SYSTEM FOR PASSIVE PERMEATION OF A BIOLOGICAL MATERIAL AND METHOD OF USING SAME

A system for passive permeation of a biological material is disclosed. The system may include a main channel extending between an upper portion and a lower portion; a main reservoir connected to the upper portion of the main channel and in fluid communication with the main channel; a bottom reservoir connected to the lower portion of the main channel and in fluid communication with the main channel; at least one secondary channel disposed in at least one position between the upper portion and the lower portion of the main channel such that fluid communication is established in the at least one position between the at least one secondary channel and the main channel; and at least one secondary reservoir respectively connected to an upper portion of each of the at least one secondary channel and in fluid communication with the respective at least one secondary channel.

TECHNICAL FIELD

In order to cryopreserve cells through vitrification, the cells must be permeated into vitrification solutions, consisting on media buffer with a high concentration of cryoprotectant agents that help the cell survive the vitrification process. The current procedure involves a highly skilled user manually aspirating and injecting the cells into various solutions of media, with increasing concentrations of cryoprotectant agents until the final concentration is reached. This procedure requires a significant amount of time by the user. The cells of interest can range in size from 50 to 200 microns, creating the need for the procedure to be performed under a microscope using micromanipulation pipettes operated by hand. Successful permeation of cells in the vitrification solutions requires careful timing and precision handling of micropipettes.

BRIEF SUMMARY OF THE INVENTION

One general aspect of the present disclosure includes a system for passive permeation of a biological material, including a main channel extending between an upper portion and a lower portion; a main reservoir connected to the upper portion of the main channel and in fluid communication with the main channel; a bottom reservoir connected to the lower portion of the main channel and in fluid communication with the main channel; at least one secondary channel disposed in at least one position between the upper portion and the lower portion of the main channel such that fluid communication is established in the at least one position between the at least one secondary channel and the main channel; and at least one secondary reservoir respectively connected to an upper portion of each of the at least one secondary channel and in fluid communication with the respective at least one secondary channel.

Another general aspect of the present disclosure includes a system for cryopreservation of a biological material, including a main channel extending between an upper end and a bottom end. The main channel includes a first section disposed proximate to the upper end of the main channel and a second section disposed proximate to the bottom end of the main channel. The first section is filled with a first liquid having a first cryoprotectant concentration and the second section is filled with a second liquid having a second cryoprotectant concentration greater than the first cryoprotectant concentration. The main channel is configured such that a biological material placed into the main channel through the upper end migrates towards the bottom end by gravity, and when the biological material reaches the bottom end, the biological material is ready for vitrification.

Another general aspect of the present disclosure includes a method of passively permeating a biological material using a system having a main channel in fluid communication with at least one secondary channel, the main channel being connected to a main reservoir at an upper portion of the main channel and being connected to a bottom reservoir at a lower portion of the main channel, and the at least one secondary channel being connected to at least one secondary reservoir. The method includes placing a first predetermined amount of a main liquid in the main reservoir; placing at least one predetermined amount of at least one secondary liquid in the at least one secondary reservoir; allowing the main liquid to flow along the main channel; allowing the at least one secondary liquid to flow along the at least one secondary channel, and then into the main channel and mix with the main liquid to form at least one mixed liquid; and placing a biological material in the main reservoir such that the biological material migrates along the main channel by gravity to travel through the at least one mixed liquid and reaches the bottom reservoir.

Other systems, methods, features and advantages of the invention will be, or will become, apparent to one with skill in the art upon examination of the following figures and detailed description. It is intended that all such additional systems, methods, features, and advantages be within the scope of the invention, and be encompassed by the following claims.

DETAILED DESCRIPTION

Various aspects are described below with reference to the drawings in which like elements generally are identified by like numerals. The relationship and functioning of the various elements of the aspects may better be understood by reference to the following detailed description. However, aspects are not limited to those illustrated in the drawings or explicitly described below. It also should be understood that the drawings are not necessarily to scale, and in certain instances details may have been omitted that are not necessary for an understanding of aspects disclosed herein, such as conventional material, construction, and assembly.

