Microfluidic device for cryoprotectant agent loading of oocytes or fertilized species

Abstract

A microfluidic device that includes a trap configured to retain an oocyte or fertilized species, a plurality of microchannels in fluid communication with the trap, and a plurality of valves, where each valve is configured to control fluid flow through a corresponding set of one or more of the plurality of microchannels. The microfluidic device further includes a plurality of fluidic timer circuits each configured to produce a corresponding first time-dependent fluid pressure signal, and a fluidic logic gate circuit configured to receive the first time-dependent pressure signals and to produce a plurality of second time-dependent fluid pressure signals, where each second time-dependent fluid pressure signal controls operation of a corresponding one of the valves.

Description

[0001] Attorney Docket No. 29539-0851 WO1 / MGH2025-035

[0002] MICROFLUIDIC DEVICE FOR CRYOPROTECTANT

[0003] AGENT LOADING OF OOCYTES OR FERTILIZED SPECIES

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS

[0005] This application claims the benefit of U.S. Provisional Application No. 63 / 712,564, filed on October 28, 2024, the disclosure of which is incorporated herein by reference in its entirety.

[0006] BACKGROUND

[0007] Approximately 1 in 6 adults worldwide experience infertility. Assisted reproductive technologies, such as in vitro fertilization (IVF) have revolutionized the landscape of reproductive medicine. The typical IVF process involves ovarian stimulation, oocyte retrieval, fertilization through intracytoplasmic sperm injection (ICSI), embryo culture, and finally embryo transfer into the uterus. Oocyte cryopreservation involves storing oocytes in a suspended state at cryogenic temperatures (e.g., in liquid nitrogen at -196 °C). As multiple oocytes can be retrieved in a single cycle, oocyte cryopreservation has been established as a component for infertility treatment. Furthermore, it enables donor oocyte banking and fertility preservation for women who are at risk of losing their ovarian functions due to cancer treatments and / or plan to delay pregnancy.

[0008] Cryopreservation by vitrification involves cooling of a liquid substance to an amorphous glassy state without the destructive formation of ice crystals. To achieve successful vitrification, fast cooling rates and loading of high concentration glass-forming cryoprotectant agents (CPAs) into cytoplasm are generally used. The CPA loading protocol is relevant to the outcome of cryopreservation. During CPA loading, the oocytes first shrink in volume due to high water permeability upon initial exposure to CPA solution of high osmolarity, then swell back as CPA and water slowly re-enter the oocyte.

[0009] To mitigate excess cell shrinkage caused by osmotic stress, multistep CPA loading protocols can be performed to allow the cell to shrink in stages. For instance, oocytes are manually transferred between several CPA droplets of increasing concentrations. After reaching the equilibrium solution (ES), the oocytes are transferred to a droplet of vitrification solution for dehydration over a brief period, then rapidly plunged into liquid nitrogen for cooling. Attorney Docket No. 29539-0851 WO1 / MGH2025-035

[0010] SUMMARY

[0011] The present disclosure relates to a microfluidic device and a method of using the same for cryogenic fertility applications, such as for autonomous CPA loading of an oocyte or for autonomous cry opreservation of a fertilized species (e.g., a fertilized egg, embryo, or blastocyst). These approaches provide a simple, low-cost, and user-friendly microfluidic device configured to automate complex multistep cryopreservation processes, such as the process of oocyte CPA loading in order to deliver cry opreservation ready oocytes or the processes for cryopreservation of a fertilized species (e.g., a fertilized egg, embryo, or blastocyst). The provided microfluidic device and method achieve autonomous, one-touch preparation of vitrification -ready oocytes, enabling achievement of IVF-on-a-chip to enhance the accessibility and affordability of IVF.

[0012] In an aspect, a microfluidic device includes a trap configured to retain an oocyte or fertilized species; a plurality of microchannels in fluid communication with the trap; a plurality of valves, wherein each valve is configured to control fluid flow through a corresponding set of one or more of the plurality of microchannels; a plurality of fluidic timer circuits each configured to produce a corresponding first time-dependent fluid pressure signal; and a fluidic logic gate circuit configured to receive the first time-dependent pressure signals and to produce a plurality of second time-dependent fluid pressure signals, wherein each second timedependent fluid pressure signal controls operation of a corresponding one of the valves.

