b shows a top view of one end of a hanging drop device for liquid exchange for use in vitrification of a specimen in accordance with an embodiment;

[0013] FIG. 3c shows a perspective view of one end of a hanging drop device for liquid exchange for use in vitrification of a specimen in accordance with an embodiment;

[0014] FIGs. 4a and 4b are schematic diagrams of designs of a microfluidic channel for a hanging drop device for liquid exchange for use in vitrification in accordance with an embodiment;

[0015] FIG. 5 illustrates a process for operation of the hanging drop device of FIG. 1 in a vitrification process in accordance with an embodiment;

[0016] FIG. 6 illustrates a method for vitrification of a specimen using a hanging drop device for liquid exchange around the specimen in accordance with an embodiment;

[0017] FIG. 7 is schematic diagram of a hanging drop device for liquid exchange for use in vitrification of a specimen in accordance with an embodiment; [0018] FIG. 8 illustrates a process for operation of the hanging drop device of FIG. 7 in a vitrification process in accordance with an embodiment;

[0019] FIG. 9 illustrates a method for vitrification of a specimen using a hanging drop device for liquid exchange around the specimen in accordance with an embodiment;

[0020] FIG. 10 is schematic diagram of a hanging drop device with a plurality microfluidic channels for liquid exchange for use in vitrification of a specimen in accordance with an embodiment;

[0021] FIG. 11 shows a top view of a hanging drop device with a plurality of microfluidic channels for liquid exchange for use in vitrification of a specimen in accordance with an embodiment;

[0022] FIGs. 12 is a schematic diagram illustrating operation processes of a hanging drop device with a plurality of microfluidic channels for liquid exchange for use in vitrification of a specimen in accordance with an embodiment;

[0023] FIG. 13 is a schematic diagram illustrating fluid flow in a hanging drop chamber for different operation processes in accordance with an embodiment;

[0024] FIG. 14 is schematic diagram of a hanging drop device with a plurality of hanging drop chambers for liquid exchange for use in vitrification of a plurality of specimens in accordance with an embodiment;

[0025] FIG. 15 shows a top view of a hanging drop device with a plurality of hanging drop chambers for liquid exchange for use in r vitrification of a plurality of specimen in accordance with an embodiment; and

[0026] FIGs. 16a-c illustrates example operation modes of a .hanging drop device with a plurality of hanging drop chambers for liquid exchange for use in vitrification of a plurality of specimen in accordance with an embodiment.

DETAILED DESCRIPTION

[0027] Various factors have contributed to a soaring market for egg or oocyte and embryo vitrification including 1) individuals choosing to wait for "the right time" to have children, and 2) a competitive employment market where more employers are offering egg freezing as a benefit. This increase in demand., however, presents fertility practices with a number of challenges. Experienced embryologists are the foremost bottleneck for fertility practices and can have a direct bearing on patient outcomes (e.g., patient outcomes are consistently high with an experienced embryologist). For example, an experienced embryologist has a minimum Bachelor of Science (BS) degree and many have PhDs., has multi-year on the job training, technical competence, and can have expensive salaries. For a manual process, highly skilled embryologists are in limited supply and can be quite expensive, the clinical outcome can be variable due to varying technical skills (e.g., between an entry-level embryologist and experienced embryologist), and the scalability can be limited due to the multi-year embryologist training process. It would, therefore, be desirable to provide a system, device and method that automates key technically challenging processes to, for example, expand laboratory bandwidth. An automated device could be operated by an entry-level embryologist (who could be employed at a lower salary, for example, at 25% of a skilled embryologist salary), the clinical outcome could be consistent and equivalent to top tier embryologists, and could have high scalability which could allow expended access in existing and new markets.

[0028] Egg (or oocyte) and embryo vitrification processes have a number of technical challenges. Egg and embryo vitrification (freezing) requires a delicate balance of timing and fluid volume control. Damage to an egg (or embryo) can occur during various critical steps of the egg vitrification process including, for example: 1) during loading of the cryoprotectant, damage to an egg may be caused by overexposure to cryoprotectant before freezing, and 2) damage to an egg may be caused by excess liquid surrounding the egg during freezing. For the step of loading the cryoprotectant, the challenges of current manual practice, which can be a multiple step process, include protocol drift, multitasking (multiple eggs requires multiple dishes and timers), and operator fatigue. For the issue of excess liquid surrounding the egg and removing the excess liquid, the challenges of the current manual practice include sub-microliter volume control by the human hand, tight timing, technical variability between individual operators results in variable outcomes, operator fatigue, and limited throughput per operator. In addition, performance of specimen (e.g., an egg or embryo) retrieval in the conventional manual process can have a variation in skill performance based on the experience of the embryologist as well as protocol drift and operator fatigue.

[0029] The present disclosure describes an integrated microfluidic platform capable of equilibrating, vitrifying, and warming eggs (or oocytes) or embryos (e.g., human eggs and embryos) on a small-format, easy-to-use system and device that can be operable by an entry level technician. By significantly reducing the level of technical skill and clinical infrastructure needed to perform reliable egg (or embryo) freezing, the disclosed platform can simultaneously improve the quality of cryopreservation and expand access to care. In particular, the present disclosure describes a hanging drop-based microfluidic system, device and method for liquid exchange for vitrification of a specimen, for example, an egg (or oocyte) or embryo (i.e., an unfertilized egg or a fertilized egg). The disclosed hanging drop device for liquid exchange advantageously provides an automated system and platform that enables taking advantage of opportunities in the fast growing area of egg vitrification where there is high demand.. The disclosed devices and processes can address a critical pain point common to all fertility practices. In some embodiments, the hanging drop device and processes can provide a fully automated and self- contained system. Advantageously, the disclosed system can be operable by entry-level technicians. In some embodiments, aspects of the system may be programable. The disclosed hanging drop device can be contactless and can optimize key vitrification steps. In addition, the disclosed system and process is versatile and in some embodiments may be utilized for eggs or embryos.

[0030] FIG. l is a schematic diagram of a hanging drop device for liquid exchange for use in vitrification of a specimen in accordance with an embodiment. The hanging drop device 100 can include a housing 102. In some embodiments, the housing can be in the form of a chip (e g., as discussed below with respect to FIGs. 3a-c). The housing 102 can include a first surface or wall 104, a second surface or wall 106 opposite the first surface 104, an opening (or open chamber) 108 in the first surface 104, an inlet (or input) 114, an outlet (or output) 116, and a microfluidic channel 112 between the inlet 114 and the outlet 116. While one inlet 114, one microfluidic channel 112, and one outlet 116 are shown in the embodiment illustrated in FIG. 1, some embodiments of a hanging drop device can advantageously include a plurality of inlets 114 and a plurality of microfluidic channels 112 as discussed below with respect to FIGs. 10- 16c. The opening 108 in the first surface 104 can be configured to form a hanging drop of a liquid provided through inlet 114 to the microfluidic channel 112 to fill (or seed) the channel 112 and the opening 108 (or open chamber) with the liquid. A droplet may be formed in the opening 108 and a hanging drop can form and create a chamber (or confined chamber ) 110 defined by a liquid-air interface 111 of the hanging drop when the first surface 104 and the opening 108 are facing towards the floor in the direction of gravity (as indicated by arrow 125). While one opening 108 is shown in the embodiment illustrated in FIG. 1, some embodiments of a hanging drop device can advantageously include a plurality of openings for a plurality of chambers 110 as discussed below with respect to FIGs. 14- 16c. In some embodiments as illustrated in FIG. 1, the hanging drop device 100 can also include an intermediate structure 118 positioned between the chamber 110 and the microfluidic channel 112. The intermediate structure 118 can form a “ceiling” of the chamber 110 formed by the hanging drop. In some embodiments, the intermediate structure 118 can be a mesh that allows fluid to pass through the structure 118 but does not allow a specimen 124 (e.g., an egg (or oocyte), or embryo) in the chamber 110 (or hanging drop) to pass through thus preventing the specimen from entering the microfluidic chamber 112. For example, one layer of nylon mesh can be bonded to the sides of the opening 108 between the chamber 110 and microfluidic channel 112, which can be used to prevent the specimen 124 from entering the microfluidic channel 112. A process and method for forming the chamber 110 (e.g., a handing drop) and loading a specimen 124 into the chamber 110 is discussed further below with respect to FIGs. 5 and 6.