Referring toFIG. 1, a system10for passive permeation of a biological material is shown. The system may include a main channel14extending between an upper portion16and a lower portion18. The main channel14may have a tubular configuration or any configuration suitable for a biological material and liquids to travel through. A main reservoir20may be connected to the upper portion16of the main channel14and in fluid communication with the main channel14. A bottom reservoir22may be connected to the lower portion18of the main channel14and in fluid communication with the main channel14. The system10may be configured such that when the bottom reservoir22is placed on a planar surface58, the main channel14may extend from the lower portion18to the upper portion16at an angle α to the planar surface58, which allows a main liquid62and a biological material12placed in the main reservoir20to migrate by gravity along the main channel14to the bottom reservoir22. In some embodiments, the angle α may be in the range of about 10 degrees to about 60 degrees. The biological material12may be various kinds of biological materials, such as an embryo or an oocyte. The main channel14may be configured to accommodate the various kinds of biological materials. For example, the main channel14may be configured to accommodate a large blastocyst-stage embryo, that is, the main channel14may be larger than about 0.3 mm and generally up to about 3 mm in width. The term “about” is specifically defined herein to include the specific value referenced as well as a dimension that is within 5% of the dimension both above and below the dimension.

The system10may include one or more spaced-apart secondary channels that are connected to the main channel14and in fluid communication with the main channel14. In some embodiments, as shown inFIG. 1, the system10may include a first secondary channel24and a second secondary channel26. The first secondary channel24may extend from a lower portion38to an upper portion36, where the lower portion38may be connected to the main channel14in a first position28such that the fluid communication between the main channel14and the first secondary channel24is established in the first position28. The second secondary channel26may extend from a lower portion42to an upper portion40, where the lower portion42may be connected to the main channel14in a second position30such that the fluid communication between the main channel14and the second secondary channel26is established in the second position30. The first and second secondary channels24,26may be spaced along a length60of the main channel14, where the first secondary channel24is disposed closer to the upper portion16of the main channel14than the second secondary channel26. In some embodiments, the vertical distance between a first outer surface98of the main channel14and the planar surface58may be between about 0.1 inch to about 9 inches. In some embodiments, a second vertical distance96between the first outer surface98of the main channel14at the second position30and the planar surface58may be about 30% to about 75% of a first vertical distance94between the first outer surface98of the main channel14at the first position28and the planar surface58.

First and second secondary reservoirs32,34may be respectively connected to the upper portions36,40of the first and second secondary channels24,26and in fluid communication with the respective first and second secondary channels24,26. The first and second secondary reservoirs32,34may be configured to receive a first secondary liquid64and a second secondary liquid66, respectively, and allow the first and second secondary liquids64,66placed therein to flow, along the respective secondary channels24,26, into the main channel14and mix with the main liquid62flowing from the main reservoir20, such that mixed liquids may be formed along the length60of the main channel14. In some embodiments, after allowing the first secondary and the second secondary liquids64,66to mix with the main liquid62for respective predetermined amount of time, first, second, and third sections50,52,54of the main channel14with the main liquid62, a first mixed liquid68, and a second mixed liquid70may be respectively established, along the length60of the main channel14, between the main reservoir20and the first secondary channel24(e.g., the first position28), between the first and second secondary channels24and26(e.g., between the first and second positions28,30), and between the second secondary channel26(e.g., the second position30) and the bottom reservoir22(e.g., the bottom56of the bottom reservoir22).

In use, a user may place predetermined amount of the main liquid62, the first secondary liquid64, and the second secondary liquid66in the main reservoir20, the first secondary reservoir32, and the second secondary reservoir34respectively. In some embodiments, the total amount of liquids used may be in the range of 1 to 10 ml total, depending on the configuration of the system10and the desired exposure time for the biological material12. The fluid dynamics of the system10will create a flow of the main liquid62that is subsequently fed by the first and second secondary liquids64,66. After a predetermined amount of time, desired first and second mixed liquids68,70may be formed in the second and third sections52,54respectively.

Then, a user may place the biological material12in the main reservoir20, such that as the biological material12migrates down the main channel14by gravity, the biological material12will be sequentially permeated with the main liquid62, the first mixed liquid68, and the second mixed liquid70for respective times of traveling from the main reservoir20to the first position28, from the first position28to the second position30, and from the second position30to the bottom56of the bottom reservoir22. The system10may be configured such that the biological material12migrating along the main channel14sequentially travels through the first, second, and third sections50,52, and54for respective first, second, and third predetermined amount of time. The respective traveling times (i.e., permeation times) of the biological material12through the first, second, and third sections50,52, and54of the main channel14may be varied as needed and/or desired by varying the length60and diameter of each of the first, second, and third sections50,52, and54, the angle α, and the concentrations of the main, first secondary, and second secondary liquids62,64and66, depending on the desired and/or needed permeation process of the biological material12. One of ordinary skill in the art with a thorough review of this disclosure will be able to optimize the length, diameter, and angles of the components of the system with merely routine optimization and without undue experimentation.