[0013] Embodiments can include one or any combination of two or more of the following features.

[0014] The plurality of microfluidic channels include an inlet microchannel configured to receive an oocyte or fertilized species; an media channel group configured to receive an equilibrium solution; a vitrification microchannel configured to receive a vitrification solution and configured for output of the oocyte or fertilized species; and a waste outlet microchannel.

[0015] The microfluidic device includes a serpentine microchannel positioned between the trap and the inlet channel, wherein the serpentine microchannel includes at least two turns. In some cases, The microfluidic device includes one or more siphoning microchannels fluidically connecting the serpentine microchannel to the waste outlet microchannel.

[0016] A width of the vitrification microchannel is between about 100 pm and about 500 pm, e.g., between about 150 pm and about 200 pm.

[0017] A width of the waste outlet microchannel is between about 100 pm and about 500 pm, e.g., between about 100 pm and about 150 pm. Attorney Docket No. 29539-0851 WO1 / MGH2025-035

[0018] The media channel group includes a media inlet microchannel; an equilibrium solution inlet microchannel; and a serpentine micromixer microchannel in fluid communication with the media inlet microchannel and the equilibrium solution inlet microchannel and disposed between the equilibrium solution inlet microchannel and the trap and between the media inlet microchannel and the trap. In some cases, the serpentine micromixer microchannel includes between 10 to 200 turns, e.g., between 40 to 80 turns.

[0019] A channel depth of the plurality of microchannels is between 100 pm and 400 pm, e.g., between 100 pm and 150 pm.

[0020] The trap includes a container region having a width of between 100 pm and 300 pm and a length of 80 pm to 300 pm; and a neck region having a width of between 5 pm and 30 pm. In some cases, the container region has a width of between 100 pm and 150 pm and a length of 80 pm to 120 pm.

[0021] Each fluidic timer circuit is a microfluidic circuit including a fluidic transistor; and a low-pass filter circuit, wherein an output pressure of the low-pass filter circuit is a gate signal for the fluidic transistor. In some cases, an output channel from each fluidic transistor is connected to an input of the fluidic logic gate circuit. In some cases, the low-pass filter circuit includes a fluidic resistor; and a fluidic capacitor, wherein a resistance of the fluidic resistor and a capacitance of the fluidic capacitor define a time constant of the low-pass filter circuit. In some cases, the fluidic capacitor includes a rigid container containing a gas with an adjustable gas volume, wherein the capacitance of the fluidic capacitor is based on the volume of gas in the rigid container. In some cases, the fluidic resistor includes a serpentine channel, wherein the resistance of the fluidic resistor is based on a length of the serpentine channel. In some cases, each fluidic timer circuit includes a first microfluidic channel and constituting at least a portion of the low-pass filter circuit; a second microfluidic channel crossing the first microfluidic channel at an intersection; and a deformable membrane separating the first microfluidic channel and the second microfluidic channel at the intersection, wherein the fluidic transistor is defined at the intersection. In some cases, the deformable membrane includes a PDMS membrane. In some cases, the first microfluidic channel is defined in a first plane and the second microfluidic channel is defined in a second plane.

[0022] The fluidic logic gate circuit include two NOT gates and two NAND gates.

[0023] The plurality of second time-dependent fluid pressure signals are configured to sequentially: open a first valve of the plurality of valves to introduce the oocyte or fertilized species into the microfluidic device through an inlet microchannel, wherein the introduced oocyte or fertilized species is retained in the trap; close the first valve and open a second valve Attorney Docket No. 29539-0851 WO1 / MGH2025-035 of the plurality of valves to flow an equilibrium solution through a media channel group, wherein the equilibrium solution is flowed past the oocyte or fertilized species retained in the trap; and close the second valve and open a third valve of the plurality of valves to flow a vitrification solution through a vitrification microchannel and to remove the oocyte or fertilized species from the microfluidic device through the vitrification microchannel.