[0031] When loaded into the chamber 110 (or hanging drop), the specimen 124 (e.g., an egg (or oocyte) or embryo) can sink due to gravity and migrate towards the liquid-air interface

111 of the chamber 110. When the specimen 124 is close to the liquid-air interface 110, the specimen 124 can be advantageously trapped by the liquid-air interface due to the surface energy and can then stay at a position at or near a center of the hanging drop (i .e., chamber

110). The chamber 110 formed in opening 108 has a connection to the microfluidic channel

112 in the housing 102, which can facilitate a liquid exchange process, for example, loading of cryoprotectant(s). Liquid can pass through the chamber 110, which is defined by the liquidair interface 111 of the hanging drop, via the microfluidic channel 112. As mentioned above, the microfluidic channel 112 is coupled to the inlet 114 and the outlet 116. Liquid exchange around the specimen 124 can occur by flowing different liquids (e.g., cryoprotectants) through inlet 114 and microfluidic channel 112 and out through outlet 116, and allowing the liquids to move through the intermediate structure 108 between the microfluidic channel 112 and the chamber 110. As discussed further below with respect to FIGs. 5 and 6, a liquid, for example, a cryoprotectant, may be loaded from the inlet 114 (as shown by arrow 120) into the microfluidic channel 112 and enter the chamber (or hanging drop) 110. In some embodiments, the liquid can be provided or delivered to the inlet 114 and to the microfluidic chamber 112 using a liquid delivery system such as, for example, a syringe pump, pneumatic regulator, or peristaltic pump. In some embodiments, the existing liquid in the chamber (or hanging drop) 110 may be withdrawn to the outlet 116 (as shown by arrow 122) simultaneously. The liquid-air interface 111 can advantageously allow accurate manipulation of a specimen 124 (e.g., an egg (or oocyte) or embryo) position.

[0032] The liquid-air interface 111 of the hanging drop and the ability to use the liquid-air interface 11 to hold the specimen 124 in one position can also have advantages for the specimen retrieval process since a device, such as, for example, a vitrification carrier could easily penetrate through the liquid-air interface 111 and retrieve the specimen 124 in the known position in the chamber 110. FIG. 2a is a schematic diagram of a hanging drop device for liquid exchange for use in vitrification of a specimen and a vitrification carrier for retrieval of the specimen in accordance with an embodiment and FIG. 2b is a schematic diagram of a hanging drop device for liquid exchange for use in vitrification of a specimen and a vitrification carrier and robotic arm for retrieval of the specimen in accordance with an embodiment. In FIG. 2a, a hanging drop device 100 includes a housing 102, a first surface or wall 104, a second surface or wall 106 (opposite the first surface 104), an opening (or open chamber) 108 in the first surface 104, a chamber 110 formed from a hanging drop and defined by the liquid-air interface 111 of the hanging drop, a microfluidic channel 112, an inlet 114, an outlet 116, and an intermediate structure (e.g., a mesh) 118 positioned between the chamber 110 and the microfluidic channel 112. A specimen 124 (e.g., an egg or embryo) may be located in the chamber 110 (or hanging drop). In some embodiments, the specimen 124 can be transported out of the chamber 110 (i.e., a specimen retrieval process) using a vitrification carrier or device 126 that is configured to penetrate the liquid-air interface 111 of the chamber 110 formed from the hanging drop and the liquid surrounding the specimen 124. In some embodiments, the vitrification carrier or device 126 can be configured to retrieve the specimen 124 and liquid surrounding the specimen 124 without physical contact with the specimen 124. In some embodiments, the vitrification carrier or device 126 may be a cryoloop device that includes, for example, a nylon loop used to suspend a film of the liquid (e.g., a cryoprotectant) containing the specimen 124 and a rod coupled to the nylon loop. As discussed further below with respect to FIGs. 5 and 6, once the specimen 124 has been retrieved from the chamber 110 using the vitrification carrier 126, the specimen 124 can be moved rapidly into a vitrification medium such as, for example, liquid nitrogen, slush nitrogen, and vapor phase liquid nitrogen, to complete the freezing process and for storage. For example, the specimen can me moved to a liquid nitrogen tank (not shown) to complete the freezing process and for storage. In some embodiments, a robotic arm 132 (shown in FIG. 2b) may be connected to the vitrification carrier 126 and used to robotically perform the retravel process for the specimen 124. As discussed further below with respect to FIG. 5 and 6, one or more cameras 128, 130 may be positioned to view the chamber (or hanging drop) 110 from different sides, for example, in FIGs. 2a and 2b, a first camera 128 can be positioned to view the chamber 110 from the bottom and a second camera 130 can be positioned to view the chamber 110 from a side of the chamber 110. The cameras 128, 130 can be utilized during, for example, the cryoprotectant loading process And specimen 124 retrieval process.

[0033] FIG. 3 a shows a perspective view of a hanging drop device for liquid exchange for use in vitrification of a specimen in accordance with an embodiment. The hanging drop device 200 includes a housing 202, a first surface or wall 204 (which includes an opening (not shown) for a hanging drop, a second surface or wall 206 opposite the first surface 204, first end 236 and a second end 238. The first end 236 include the location 234 of the opening for the hanging drop chamber and towards the second end 236 is an inlet 214 and an outlet 216. A microfluidic channel 212 connected to and between the inlet 214 and the outlet 216 connects to the opening for the hanging drop chamber. FIG. 3b shows a top view of one end of a hanging drop device for liquid exchange for use in vitrification of a specimen in accordance with an embodiment. A first end 236 of the hanging drop device 200 is shown in FIG. 3b, in particular, a top view of the second surface 206 (also shown in FIG. 3a) is illustrated and the location 234 of the opening for a hanging drop chamber at the first end 236. The housing 202 includes the microfluidic channel 212 which is connected to the opening for a hanging drop chamber at the first end 236. FIG. 3c shows a perspective view of one end of a hanging drop device for liquid exchange for use in vitrification of a specimen in accordance with an embodiment. A first end 236 of the hanging drop device 200 is shown in FIG. 3c, in particular, a perspective view of the first surface 204 (also shown in FIG. 3a) is illustrated. An opening 208 for a hanging drop chamber is located at the first end 236 and is connected to the microfluidic channel 212. In some embodiments, a rim 240 or other barrier may be provided around the opening 208 to provide additional stability and, for example, to help prevent liquid in the opening 208 from spreading. In some embodiments, the hanging drop device (100, 200) can be a disposable device. In some embodiments, the hanging drop device 200 can be made of a material with good chemical resistance and biocompatibility for various applications such as, for example, cyclic olefin copolymer (COC). In some embodiments, the overall hanging device 200 can have dimension of 50 x 7 mm. In some embodiments, the chamber 208 can have, for example, dimensions from 1.5mm to 5mm. In one example, a 3 mm diameter chamber 208 can be used with a rim 240 height of 1mm and in this design, a hanging drop (i.e., chamber 110 shown in FIGs. l-2b) can be generated in the opening 208 with volume ranging from 5 ul to 25 ul.

[0034] FIGs. 4a and 4b are schematic diagrams of designs of a microfluidic channel for a hanging drop device for liquid exchange for use in vitrification in accordance with an embodiment. As discussed above with respect to FIGs. l-3c, a hanging drop device for vitrification of a specimen (e.g., an egg (or oocyte) or embryo) can includes a microfluidic channel 212 between an inlet 214 and outlet 216. The microfluidic channel is also connected to an opening 208 that can be used for form a chamber (or hanging drop). Liquid can be provided through the inlet 214 and microfluidic channel 212 to the chamber in the opening 208 and can leave the chamber in the opening 208 via the microfluidic channel 212 to the outlet 216. FIGs. 4a and 4b illustrate different arrangements for the microfluidic channel as it enters the opening 208 and a hanging drop chamber in the opening 208. In FIG. 4a, the microfluidic channel 212 can be arranged as a semi-circle 252 around the opening 208 to provide liquid to a chamber in the opening 208. Accordingly, the microfluidic channel 212 has a much larger input area and liquid can enter a chamber (or hanging drop) in the opening 208 from around the chamber which can provide a more even distribution of the liquid in the chamber (or hanging drop) in the opening 208. A portion of microfluidic channel 212 connected to the outlet 216 can be arranged as a single “point” in the chamber for exit of the fluid from the chamber to the outlet 216. In the embodiment illustrated in FIG. 4b, a portion of the microfluidic channel 212 connected to the inlet 214 can enter the opening 208 (and a chamber formed in the opening by a hanging drop) arranged as a single “point” and a portion of microfluidic channel 212 connected to the outlet 216 can be arranged as a single “point” in the chamber for exit of the fluid from the chamber to the outlet 216. Accordingly, as illustrated in FIG. 4b, a “point-to-point” arrangement 254 may be used for the portion of the microfluidic channel 212 entering the opening 208 (and a chamber or hanging drop formed in the opening 208) and the portion of the microfluidic channel exiting the opening 208 (and a chamber or hanging drop formed in the opening 208).