In use, a user may observe the bottom reservoir22under a microscope to wait for the biological material12to arrive at the bottom reservoir22. In some embodiments, the system10may be made of clear plastic that does not react to the liquids used with the system10, such that a process of the biological material12migrating from the main reservoir20to the bottom reservoir22is visible to a user. In some embodiments, the system10may be configured such that the biological material12travels slowly enough for the user to follow it using the microscope as it travels down the main channel14. In some embodiments, as shown inFIG. 2, the system10may be incorporated into a device72, where the device72is manufactured of a clear plastic and the dimensions of the device72may be between about 1 and 3 inches in either direction.

The first and second secondary channels24,26and the first and second secondary reservoirs32,34may be configured (e.g., shape, length, dimension) such that the first and second secondary channels24,26are always at higher pressure than the main channel14such that the biological material12migrates along the main channel14towards the bottom56of the bottom reservoir22without migrating towards the first and second secondary channels24,26. In some embodiments, the differences in pressure are achieved via a combination of height and channel length. The longer a channel is, the higher the pressure drop that the flow experiences, due to head loss through the channel (friction). In some embodiments, the main channel14may have a length between about 1 inch and about 12 inches, and the lengths of the first and second secondary channels24,26each may be smaller than half of the length of the main channel14. Using this, the main channel14may be configured such that the first and second secondary channels24,26are at higher pressure than the main channel14so that the first and second secondary liquids64,66merge into the main liquid62. In the meantime, the elevation of the first and second secondary liquids64,66in the first and second secondary reservoirs32,34may be higher than the elevation of the main liquid62in the main reservoir20and the density of the first and second secondary liquids64,66in the first and second secondary reservoirs32,34may be greater than the density of the main liquid62in the main reservoir20. In some embodiments, valves (e.g., one-way check-valve) may be provided at the lower portions38,42of the respective first and second secondary channels24,26to facilitate preventing the main liquid62and the biological material12from migrating towards the first and second secondary channels24,26.

The system10provides the ability to automate the passive permeation process of a biological material by using gravity, thereby reducing the amount of time needed to perform the permeation process, increasing accuracy of the process, and eliminating the need for careful timing and precision handling of micropipettes.

Although a system10with two secondary channels are specifically depicted and described above, it will be appreciated that the number of secondary channels may be varied as desired and/or needed, without departing from the scope of the present invention, to achieve a desired permeation process of the biological material12. For example, a system10having a greater number of secondary channels may allow the biological material12placed in the main reservoir20to be permeated with a greater number of mixed liquids. As described above, the configuration and spacing of the two or more secondary channels may be varied as needed and/or desired depending on the respective desired permeation times in the main liquid62and the two or more mixed liquids.

In some embodiments, the system10may include a port90configured such that the biological material12can be flushed out of the system10. In some embodiments, a second outer surface92of the system10may be removable such that the biological material12disposed in the system10can be manually retrieved by a user.

In some embodiments, as shown inFIG. 3, the system10may include a single main channel14extending between an upper end74to a bottom end76. The single main channel14may include two or more sections with different liquids, such that a biological material12placed in the single main channel14through the upper end74may migrate down the single main channel14by gravity, thereby the biological material12is permeated with each of the different liquids for a predetermined amount of time, depending on the length of each section, to achieve a desired permeation process of the biological material12. In some embodiments, as shown inFIG. 4, the bottom end76of the single main channel14may be connected to a petri dish that sits on a supporting surface82of a microscope84. The angle φ of the single main channel14relative to the supporting surface82may be configured (up to fully vertical) such that desired traveling/permeation time of the biological material12through the single main channel14may be achieved.

In some embodiments, the system10may be used with cryoprotectant solutions for cryopreservation of a biological material such that the biological material is ready for vitrification. While a system10for passive permeation of an embryo for cryopreservation of the embryo is specifically described herein, the system10may be successfully implemented for use with other types of liquids and/or other types of biological materials (e.g., oocytes) for other medical and/or experimental uses. For the sake of brevity, a system disclosed herein is described and depicted as a system for cryopreservation of an embryo, one of ordinary skill in the art, with a thorough review of the subject specification and figures, would readily comprehend how the system may be implemented for convenient passive permeation of other types of biological materials with the same or other types of liquids for the same or other medical and/or experimental uses, and would comprehend which other types of biological materials, liquids, and uses might be suitable without undue experimentation.