[0024] The plurality of second time-dependent fluid pressure signals are configured to close a waste outlet valve concurrently with opening the third valve.

[0025] The microfluidic device includes a first microfluidic chip, wherein the trap and the plurality of microchannels are defined in the first microfluidic chip; a second microfluidic chip, wherein the plurality of fluidic timer circuits and the fluidic logic gate circuit are defined in the second microfluidic chip; and fluid flow passages connecting the first microfluidic chip and the second microfluidic chip.

[0026] In an aspect, combinable with the prior aspect, a method includes producing, by each of a plurality of fluidic timer circuits of a microfluidic device, a respective first time-dependent fluid pressure signals; generating, by a fluidic logic gate circuit of a microfluidic device, a plurality of second time-dependent fluid pressure signals responsive to receiving the first timedependent fluid pressure signals as inputs; and by each second time-dependent fluid pressure signal, controlling operation of a corresponding valve of a plurality of valves of the microfluidic device, wherein each valve is configured to control fluid flow through a corresponding one of a plurality of microchannels of the microfluidic device, and wherein the plurality of microchannels are in fluid communication with an trap of the microfluidic device that is configured to retain an oocyte or fertilized species.

[0027] Embodiments can include one or any combination of two or more of the following features.

[0028] Controlling operation of the valves of the microfluidic device includes controlling an opening and closing sequence of the valves.

[0029] Controlling operation of the valves of the microfluidic device includes controlling a timing of opening and closing of each of the valves.

[0030] Controlling operation of the valves of the microfluidic device includes sequentially: opening a first valve to introduce an oocyte or fertilized species into the microfluidic device through an inlet microchannel, wherein the introduced oocyte or fertilized species is retained in the trap; closing the first valve and opening a second valve to flow an equilibrium solution through a media channel group, wherein the equilibrium solution is flowed past the oocyte or fertilized species retained in the trap; and closing the second valve and opening a third valve Attorney Docket No. 29539-0851 WO1 / MGH2025-035 to flow a vitrification solution through a vitrification microchannel to remove the oocyte or fertilized species from the microfluidic device through the vitrification microchannel. In some cases, controlling operation of the valves of the microfluidic device includes closing a waste outlet valve concurrently with opening the third valve. In some cases, the method includes, when the first valve is opened, flowing a fluid containing the oocyte or fertilized species through a serpentine microchannel connecting the inlet microchannel and the trap. In some cases, the method includes flowing at least some of the fluid from the serpentine microchannel to a waste outlet microchannel. In some cases, the method includes closing the first valve and opening the second valve about 200 seconds after opening the first valve; and closing the second valve and opening the third valve about 900 seconds after opening the second valve. In some cases, the method includes, when the first valve is closed and the second valve is open, flowing a fluid through the media channel group, including: flowing media through a media inlet microchannel; flowing equilibrium solution through an equilibrium solution inlet microchannel; and mixing the media and the equilibrium solution in a serpentine micromixer microchannel in fluid communication with the media inlet microchannel and the equilibrium solution inlet microchannel and disposed between the equilibrium solution inlet microchannel and the trap and between the media inlet microchannel and the trap. In some cases, the method includes, by mixing the media and the equilibrium solution in the serpentine micromixer microchannel, gradually increasing a concentration of the equilibrium solution in fluid that flows past the trap.

[0031] Controlling operation of the valves occurs without external electronic control.

[0032] Producing a time-dependent fluid pressure signal by a fluidic timer circuit includes gating a fluidic transistor with an output pressure of a low-pass filter circuit. In some cases, an output channel from each fluidic transistor is connected to an input of the fluidic logic gate circuit. In some cases, the method includes operating the low-pass filter circuit, including supplying the output pressure of the low-pass filter circuit for a time defined by a resistance of a fluidic resistor of the low-pass filter circuit and a capacitance of a fluidic capacitor of the low-pass filter circuit.

[0033] The fluidic logic gate circuit includes two NOT gates and two NAND gates configured to output the plurality of second time-dependent fluid pressure signals.