[0035] FIG. 5 illustrates a process for operation of the hanging drop device of FIG. 1 in a vitrification process in accordance with an embodiment. As mentioned above, the hanging drop device with a microfluidic channel can be used for liquid exchange (e.g., cryoprotectant loading) with a specimen (e.g., an egg (or oocyte) or embryo) for vitrification. In a first step 370, a first surface 304 of the hanging drop device 200 can be facing upwards, away from the direction of gravity (as indicated by arrow 325), and a second surface 306 of the hanging drop device 300 can be facing downwards towards the ground (or floor) and the direction of gravity 325. A microfluidic channel 312 and an opening 308 of the hanging drop device 300 can be filled with a liquid, for example, a basic solution (BS) to facilitate loading of a specimen 324 into the hanging drop device 300 and the formation of a hanging drop in the opening 308 in the second step 372. In some embodiments, the basic solution can be a culture medium. The opening 308 can be formed in the first surface 304 of a housing of the hanging drop device 300. The basic solution may be loaded into the microfluidic channel 312 and opening 308 via an inlet 314. In some embodiments, the liquid can be provided or delivered to the inlet 314 and to the microfluidic chamber 312 using a liquid delivery system such as, for example, a syringe pump, pneumatic regulator, or peristaltic pump. When the liquid fills the opening 308, a droplet can be formed by surface tension. The specimen (e.g., an egg or embryo) 324 may then be transferred into the liquid (or droplet) in the opening 308, for example, the specimen 324 may be injected into the droplet in the opening 308 using a pipette 360. In an example, the pipette volume can range from 0.5 ul to 5 ul.

[0036] Then, in a second step 372, the hanging drop device 300 can be flipped over so that the first surface 304 and opening 308 is facing towards the ground (e.g., the floor) in the direction of gravity 325. A hanging drop, or chamber 310, can be formed from the liquid (e.g., droplet) in the opening 308 due to the balance between surface tension, gravity, and the pressure difference with the microfluidic channel 312. The specimen 324 in the chamber 310 can then sink due to gravity and migrate towards a liquid-air interface 311 that defines the hanging drop forming the chamber 310. Once the specimen 324 is close to the liquid-air interface 311, the specimen 324 can be captured (or trapped) by the liquid air interface 311. In some embodiments, the specimen 324 can be captured by the liquid-air interface 311 in a short time (e.g., < 10s). In some embodiments, the liquid-air interface 31 1 can be used advantageously maintain the specimen 324 in one position (e.g., near or at the center of the chamber 310) during the liquid exchange (this step 372) and specimen retrieval (fourth step 376). In some embodiments, one or more cameras (e.g., cameras 128, 130 shown in FIGs. 2a and 2b) can be positioned to view the chamber 310 formed by the hanging drop from different sides, for example, the bottom and the side. In some embodiments, a side view camera can help evaluate the hanging drop (i.e., chamber 310) volume and shape for feedback flow control process. A bottom view camera can be used to identify specimen 324 position in the chamber 310.

[0037] In a third step 374, the inlet 314, microfluidic channel 312, chamber 310 and outlet 316 can be used for a liquid exchange process. Liquid exchange around the specimen 324, for example, for cryoprotectant loading, can be performed through the microfluidic channel 312 which is connected to the chamber 310. Liquid can be provided to the inlet 314 (as indicated by arrow 320) and pass from the inlet 314 into the microfluidic channel 312 and from the microfluidic chamber 312 into the chamber 310 (i.e., the hanging drop) through the intermediate structure 318 (e.g., a mesh). The liquid can also pass out of the chamber 310 to the microfluidic channel 312 through the intermediate structure 318 and then from the microfluidic channel 312 to the outlet 316 (as indicated by arrow 322). In some embodiments, the liquid can be provided or delivered to the inlet 314 and to the microfluidic chamber 312 using a liquid delivery system such as, for example, a syringe pump, pneumatic regulator, or peristaltic pump. As mentioned, cryoprotectant (or a cryoprotective agent (CPA)) loading can be performed through the microfluidic channel 312. In some embodiments, two types of cryoprotectant can be loaded during the liquid exchange process (step 374), namely, an equilibration solution (ES) and vitrification solution (VS). In some embodiments, both of the equilibration solution and the vitrification solution are composed of cryoprotectant components such as, for example, ethylene glycol and dimethyl sulfoxide, and the equilibration solution has a lower concentration of cryoprotectant. For example, in some embodiments, the equilibration solution can be composed of 50% cryoprotectant and the vitrification solution can be composed of 100% cryoprotectant. In some embodiments, loading of the equilibration solution can be performed by a gradual increase of equilibration solution by loading mixes of basic solution and equilibration solution. In some embodiments, vitrification loading can be performed utilizing a high flow rate to minimize the time needed for complete liquid exchange inside the chamber 310 (i.e., the hanging drop). In a hanging drop device 300 with one microfluid channel, in some embodiments each liquid (e.g., BS, ES (or BS/ES mixture), VS) can be loaded into the hanging drop device 300 (and chamber 310) sequentially. Cryoprotectant concentration and time control are key parameters affecting specimen (e.g., an egg (or oocyte) or embryo) survival during vitrification. Liquid exchange efficiency inside the disclosed hanging drop device 300 can be realized at a fast speed. In some embodiments, VS loading can be realized within 5s, which allows the precise control of solution concentration and exposure time.

[0038] As mentioned above, in some embodiments, one or more cameras (e.g., cameras 128, 130 shown in FIGs. 2a and 2b) can be positioned to view the chamber 310 formed by the hanging drop from different sides, for example, the bottom and the side. In some embodiments, a side view camera can be used to demonstrate the mixing efficiency during the cryoprotectant loading process. A bottom view camera can be used to identify specimen 324 position in the chamber 310 and evaluate the condition of the specimen 324 during the liquid exchange (e.g., cryoprotectant loading) process.

[0039] When the cryoprotectant loading is complete, egg (or oocyte) retrieval can be performed in a fourth step 376 using, for example, a vitrification carrier 326. As mentioned above, in some embodiments, the position of the specimen 324 in the chamber 310 can be monitored by one or more cameras (e.g., cameras 128, 130 shown in FIGs. 2a and 2b) that are positioned to view the chamber 310 formed by the hanging drop from different sides, for example, the bottom and the side. In addition, a bottom view and side view camera can be used to monitor the motion of a vitrification carrier 326 used to retrieve the specimen 324 from the chamber 210 (i.e., the hanging drop). In some embodiments, the vitrification carrier 326 may be a cryoloop device that includes, for example, a nylon loop used to suspend a film of the liquid (e.g., a cryoprotectant) containing the specimen 324 and a rod coupled to the nylon loop. In some embodiments, when the vitrification carrier 326 interacts with the liquid-air interface 311 of chamber 310, the affinity of the liquid to the vitrification carrier 326 leads to the removal of a small portion of the liquid or solution from the chamber 310 (i.e., the hanging drop).

Accordingly, in such embodiments, the specimen 324 (e.g., an egg (or oocyte) or embryo) can be carried by the liquid and retained on the vitrification device without touching the vitrification carrier 326. After retrieving the specimen 324 from the chamber in step 376, in some embodiments the vitrification carrier 326 can be immersed into a vitrification medium such as, for example, liquid nitrogen, slush nitrogen, and vapor phase liquid nitrogen, to finish the verification process.