When the system10with first and second secondary channels24and26is used for cryopreservation of an embryo12, the system10may be provided with cryoprotectant solutions with different concentrations. In some embodiments, for example, cryoprotectant solutions with increasing cryoprotectant concentrations may be respectively placed in the main reservoir20, the first secondary reservoir32, and the second secondary reservoir34. After allowing the first and the second secondary liquids64,66to mix with the main liquid62for respective predetermined amount of time, first, second, and third sections50,52, and54of the main channel14with increasing cryoprotectant concentrations may be respectively established, along the length60of the main channel14, between the main reservoir20and the first secondary channel24, between the first and second secondary channels24and26, and between the second secondary channel26and the bottom reservoir22.

Then, a user may place an embryo12in the main reservoir20, and the embryo12will migrate down the main channel14by gravity to be permeated with the cryoprotectant solutions with increasing concentrations. The system10may be configured such that the embryo12migrating along the main channel14sequentially travels through the first, second, and third sections50,52, and54for respective first, second, and third predetermined amount of time. The first, second, and third predetermined amount of time may be selected such that when the embryo12migrating from the main reservoir20reaches the bottom reservoir22, the embryo12is ready for vitrification. In some embodiments, the system10is configured such that the embryo12may spend no more than 15 minutes in the system10. For example, the system10may be configured such that the respective traveling time of the embryo12through each of the first, second, and third sections50,52, and54may range from 30 seconds to 5 minutes. In some other embodiments, the system10may be configured such that the respective traveling time of the embryo12through each of the first, second, and third sections50,52, and54may range from 30 seconds to 2 minutes. For example, the system10may be configured such that the embryo12may travel in the first section50for about 1 minute, and then travel in the second section52for about 2 minutes, and then travel in the third section54for about 20 seconds to about 30 seconds. When the embryo12reaches the bottom56of the bottom reservoir22, the user may confirm the presence of the embryo12using a microscope. Then the user may extract the embryo12from the bottom56of the bottom reservoir22and places the embryo12in a device for vitrification.

As discussed above, each of the second and third sections52and54may have gradually increasing cryoprotectant concentration, such that the embryo12migrating down the main channel14may be permeated with gradually increasing cryoprotectant concentrations. The increasing pattern of the cryoprotectant concentrations in the second and third sections52and54may be varied as desired and/or needed by varying the configuration of the system10(e.g., the length60of main channel14, the location of the first and second positions28and30, the height of first and second secondary reservoirs32and34, the diameter of the main channel14and the first and second secondary channels24and26, and the cryoprotectant concentration of the first and second secondary liquids64and66) such that desired and/or needed mixing rates of the main liquid62and the first and second secondary liquids64and66may be achieved. In some embodiments, for example, the system10is configured such that the achieved greatest cryoprotectant concentrations in the first, second, and third sections50,52, and54of the main channel14may be 0%, 17%, and 55% by volume respectively.

The increasing pattern of the cryoprotectant concentrations in the second and third sections52and54may determine how gradually the embryo12experiences the changes in cryoprotectant concentration. In some embodiments, as shown inFIG. 5, when the first and second secondary liquids64,66mix with the main liquid62fast, the embryo12may experience two fast increases in cryoprotectant concentration (e.g., as shown as the curve78). When first and second secondary liquids64,66mix with the main liquid62slowly, the embryo12may experience two gradual increases in cryoprotectant concentration (e.g., as shown as the curve80).

In some embodiments, after desired cryoprotectant concentrations are achieved in the first, second, and third sections50,52, and54of the main channel14, respectively, a system for detaching the main channel14from the first and second secondary channels24,26may be used, such that a single main channel14with desired concentration gradient already present may be formed (e.g., as shown inFIG. 3), in which the embryo12may travel down gradually increasing cryoprotectant concentrations. After the embryo12reaches the bottom end76of the single main channel14, the embryo12is ready for vitrification, and the user may also use this single main channel14for vitrification by plunging it into a vitrification solution, such as liquid nitrogen.

While various embodiments of the present disclosure have been described, the present disclosure is not to be restricted except in light of the attached claims and their equivalents. One skilled in the relevant art will recognize that numerous variations and modifications may be made to the embodiments described above without departing from the scope of the present invention, as defined by the appended claims. Moreover, the advantages described herein are not necessarily the only advantages of the present disclosure and it is not necessarily expected that every embodiment of the present disclosure will achieve all of the advantages described.