[0034] The microfluidic device further includes a serpentine channel positioned between the trap and the inlet, wherein the serpentine channel includes at least two turns. In some cases, the serpentine channel includes a plurality of siphoning channels that allows a portion of fluid Attorney Docket No. 29539-0851 WO1 / MGH2025-035 flow in the serpentine channel to pass to the waste outlet, wherein the plurality of siphoning channels are configured to reduce flow velocity through the trap.

[0035] The microfluidic device further includes: a media inlet in fluid communication with the trap; and a serpentine micromixer in fluid communication with the media inlet and the equilibrium solution inlet, wherein the serpentine micromixer is positioned between the equilibrium solution inlet and the trap and between the media inlet and the trap, and wherein the serpentine micromixer is configured to mix an equilibrium solution and a media solution. In some cases, the serpentine micromixer includes from 10 to 200 turns, e.g., from 40 to 80 turns, to achieve mixing of the equilibrium solution and the media solution.

[0036] The techniques described here can have one or more of the following advantages.

[0037] The oocyte preparation techniques are robust, low cost, and user-friendly and provide a fully automated solution to complex multi-step oocyte CPA loading processes. Automating oocyte CPA loading improves standardization, minimizes variability and reduces cost, enhancing accessibility of IVF treatment by enabling oocyte vitrification in satellite clinics and / or remote settings, rather than solely in embryology labs. For instance, this can allow safe transport of oocytes to central embryology labs for fertilization or to long-term storage facilities for preservation. In addition, automation improves reliability and consistency, e.g., as compared to manual processing methods.

[0038] The approaches to oocyte preparation are performed using fluidic timing circuits that rely on constant pressure supplies, and without electronics, external pressure sensors, syringe pumps, optical systems, or other complex and expensive external components. Pairing the transistor’s ability to amplify pressure and flowrate signals with internal feedback systems allows for stable devices with very simple inputs. For instance, there is no need to drive each valve individually or have an external port for each input and output need. The size and complexity of control systems are decreased as optical, computer vision systems and sensors are replaced with on-chip fluidic feedback systems. Accordingly, this technology can benefit a wide range of applications that demand precise deterministic control over fluids and biologies.

[0039] The approaches described here provided an automated approach to gradually increasing CPA loading of the oocyte, which can help avoid high compressive strain rates that can occur in conventional step loading, thus avoiding membrane damage and compromised developmental competence.

[0040] The approaches described here can be multiplexed to handle more oocytes, thus further improving efficiency with scalability. For instance, the timing circuitry can be scaled to Attorney Docket No. 29539-0851 WO1 / MGH2025-035 generate copious functional digital pressure outputs with additional fluidic computing components.

[0041] DESCRIPTION OF THE DRAWINGS

[0042] The following drawings illustrate certain embodiments of the features and advantages of this disclosure. These embodiments are not intended to limit the scope of the appended claims in any manner. Like reference symbols in the drawings indicate like elements.

[0043] FIG. l is a schematic illustration of a microfluidic device.

[0044] FIG. 2A is a schematic illustration of a portion of an cryopreservation module.

[0045] FIG. 2B is a table of phase of operation of a microfluidic device.

[0046] FIG. 3 is a schematic illustration of an cryopreservation module.

[0047] FIG. 4Ais an electrical schematic diagram and a physical schematic diagram of a timing circuit of a microfluidic transistor module.

[0048] FIG. 4B is a graph illustrating pressure measurement of Pcas compared with models that employ either constant or varying capacitance.

[0049] FIG. 4C is a schematic illustration of a fluidic capacitor and a graph illustrating fluidic capacitance that varies with the initial gas volume in a fluidic capacitor.

[0050] FIG. 4D is a graph that illustrates the relationship between duration of the timer of a timing circuit and the gas volume in a fluidic capacitor.

[0051] FIG. 5 A is a truth table for the pressure states of the logic gate inputs (Pl, P2, and P3) and outputs (A, B, C, D) of a logic gate circuit at different timepoints.

[0052] FIG. 5B is a schematic diagram of a logic gate circuit.

[0053] FIG. 5C is an electrical schematic diagram of a microfluidic transistor module having three timer circuits and a logic gate circuit.

[0054] FIG. 5D is a physical schematic diagram of a microfluidic transistor module.