[0040] In the traditional manual treatment process for egg or embryo vitrification, the identification and handling of eggs are the most challenging processes. The manipulation of a single egg is hard due to the small dimension and transparency, while egg recovery rate is critical due to the precious value of egg. Utilizing the disclosed hanging drop device 100, 200, 300 and a vitrification carrier or device 126, 326 (e.g., a cryoloop device), the specimen (e.g., an egg (or oocyte) or embryo) has no contact with the vitrification carrier or device 126, 326 boundaries, and the specimen (e.g., an egg or embryo) trapping/adhesion problem can be avoided. As discussed above with respect to FIGs. 1, 2a and 5, the specimen 124, 324 will be aligned to the bottom of hanging drop (i.e., chamber 110, 310) automatically, which simplifies the specimen searching process in the solution in the chamber 110., 310. Based on the surface energy trapping mechanism, the external shear stresses applied on the specimen 124, 324 can be avoided.

[0041] FIG. 6 illustrates a method for vitrification of a specimen using a hanging drop device for liquid exchange around the specimen in accordance with an embodiment. The process illustrated in FIG. 6 is described below with reference to FIGs. 1, 2a, 2b and 5. Although the blocks of the process are illustrated in a particular order, in some embodiments, one or more blocks may be executed in a different order than illustrated in FIG. 6 or may be bypassed. While the process of for vitrification of a specimen using a hanging drop device is described below with respect to embodiments of a hanging drop device 100, 300 with one inlet 114, 314, one microfluidic channel 112, 312, one outlet 116, 316, and one opening 108., 308 (as shown in FIGs. 1, 2a, 2b, and 5), it should be understood that in some embodiments the vitrification process illustrated in FIG. 6 can be performed using a hanging drop device that advantageously includes a plurality of inlets 114, 314, a plurality of microfluidic channels 112, 312, and a plurality of openings 108, 308 (or hanging drop chambers) as discussed below with respect to FIGs. 10-16c.

[0042] At block 402, a basic solution may be provided into a microfluidic channel 112, 312 and opening 108, 308 of a hanging drop device 100, 300, in a first position with the opening 108, 308 that is formed in a first surface 104, 304 of the hanging drop device 100, 300 facing upwards (i.e., away from the direction of gravity 125, 325). The microfluidic channel 112, 312 and the opening 108, 308 of the hanging drop device 100, 300 can thus be filled with the basic solution. In some embodiments, the liquid (e.g., the basic solution) in the opening 108, 308 can form a droplet by surface tension. In some embodiments, the liquid can be provided or delivered to the inlet 314 and to the microfluidic chamber 312 using a liquid delivery system such as, for example, a syringe pump, pneumatic regulator, or peristaltic pump. At block 404, a specimen (e g., an egg or embryo) 124, 324 may be loaded into the chamber 110, 310 and the liquid (e.g., the basic solution) in the opening 108, 308. As mentioned above, in some embodiments, the specimen 124, 324 can be injected into a droplet of liquid in the opening 108, 308 using a pipette 360.

[0043] At block 406, the hanging dop device 100, 300 can be flipped to a second position with the opening 108, 308 and the first surface 104, 304 of the hanging drop device 100, 300 facing downwards towards the ground (e.g., the floor) to form the hanging drop that defines a chamber 110, 310 that includes the specimen 124, 324. In the second position, the opening 108, 308 and first surface 104, 304 are facing the direction of gravity 125, 325. Accordingly, the hanging drop (i.e., chamber 110, 310) can be formed due to the balance between surface tension, gravity, and the pressure difference with the microfluidic channel 112, 312 which is connected to the opening 108, 308 and the chamber 110, 310 formed by the hanging drop. The specimen 124, 324 in the chamber 110, 310 can then sink due to gravity and migrate towards a liquid-air interface 111, 311 that defines the hanging drop forming the chamber 110, 310. The specimen 324 can then advantageously be captured (or trapped) by the liquid air interface 311.

[0044] At block 408, liquid including, for example, cryoprotectant, can be loaded in the hanging drop device 100, 300 using the microfluidic channel 112, 312 of the hanging drop device 100, 300 to enable a liquid exchange process with the chamber 110, 310 and around the specimen 124, 324 in the chamber 110, 310. In some embodiments, the liquid can be provided or delivered to the inlet 314 and to the microfluidic chamber 312 using a liquid delivery system such as, for example, a syringe pump, pneumatic regulator, or peristaltic pump. In some embodiments, the liquid-air interface 311 of the chamber 110, 310 (i.e., the hanging drop) can be used to advantageously maintain the specimen 124, 324 in one position (e.g., near or at the center of the chamber 310) during the liquid exchange. In some embodiments, the liquid can pass from an inlet 114, 314 of the hanging drop device 100, 200 to an outlet 116, 316 of the hanging drop device 100, 300 via the microfluidic channel 112, 312. The microfluidic channel 112, 312 can be connected to the chamber 110, 310 and the liquid can pass from the microfluidic chamber 112, 312 into the chamber 110, 310 through an intermediate structure 1 18, 318. During the liquid exchange process, the liquid can also pass out of the chamber 110, 310 to the microfluidic channel 112, 312 through the intermediate structure 118, 318. As discussed above with respect to FIG. 5, the cryoprotectants loaded in the hanging drop device 100, 300 can include, for example, an equilibration solution (ES) and vitrification solution (VS). In some embodiments, each liquid (e.g., BS, ES (or BS/ES mixture), VS) can be loaded into the hanging drop device 100, 300 (and chamber 110, 310) sequentially. In some embodiments, loading of a cryoprotectant using microfluidic channel(s) can be performed using a hanging drop device that advantageously includes a plurality of inlets 114, a plurality of microfluidic channels 114, and a plurality of openings 108 as discussed below with respect to FIGs. 10-16c. As discussed further below with respect to FIGs. 10- 16c, in some embodiments, a hanging drop device which includes a plurality of inlets and a plurality of microfluidic channels can advantageously enable, for example, simultaneous loading of liquids and mixing of liquids in the hanging drop chamber.

[0045] At block 410, the specimen 124, 324 may be retrieved from the chamber 110, 310 of the hanging drop device 100, 300 through the liquid-air interface 111, 311 of the chamber 110, 310. In some embodiments, the liquid-air interface 311 can be used to advantageously maintain the specimen 124, 324 in one position (e.g., near or at the center of the chamber 310) during the specimen retrieval. In some embodiments, a vitrification carrier 126, 326 (e.g., a cryoloop device) can be used to penetrate the liquid-air interface 111, 311 and retrieve the specimen 124, 324. At block 412, after the specimen 124, 324 has been retrieved from the chamber 110, 310 using the vitrification carrier 126, 326, the vitrification carrier 126, 326 can be immersed into a vitrification medium such as, for example, liquid nitrogen, slush nitrogen, or vapor phase liquid nitrogen, to finish the verification process. In some embodiment, the liquid nitrogen may be provided in a tank that may also be used for storage of the specimen 124, 324.

[0046] FIG. 7 is schematic diagram of a hanging drop device for liquid exchange for use in vitrification of a specimen in accordance with an embodiment. The hanging drop device 500 can include a housing 502. In some embodiments, the housing 502 can be in the form of a chip. The housing 502 can include a first surface or wall 504, a second surface or wall 506 opposite the first surface 504, an opening (or open chamber) 508 in the first surface 504, an inlet (or input) 514, an outlet (or output) 516, and a microfluidic channel 512 between the inlet 514 and the outlet 516. While one inlet 514, one microfluidic channel 512, and one outlet 516 are shown in the embodiment illustrated in FIG. 7, some embodiments of a hanging drop device can advantageously include a plurality of inlets 514 and a plurality of microfluidic channels 512 as discussed below with respect to FIGs. 10-16c. The opening 508 in the first surface 504 can be configured to form a hanging drop of a liquid provided through inlet 514 to the microfluidic channel 512 to fill (or seed) the channel 512 and the opening 508 (or open chamber) with the liquid. A hanging drop can form and create a chamber (or confined chamber) 510 defined by a liquid-air interface 511 of the hanging drop when the first surface 504 and the opening 508 are facing towards the floor in the direction of gravity (as indicated by arrow 525). A rim 540 or other barrier may be provided around the opening 208 to provide additional stability and, for example, to help prevent liquid in the opening 508 from spreading. The hanging drop deice 500 can also include a through hole or opening 580 that is formed in the second surface 506 of the housing 502 above and corresponding to the opening 508. The through hole 580 can be configured to allow a specimen 524 to be directly loaded into the chamber 510 (i.e., the hanging drop), for example, using a pipette. Surface tension of the liquid provided to the microfluidic channel 512 to form the chamber 110 (i.e., the hanging drop) can also create a second liquid-air interface 582 (e.g., a convex shaped liquid-air interface) in the through hole 580 of the second surface 506 that can prevent liquid from moving out of the microfluidic channel 512 and the chamber 510. While one opening 508 and one corresponding through hole 580 is shown in the embodiment illustrated in FIG. 7, some embodiments of a hanging drop device can advantageously include a plurality of openings 508 for a plurality of chambers 510 and a plurality of corresponding through holes 580 as discussed below with respect to FIGs. 14-16c. A process and method for forming the chamber 510 (e.g., a handing drop) and loading a specimen 524 into the chamber 510 using the through hole 582 is discussed further below with respect to FIGs. 8 and 9.

[0047] When loaded into the chamber 510 (or hanging drop) using the through hole 580, the specimen 524 (e.g., an egg (or oocyte) or embryo) can sink to the bottom of the chamber 510 due to gravity and can be captured by the liquid-air interface 511 due to the surface energy. In some embodiments, the specimen 524 can then stay at a position at or near a center of the hanging drop (i.e., chamber 510). The chamber 510 formed in opening 508 has a connection to the microfluidic channel 512 in the housing 502, which can facilitate a liquid exchange process, for example, loading of cryoprotectant(s). Liquid can pass through the chamber 510, which is defined by the liquid-air interface 151 of the hanging drop, via the microfluidic channel 512. As mentioned above, the microfluidic channel 512 is coupled to the inlet 514 and the outlet 516. Liquid exchange around the specimen 524 can occur by flowing different liquids (e.g., cryoprotectants) through inlet 514 and microfluidic channel 512 and out through outlet 516, and allowing the liquids to move into the chamber 510 from the microfluidic channel 512 and out of the chamber 510 to the microfluidic channel 512. In some embodiments, the liquid can be provided or delivered to the inlet 514 and to the microfluidic chamber 512 using a liquid delivery system such as, for example, a syringe pump, pneumatic regulator, or peristaltic pump. As discussed further below with respect to FIGs. 8 and 9, a liquid, for example, a cryoprotectant, may be loaded from the inlet 514 (as shown by arrow 520) into the microfluidic channel 512 and enter the chamber (or hanging drop) 510. In some embodiments, the existing liquid in the chamber (or hanging drop) 510 may be withdrawn to the outlet 516 (as shown by arrow 522) simultaneously. The liquid-air interface 511 can advantageously allow accurate manipulation of a specimen 524 (e.g., an egg or embryo) position. The through hole 580 can also provide additional hanging drop stability during the loading of cryoprotectants (or cryoprotective agents (CPAs)). Because a liquid-air interface is located on both sides of the chamber 510 (i.e., liquid-air interface 511 defining the hanging drop and liquidair interface 582 in the through hole 580), the pressure inside the chamber 510 is balanced by itself. As a result, high precise pressure control over the cryoprotectant loading process may not be required and the maximum operating flow rate of the hanging drop device can be increased. [0048] In some embodiments, the specimen 524 can be transported out of the chamber 510 (i.e., a specimen retrieval process) using a vitrification carrier or device (not shown) that is configured to penetrate the liquid-air interface 511 of the chamber 510 formed from the hanging drop and the liquid surrounding the specimen 524. In some embodiments, the vitrification carrier or device (e.g., vitrification carrier 126 shown in FIG. 2a) can be configured to retrieve the specimen 524 and liquid surrounding the specimen 524 without physical contact with the specimen 524. In some embodiments, the vitrification carrier or device may be a cryoloop device that includes, for example, a nylon loop used to suspend a film of the liquid (e.g., a cryoprotectant) containing the specimen 124 and a rod coupled to the nylon loop. As discussed further below with respect to FIGs. 8 and 9, once the specimen 524 has been retrieved from the chamber 510 using the vitrification carrier, the specimen 524 can be moved rapidly into a vitrification medium such as liquid nitrogen, for example, a liquid nitrogen tank (not shown) to complete the freezing process and for storage. In some embodiments, a robotic arm may be connected to the vitrification carrier and used to robotically perform the retravel process for the specimen 524.

[0049] FIG. 8 illustrates a process for operation of the hanging drop device of FIG. 7 in a vitrification process in accordance with an embodiment. As mentioned above, the hanging drop device with a microfluidic channel can be used for liquid exchange (e.g., cryoprotectant loading) with a specimen (e.g., an egg (or oocyte) or embryo) for vitrification. In a first step 690, a first surface 604 of the hanging drop device 600 and an opening 608 in the first surface 604 can be facing downwards towards the ground (or floor) and the direction of gravity (as indicated by arrow 625), and a second surface 606 and a through hole 680 in the second surface 606 of the hanging drop device 600 can be facing upwards away from the direction of gravity 625. A microfluidic channel 612 and the opening 608 of the hanging drop device 600 can be filled with a liquid, for example, a basic solution (BS) to facilitate the formation of a hanging drop in the opening 608, the formation of a liquid-air interface 682 in the through hole 680, and loading of a specimen 624 into the chamber 610. In some embodiments, the basic solution can be a culture medium. The basic solution may be loaded into the microfluidic channel 612 and opening 608 via an inlet 614. In some embodiments, the liquid can be provided or delivered to the inlet 614 and to the microfluidic chamber 612 using a liquid delivery system such as, for example, a syringe pump, pneumatic regulator, or peristaltic pump. A hanging drop, or chamber 610, can be formed from the liquid in the opening 608 due to the balance between surface tension, gravity, and the pressure difference with the microfluidic channel 612. The specimen (e.g., an egg (or oocyte) or embryo) 624 may then be transferred into the chamber 610, for example, in some embodiments a pipette 660 (e.g., the tip of the pipette) may be directly inserted into the chamber 610 (i.e., the hanging drop) via the through hole 680 and the specimen 624 may be released into the chamber 610. The specimen 624 can then settle down to the bottom of the chamber 610 and may be captured by the liquid-air interface 611 of the chamber 610. In some embodiments, the specimen 624 may be captured by the liquid-air interface 611 defining the chamber 610 within seconds. In some embodiments, the liquid-air interface 611 can be used advantageously maintain the specimen 624 in one position (e.g., near or at the center of the chamber 610) during the liquid exchange (this step 692) and specimen retrieval (fourth step 694).

[0050] In a second step 692, the inlet 614, microfluidic channel 612, chamber 610 and outlet 616 can be used for a liquid exchange process. Liquid exchange around the specimen 624, for example, for cryoprotectant loading, can be performed through the microfluidic channel 612 which is connected to the chamber 610. Liquid can be provided to the inlet 614 (as indicated by arrow 620) and pass from the inlet 614 into the microfluidic channel 612 and from the microfluidic chamber 612 into the chamber 610 (i.e., the hanging drop). In some embodiments, the liquid can be provided or delivered to the inlet 614 and to the microfluidic chamber 612 using a liquid delivery system such as, for example, a syringe pump, pneumatic regulator, or peristaltic pump The liquid can also pass out of the chamber 610 to the microfluidic channel 612 and then from the microfluidic channel 612 to the outlet 616 (as indicated by arrow 622). As mentioned, cryoprotectant (or a cryoprotective agent (CPA)) loading can be performed through the microfluidic channel 612. As discussed above with respect to FIG. 5, in some embodiments, two types of cryoprotectant can be loaded during the liquid exchange process (step 692), namely, an equilibration solution (ES) and vitrification solution (VS). In a hanging drop device 600 with one microfluid channel, in some embodiments each liquid (e.g., BS, ES (or BS/ES mixture), VS) can be loaded into the hanging drop device 600 (and chamber 610) sequentially.