[0055] FIG. 5E is a photograph of an example 4-layer PDMS microfluidic transistor module filled with green dye.

[0056] FIG. 5F is a graph illustrating pressure outputs of a microfluidic transistor module.

[0057] FIG. 6A illustrates a mouse oocyte is held in the trap.

[0058] FIG. 6B is a schematic diagram of an RC charging circuit for mixing of ES and culture media.

[0059] FIG. 6C is a fluorescent image of a serpentine mixer showing uniform mixing of ES and media.

[0060] FIG. 6(D-G) are graphs illustrating measured flow rates at various inlets and outlets. Attorney Docket No. 29539-0851 WO1 / MGH2025-035

[0061] FIG. 6H is a graph illustrating normalized volume of oocytes when exposed to step loading vs. linear loading of 7.5% EG + 7.5% DMSO.

[0062] FIG. 61 are images of the mouse oocytes showing initial volume and minimal volumes under step and linear loading conditions.

[0063] FIG. 7 is a flow chart.

[0064] FIG. 8 is a schematic illustration of dimensions of a cryopreservation module.

[0065] FIG. 9 is a schematic illustration of simulated streamlines around a trap indicating that the majority of flow passes through the trap.

[0066] FIG. 10 is an image illustrating deformation of an oocyte in a trap without the presence of a siphoning channel.

[0067] FIG. 11 is a simulated velocity field of a portion of a cry opreservation module with a parallel siphoning channel to slow down the oocyte.

[0068] FIG. 12 is a fluorescent image showing that the equilibrium solution flows over the trap and is not blocked by the trapped oocyte.

[0069] FIG. 13 are images of two oocytes (top and bottom row) during step loading of ES. Each column refers to the oocyte before ES loading, the oocyte with minimal volume and the oocyte with blebbing.

[0070] FIG. 14 is a graph illustrating normalized volume (% of initial volume) of the oocytes during different ES loading protocols.

[0071] FIG. 15 is an illustration of flow control in the oocyte chip through Quake-style valve using the output pressures from the microfluidic transistor chip.

[0072] Like reference numbers and designations in the various drawings indicate like elements.

[0073] DETAILED DESCRIPTION

[0074] The present disclosure relates to a microfluidic device and a method of using the same for cryogenic fertility applications, such as for autonomous CPA loading of an oocyte or for autonomous cry opreservation of a fertilized species (e.g., a fertilized egg, embryo, or blastocyst). These approaches provide a simple, low-cost, and user-friendly microfluidic device configured to automate complex multistep cryopreservation processes, such as the process of oocyte CPA loading in order to deliver cry opreservation ready oocytes or the processes for cryopreservation of a fertilized species (e.g., a fertilized egg, embryo, or blastocyst). The provided microfluidic device and method achieve autonomous, one-touch Attorney Docket No. 29539-0851 WO1 / MGH2025-035 preparation of vitrification-ready oocytes, enabling achievement of IVF-on-a-chip to enhance the accessibility and affordability of IVF.

[0075] The microfluidic device includes a microfluidic transistor module having fluidic timer circuits and fluidic logic gate circuits, which generate autonomously timed pressure outputs that control deformable valves in an oocyte module, thereby regulating fluid flow over the oocyte. Using constant pressure supplies (e.g., without external electronic control or syringe pumps), these approaches achieve autonomous oocyte trapping, gradual loading of the oocyte with equilibrium solution in increasing concentration, dehydration with vitrification solution, and oocyte extraction from the microfluidic device. Specifically, microfluidic transistor technology was used to create fluid timers with long durations (> 15 minutes) and to generate time-dependent digital pressure outputs with fluidic logic gates. These digital pressure outputs control the opening and closing of embedded valves, therefore allowing precise regulation of flow conditions over the oocyte. In addition, microfluidic logic was used to produce linear equilibrium solution concentration gradients over a desired time frame using the fluidic capacitor. Once an oocyte is loaded into the device, the sequential delivery of different solutions at designated timepoints occurs automatically, without human intervention, bulky syringe pumps or electronics. Following oocyte preparation, the CPA-loaded oocyte can be extracted from the device for cryopreservation. These approaches enable automated oocyte processing while limiting exposure time of the oocyte to the vitrification solution to reduce toxicity, and enable ready extraction of the oocyte from the device.