[0051] When the cryoprotectant loading is complete, egg retrieval can be performed in a third step 694 using, for example, a vitrification carrier 626. As mentioned above, in some embodiments, the position of the specimen 624 in the chamber 610 can be monitored by one or more cameras (e g., cameras 128, 130 shown in FIGs. 2a and 2b) that are positioned to view the chamber 610 formed by the hanging drop from different sides, for example, the bottom and the side. In addition, a bottom view and side view camera can be used to monitor the motion of a vitrification carrier 626 used to retrieve the specimen 624 from the chamber 610 (i.e., the hanging drop). In some embodiments, the vitrification carrier 626 may be a cryoloop device that includes, for example, a nylon loop used to suspend a film of the liquid (e.g., a cryoprotectant) containing the specimen 624 and a rod coupled to the nylon loop. In some embodiments, when the vitrification carrier 626 interacts with the liquid-air interface 611 of chamber 610, the affinity of the liquid to the vitrification carrier 626 leads to the removal of a small portion of the liquid or solution from the chamber 610 (i.e., the hanging drop). Accordingly, in such embodiments, the specimen 624 (e.g., an egg or embryo) can be carried by the liquid and retained on the vitrification device without touching the vitrification carrier 626. After retrieving the specimen 624 from the chamber in step 694, in some embodiments the vitrification carrier 626 can be immersed into a vitrification medium such as, for example, liquid nitrogen, slush nitrogen, and vapor phase liquid nitrogen, to finish the verification process.

[0052] FIG. 9 illustrates a method for vitrification of a specimen using a hanging drop device for liquid exchange around the specimen in accordance with an embodiment. The process illustrated in FIG. 9 is described below with reference to FIGs. 7 and 8. Although the blocks of the process are illustrated in a particular order, in some embodiments, one or more blocks may be executed in a different order than illustrated in FIG. 9 or may be bypassed. While the process for vitrification of a specimen using a hanging drop device is described below with respect to embodiments of a hanging drop device 500, 600 with one inlet 14, 614, one microfluidic channel 512, 612, one outlet 516, 616, one opening 508, 608 (as shown in FIGs. 7 and 8), it should be understood that in some embodiments the vitrification process illustrated in FIG. 9 can be performed using a hanging drop device that advantageously includes a plurality of inlets 514, 614, a plurality of microfluidic channels 512, 612, and a plurality of openings 508, 608 (or hanging drop chambers) as discussed below with respect to FIGs. 10- 16c.

[0053] At block 702, a liquid, for example, a basic solution, may be provided into a microfluidic channel 512, 612 and opening 508, 608 of a hanging drop device 500, 600, in a position with the opening 508, 608 that is formed in a first surface 504, 604 of the hanging drop device 500, 600 facing downwards toward the ground (e.g., the floor) in the direction of gravity 525, 625. In some embodiments, the liquid can be provided or delivered to the inlet 514, 614 and to the microfluidic chamber 512, 612 using a liquid delivery system such as, for example, a syringe pump, pneumatic regulator, or peristaltic pump. The microfluidic channel 512, 612, the opening 508, 608, and a through hole 580, 680 corresponding to the opening 508, 608 can thus be filled with the basic solution. The through hole 580, 680 can be formed in a second surface 506, 606 of the hanging drop device 500, 600 and be positioned above the opening 508, 608. Filling the microfluidic channel 512, 612 and the opening 508, 608 of the hanging drop device 500, 600 with the liquid, for example, a basic solution (BS) can facilitate the formation of a hanging drop in the opening 508, 608, the formation of a liquid-air interface 582, 682 in the through hole 580, 680, and loading of a specimen 524, 624 into the chamber 520, 610. The hanging drop, or chamber 510, 610, can be formed from the liquid in the opening 508, 608 due to the balance between surface tension, gravity, and the pressure difference with the microfluidic channel 512, 612. Surface tension of the liquid provided to the microfluidic channel 512, 612 to form the chamber 110 (i.e., the hanging drop) can also create the second liquid-air interface 582, 682 (e.g., a convex shaped liquid-air interface) in the through hole 580, 680 in the second surface 506, 606 that can prevent liquid from moving out of the microfluidic channel 512, 612 and the chamber 510, 610. At block 704, a specimen (e.g., an egg (or oocyte) or embryo) 524, 624 may be loaded into the chamber 510, 610 (i.e., the hanging drop) using the through hole 508, 608. For example, in some embodiments, a pipette 660 (e.g., the tip of the pipette) may be directly inserted into the chamber 610 (i.e., the hanging drop) via the through hole 680 and the specimen 624 may be released into the chamber 610. The specimen 624 can then settle down to the bottom of the chamber 610 and may be captured by the liquid-air interface 611 of the chamber 610.

[0054] At block 706, liquid including, for example, cryoprotectant, can be loaded in the hanging drop device 500, 600 using the microfluidic channel 512, 612 of the hanging drop device 500, 600 to enable a liquid exchange process with the chamber 510, 610 and around the specimen 524, 624 in the chamber 510, 610. In some embodiments, the liquid can be provided or delivered to the inlet 514, 614 and to the microfluidic chamber 512, 612 using a liquid delivery system such as, for example, a syringe pump, pneumatic regulator, or peristaltic pump. In some embodiments, the liquid-air interface 511, 611 of the chamber 510, 610 (i.e., the hanging drop) can be used to advantageously maintain the specimen 524, 624 in one position (e.g., near or at the center of the chamber 510, 610) during the liquid exchange. In some embodiments, the liquid can pass from an inlet 514, 614 of the hanging drop device 500, 600 to an outlet 516, 616 of the hanging drop device 500, 600 via the microfluidic channel 512, 612. The microfluidic channel 512, 612 can be connected to the chamber 510, 610 and the liquid can pass from the microfluidic chamber 512, 612 into the chamber 510, 610. During the liquid exchange process, the liquid can also pass out of the chamber 510, 610 to the microfluidic channel 512, 612. As discussed above with respect to FIGs. 5 and 8, the cryoprotectants loaded in the hanging drop device 500, 600 can include, for example, an equilibration solution (ES) and vitrification solution (VS). In some embodiments, each liquid (e.g., BS, ES (or BS/ES mixture), VS) can be loaded into the hanging drop device 500, 600 (and chamber 510, 610) sequentially. In some embodiments, loading of a cryoprotectant using microfluidic channel(s) can be performed using a hanging drop device that advantageously includes a plurality of inlets 514, 614, a plurality of microfluidic channels 512, 612 and a plurality of openings 508, 608 as discussed below with respect to FIGs. 10-16c. As discussed further below with respect to FIGs. 10-16c, in some embodiments, a hanging drop device which includes a plurality of inlets and a plurality of microfluidic channels can advantageously enable, for example, simultaneous loading of liquids and mixing of liquids in the hanging drop chamber.

[0055] At block 708, the specimen 524, 624 may be retrieved from the chamber 510, 610 of the hanging drop device 500, 600 through the liquid-air interface 511, 611 of the chamber 510, 610. In some embodiments, the liquid-air interface 511, 611 can be used to advantageously maintain the specimen 524, 624 in one position (e.g., near or at the center of the chamber 510, 610) during the specimen retrieval. In some embodiments, a vitrification carrier 626 (e g., a cryoloop device) can be used to penetrate the liquid-air interface 511, 611 and retrieve the specimen 524, 624. At block 710, after the specimen 524, 624 has been retrieved from the chamber 510, 610 using the vitrification carrier 626, the vitrification carrier 626 can be immersed into a vitrification medium such as, for example, liquid nitrogen, slush nitrogen, or vapor phase liquid nitrogen, to finish the verification process. In some embodiment, the liquid nitrogen may be provided in a tank that may also be used for storage of the specimen 524, 624.

[0056] As mentioned above, in some embodiments, a hanging drop device may advantageously include a plurality of microfluidic channels. FIG. 10 is schematic diagram of a hanging drop device with a plurality of microfluidic channels for liquid exchange for use in vitrification of a specimen in accordance with an embodiment. The hanging drop device 800 illustrated in FIG. 10 can be formed as a chip constructed using a plurality of layers 802-810. In some embodiments, each layer may be formed from a COC sheet. In FIG. 10, a top view of each layer 802-810 is shown to illustrate the elements in each layer. In the embodiment illustrated in FIG. 10, the hanging drop device 800 includes three inlets 812, 814, 816, one general outlet 818, an opening (or open chamber) 828 used to form a chamber (i.e., a hanging drop), and a plurality of microfluidic channels 830-838. The opening 828 can have a plurality of connections to the plurality of microfluidic channels 830-838, for example, in FIG. 10, the opening 828 has six connections with six microfluidic channels 820-838. A first layer 802 can include the opening 828, a first inlet 812, a second inlet 814, a third inlet 816, and the general outlet 818. A second layer 804 can include the opening 828, the first inlet 812, the second inlet 814, the third inlet 816, and the general outlet 818. In addition, in the second layer a microfluidic channel 830 can be connected to and positioned between the first inlet 812 and the opening 828, a microfluidic channel 832 can be connected to and positioned between the second inlet 814 and the opening 828, and two microfluidic channels 834, 836 can be connected to and positioned between the outlet 818 and the opening 828. A third layer 806 can include the opening 828 and the third inlet 816. A fourth layer 808 can include the opening 828 and the third inlet 816. In addition, in the fourth layer two microfluidic channels 838 can be connected together and each of the two channels 838 can be connected to an positioned between the third inlet 816 and the opening 828. A fifth layer 810 can include the opening 828. When assembled, the various openings 828, inlets 812-816, and outlet 818 in each layer will be aligned. In addition, when assembled, the first layer 802 can be configured to seal the microfluidic channels 830-836 in the second layer 804 and the third layer 806 can be configured to seal the microfluidic channels 838 in the fourth layer. In some embodiments, the opening 828 can be implemented with an intermediate structure such as described above with respect to FIGs. 1-6. In some embodiments, the hanging drop device 800 can include a through hole that can be utilized to load a specimen in a chamber (or hanging drop) formed in the opening 828 as described above with respect to FIGs. 7-9.

[0057] In some embodiments, for a vitrification process various liquids (e.g., cryoprotectants) may be provided to the hanging drop device 800 including a chamber (not shown) formed by a hanging dop in the opening 828 using the first 812, second 814, and third 816 inlets. A chamber (or hanging drop) can be formed in the opening 828 using a liquid, for example, a basic solution, as described above with respect to, for example, FIGs. 5 and 8. Advantageously, in some embodiments, each inlet 812, 814, 816 can be used to provide a different type of liquid, for example, the first inlet 812 may be used to provide a basic solution (BS), the second inlet 814 may be used to provide an equilibration solution (ES) and a third inlet 816 can be used to provide a vitrification solution (VS). Different operation modes and fluid flow for the hanging drop device 800 using the plurality of inlets and the plurality of microfluidic channels are described further below with respect to FIGs. 12 and 13. The outlet 818 can be configured for waste retrieval from the chamber formed in the opening 828 during, for example, a cryoprotectant (or cryoprotective agent (CPA)) loading process. Each different type of liquid (e.g., cryoprotectant) has direct access to the chamber (or handing drop) formed in the opening 828 via a separate inlet 812, 814, 816 and one or more microfluidic channels corresponding to the particular inlet which can result in minimized dead volume and a fast response time. Advantageously, the plurality of inlets and microfluidic channels can enable, for example, mixing of different CPA components directly inside the chamber (or hanging drop) in the opening 828 via the different inlets 812, 814, 816 simultaneously as discussed further below with respect to FIGs. 12 and 13. In some embodiments, the CPA concentration inside the chamber (or handing drop) in opening 828 may be adjusted in real-time without delay time. The hanging drop device 800 with a plurality of inlets 812, 814, 816 and a plurality of microfluidic channels 830-838 can result in an improved mixing process and flow stability.

[0058] FIG. 11 shows a top view of a hanging drop device with a plurality of microfluidic channels for liquid exchange for use in vitrification of a specimen in accordance with an embodiment. Hanging drop device 800 is formed as a chip and can include a first inlet 812, a second inlet 814, a third inlet 816, an outlet 818. Hanging drop device 800 can also include n opening 828 which can be configured to form a chamber (or hanging drop) from a liquid provided to the hanging drop device 800 using one of the inlets 812-816. As discussed above with respect to FIG. 10, each inlet 812, 814, 816 and the outlet 818 can be in fluid communication with the opening 818 via a plurality of microfluidic channels.

[0059] FIG. 12 is a schematic diagram illustrating operation processes of a hanging drop device with a plurality of microfluidic channels for liquid exchange for use in vitrification of a specimen in accordance with an embodiment. FIG. 12 illustrates the flow of liquid into an out of a chamber 840 (or hanging drop) formed in an opening 828 (shown in FIGs. 10 and 11) of a hanging drop device 800 (show in FIGs. 10 and 11) with a plurality of inlets, a plurality of microfluidic channels, and implemented using a plurality of layers. In some embodiments, the chamber 840 may be used for CPA mixing 850. For example, a microfluidic channel 830 can be used to provide a basic solution (BS) to the chamber 840 and a different microfluidic channel 832 may be used to provide an equilibration solution (ES) to the chamber 840 simultaneously with the basic solution in order to mix the BS and ES. Two microfluidic chambers 834, 836 can be used to allow fluid to flow out of the chamber 840 to an outlet (e.g., outlet 818 shown in FIG. 10). For an ED loading process 852, a microfluidic channel 832 specifically used for ES can be used to provide the ES to the chamber 840 and two microfluidic channels 834, 836 can be used to allow the ES to flow out of the chamber 840 to an outlet (e.g., outlet 818 shown in FIG. 10). As discussed above with respect to FIG. 10, in some embodiments, the BS inlet 812 (not shown) and its corresponding microfluidic channel 830, the ES inlet 814 (not shown) and its corresponding microfluidic channel 832, and the outlet 818 (not shown) and its two corresponding microfluidic channels 834, 836 can be implemented on the same layer (e.g., layer 2) of the hanging drop device 800. For a vitrification solution (VS) loading process 854, the VS can be loaded into the chamber 840 using two microfluidic channels 838 and two microfluidic channels 834, 836 can be used to allow the VS to flow out of the chamber 840 to an outlet (e.g., outlet 818 shown in FIG. 10). In some embodiments, the VS inlet 816 (not shown) and its corresponding microfluidic channels 838 can be implemented on the same layer (e.g., layer 4) of the hanging drop device 800 and the outlet 818 (not shown ) and its corresponding microfluidic channels 824, 836 can be implemented on a different layer (e.g., layer 2).

[0060] FIG. 13 is a schematic diagram illustrating fluid flow in a hanging drop chamber for different operation processes in accordance with an embodiment. As discussed above with respect to FIG. 10, an opening 828 in a hanging drop device 800 can be configured to form a chamber (or hanging drop) from a liquid (e.g., a basic solution) provided to the opening 828. FIG. 13 shows cross-sectional view of a chamber (or hanging drop 840 formed in an opening 828 and the various microfluidic channels 830-838 connected to the chamber 840 to illustrate fluid flow for various operation modes. For a CPS mixing mode 860, a liquid, for example, BS may be provided to the chamber 840 via a microfluidic channel 830 simultaneously with, for example, ES provided from a microfluidic channel 832. The BS and ES can circulate and mix in the chamber 840 as indicated by arrows 870, 872, respectively, and exit the chamber 840 via the microfluidic channels 834, 836 connected to an outlet 818 (not shown). For an ES loading mode 862, ES can be provided to the chamber 840 via a microfluidic channel 832. The ES can then circulate in the chamber 840 as indicated by arrows 874, 876 and exit the chamber 840 via the microfluidic channels 834, 836 connected to an outlet 818 (not shown). For a VS loading mode, VS can be provided to the chamber 840 via two microfluidic channels 838. The VS can then circulate in the chamber 840 as indicated by arrows 878, 880 and exit the chamber 840 via the microfluidic channels 834, 836 connected to an outlet 818 (not shown).

[0061] As mentioned above, in some embodiments, a hanging drop device for vitrification of a specimen can include a plurality of hanging drop chambers. FIG. 14 is schematic diagram of a hanging drop device with a plurality of hanging drop chambers for liquid exchange for use in vitrification of multiple specimens in accordance with an embodiment. The hanging drop device 900 illustrated in FIG. 14 can be formed as a chip constructed using a plurality of layers 902-906. In some embodiments, each layer may be formed from a COC sheet. In FIG. 14, a top view of each layer 902-906 is shown to illustrates the elements in each layer. In the embodiment illustrated in FIG. 14, the hanging drop device 900 includes four openings (or open chambers) 908, 910, 912, and 914 and each opening 908, 910, 912, 914 can be used to form a chamber (i.e., a hanging drop). The hanging drop device 900 can also include four inlets 920, 922, 924, 926, one general outlet 930, and a plurality of microfluidic channels 940- 946, 950 and 960-966. Each opening 908, 910, 912, and 914 can have a plurality of connections to at least two of the plurality of microfluidic channels 940-946, 950 and 960- 966. A first layer 902 can include the openings 908-914, the inlets 920-926, and the outlet 930. A second layer 904 can include the openings 908-914, the inlets 920-926, and the outlet 930. In the second layer 904, a microfluidic channel 940 can be connected to and positioned between the first inlet 920 and the opening 908, a microfluidic channel 942 can be connected to and positioned between the second inlet 922 and the opening 910, a microfluidic channel 944 can be connected to and positioned between the third inlet 924 and the opening 912, a microfluidic channel 946 can be connected to and positioned between the fourth inlet 926 and the opening 914, and a microfluidic channel 930 can be connected to all of the openings 908- 914 and the outlet 930. In addition, the openings 908-914 can be connected to each other using microfluidic channels 960-966. A third layer 906 can include the openings 908, 910, 912, and 914. When assembled, the various openings 908-914, inlets, 920-926, and outlet 930 in each layer will be aligned. In addition, when assembled, the first layer 902 can be configured to seal the microfluidic channels 940-946, 950 and 960-966 in the second layer 904. In some embodiments, each opening 908-914 can be implemented with an intermediate structure such as described above with respect to FIGs. 1-6. In some embodiments, the hanging drop device 900 can include a through hole for each opening 908-914 that can be utilized to load a specimen in a chamber (or hanging drop) formed in the corresponding opening as described above with respect to FIGs. 7-9

[0062] In some embodiments, a different specimen (not shown) can be loaded into each chamber formed in each opening 908, 910, 912, 914. Accordingly, in some embodiments, the hanging drop device 900 can be used to perform a vitrification process for more than one specimen, for example, up to four specimens. In some embodiments, a hanging drop device 900 with multiple openings 908-914 that can be used to form multiple chambers (or hanging drops) can enable sequence loading a distribution processes to arrange the loading reagent content (e.g., cryoprotectants) in different chambers corresponding to different openings. Various operation modes of the hanging drop device 900 are discussed below with respect to FIGs. 16a-c.

[0063] FIG. 15 shows a top view of a hanging drop device with a plurality hanging drop chambers for liquid exchange for use in vitrification of a plurality of specimens in accordance with an embodiment. Hanging drop device 900 can be formed as a chip and can include four openings (or open chambers) 908, 910, 912, and 914 and each opening 908, 910, 912, 914 can be used to form a chamber (i.e., a hanging drop). Hanging drop device can also include four inlets 920, 922, 924, 926, one general outlet 930, and a plurality of microfluidic channels as discussed above with respect to FIG. 14.

[0064] FIGs. 16a-c illustrates example operation modes of a hanging drop device with a plurality of hanging drop chambers for liquid exchange for use in vitrification of a plurality of specimens in accordance with an embodiment. In a parallel processing operation mode 1002, CPA can be loaded for each chamber corresponding to each opening 908-914 and the shared outlet 830 can be used for waste collection. In a sequence processing operation mode 1004, reagent (e.g., a cryoprotectant) can be loaded in one chamber corresponding to one of the openings 908-914, and then the flow of the liquid will pass through the chamber and move to a second chamber corresponding to a different opening 90-914. In a distribution processing operation mode, the flow of liquid is distributed from a first chamber corresponding to a first opening (e.g., opening 908) to the rest of the chambers corresponding to each of the remaining openings 910-914.

[0065] The present invention has been described in terms of one or more preferred embodiments, and it should be appreciated that many equivalents, alternatives, variations, and modifications, aside from those expressly states, are possible and within the scope of the invention.

Claims

CLAIMS:

1. A hanging drop device for liquid exchange for use in vitrification of a specimen, the device comprising; a housing; a plurality of microfluidic channels disposed in the housing and configured to facilitate liquid exchange around the specimen; a plurality of inlets disposed in the housing, each inlet connected to and in fluid communication with at least one of the plurality of microfluidic channels; an outlet disposed in the housing and connected to and in fluid communication with at least one of the plurality of microfluidic channels; and an opening disposed in the housing and connected to and in fluid communication with each of the plurality of microfluidic channels, the opening configured to form a hanging drop chamber from a liquid loaded into the opening, the hanging drop chamber configured to receive the specimen.

2. The hanging dop device according to claim 1, wherein the housing comprises a plurality of layers.

3. The hanging drop device according to claim 2, wherein at least one of the plurality of inlets and at least one of the plurality of microfluidic channels are disposed in different layers of the housing.

4. The hanging drop device according to claim 1, wherein the specimen is one of an oocyte or an embryo.

5. The hanging drop device according to claim 1, wherein the opening further comprises an intermediate structure positioned between the opening and the plurality of microfluidic channels.

6. The hanging drop device according to claim 1, wherein the housing further comprises a through hole positioned above the opening and configured to enable loading of the specimen into the hanging drop chamber formed by the opening from a liquid loaded into the opening.,

7. The hanging drop device according to claim 1, wherein each inlet in the plurality of inlets can be configured to receive a different type of liquid.

8. The hanging drop device according to claim 1, further comprising a plurality of openings connected to and in fluid communication with the plurality of microfluidic channels, the plurality of inlets and the outlet.

9. The hanging drop device according to claim 1, wherein at least two inlets of the plurality of inlets and at least two of the plurality of microfluidic channels corresponding to the at least two inlets are configured to receive different liquids simultaneously.

10. A method for vitrification of a specimen using a hanging drop device for liquid exchange around the specimen, the method comprising: receiving a basic solution in at least one microfluidic channel of a hanging drop device to form a hanging drop chamber in an opening of the hanging drop device, the hanging drop chamber defined by a liquid-air interface; receiving the specimen in the hanging drop chamber, wherein the specimen is supported by the liquid-air interface of the hanging drop chamber; and loading, using the at least one microfluidic channel, a plurality of liquids into the hanging drop chamber to facilitate liquid exchange around the specimen, wherein at least one liquid of the plurality of liquids is a cryoprotectant.

11. The method according to claim 10, further comprising: retrieving the specimen from the hanging drop chamber; and immersing the specimen in a vitrification medium.

12. The method according to claim 10, wherein the specimen is one of an oocyte or an embryo.

13. The method according to claim 11, wherein the vitrification medium is one of liquid nitrogen, slush nitrogen, or vapor phase liquid nitrogen.

14. The method according to claim 10, wherein the plurality liquids includes one or more of a basic solution, an equilibration solution, or a vitrification solution,.

15. The method according to claim 10, wherein the at least one microfluidic channel comprises a plurality of microfluidic channels and the hanging drop chamber is connected to an in fluid communication with each microfluidic channel in the plurality of microfluidic channels.

16. The method according to claim 10, wherein loading a plurality of liquids into the hanging drop chamber to facilitate liquid exchange around the specimen comprises mixing two or more of the plurality of liquids in the hanging drop chamber.

17. The method according to claim 16, wherein the two or more of the plurality of liquids includes a basic solution and an equilibration solution.

18. A hanging drop device for liquid exchange for use in vitrification of a specimen, the device comprising; a housing; a plurality of microfluidic channels disposed in the housing and configured to facilitate liquid exchange around the specimen; a plurality of inlets disposed in the housing, each inlet connected to and in fluid communication with at least one of the plurality of microfluidic channels; an outlet disposed in the housing and connected to and in fluid communication with at least one of the plurality of microfluidic channels; and a plurality of openings disposed in the housing, each opening connected to and in fluid communication with at least one other opening in the plurality of openings using at least one of the plurality of microfluidic channels and each opening connected to and in fluid communication with at least one inlet in the plurality of inlets and the outlet, wherein each opening is configured to form a hanging drop chamber from a liquid loaded into the opening, the hanging drop chamber configured to receive one specimen.

19. The hanging dop device according to claim 18, wherein the housing comprises a plurality of layers.

20. The hanging drop device according to claim 18, wherein each inlet in the plurality of inlets can be configured to receive a different type of liquid.