[0076] FIG. 1 is a schematic illustration of an example microfluidic device 100 that includes an cryopreservation module 102, deformable valves 104-110, and a microfluidic transistor module 115. In some examples, the cry opreservation module 102 and the microfluidic module 115 are two distinct microfluidic chips connected by fluid flow pathways such as tubing. In some examples, the cry opreservation module 102 and the microfluidic transistor module 115 are formed on a single microfluidic chip.

[0077] Referring to FIG. 1, the cryopreservation module 102 includes an trap 114 configured to retain an oocyte 116 or a fertilized species such as a fertilized egg, embryo, or blastocyst. An oocyte is an immature female gamete derived from an oogonium through the process of oogenesis and can be collected from an ovary before fertilization. An oocyte is a haploid cell that, after completing meiosis and being fertilized by a sperm cell, becomes a zygote. When the microfluidic device 100 is used for cry opreservation of a fertilized species (e.g., a fertilized egg, embryo, or blastocyst), rather than for CPAloading of an oocyte, the trap 114 is configured (e.g., sized) to retain a fertilized egg, embryo, or blastocyst. In some examples, the size of the Attorney Docket No. 29539-0851 WO1 / MGH2025-035 trap 114 and the channels of the cryopreservation module 102 is the same regardless of the intended use of the oocyte module for oocyte CPA loading or for fertilized egg or embryo cry opreservation, e.g., because at early stages after fertilization, the size of a fertilized egg or embryo is generally consistent with the size of an oocyte. In some examples, e.g., for use with cryopreservation of blastocysts, the trap and channels of the oocyte module are larger. Description herein of the oocyte module 102 is generally provided in the context of CPA loading of oocytes, but a similar structure and operation is applicable to cryopreservation of fertilized species (e.g., fertilized eggs, embryos, or blastocysts).

[0078] The cryopreservation module 102 includes multiple fluid inlet microchannels 118-122 (sometimes referred to as fluid inlets) and fluid outlet microchannels 124-126 (sometimes referred to as fluid outlets) in fluid communication with the trap 114. Microchannels are channels having at least one dimension that is less than 1 mm. A channel is a gap through which fluid may flow, such as a capillary, conduit, or chamber. The fluid inlets 118-122 include an inlet 118 for receiving the oocyte or fertilized species, a media solution inlet group 120 for receipt of equilibrium solution and culture media, and a vitrification solution inlet 122 for receipt of vitrification solution. The fluid outlets 124-126 include a waste outlet 126 and an oocyte collection outlet 124 for receiving the expelled, CPA-loaded oocyte or fertilized species. Fluid flow through the fluid inlets and outlets 118-126 is controlled by the deformable valves 104-110.

[0079] The operation of the valves 104-110 is controlled by the microfluidic transistor module 115, which acts as a fluidic controller. Specifically, the microfluidic transistor module 115 generates four time-dependent pressure signals from constant pressure supplies. The microfluidic transistor chip contains three fluidic timers 138 with preset durations, e.g., durations of 200, 1100 and 1175 seconds. The output pressure of these timers is fed to a logic circuit 140 composed of four fluidic logic gates to produce digital pressure signals 141 that alternate between high and low pressure at designated time points. These digital pressure outputs are employed to open and close the valves 104-110, thereby managing the flow of various solutions in the oocyte chip.

[0080] In some examples, the deformable valves 104-110 are Quake-style valves that include a deformable component (e.g., silicone membrane) that is sandwiched between two layers of the microfluidic device 100. FIG. 15 illustrates a non-limiting example of a Quake-style valve. Upon application of an output pressure provided by the microfluidic transistor module 115, the elastic member deform to allow or prohibit fluid flow within the channel(s) controlled by that valve. Attorney Docket No. 29539-0851 WO1 / MGH2025-035

[0081] Referring to FIGS. 1-3, for CPA loading of an oocyte, the inlet 118 receive an oocyte solution from an oocyte source 128. The oocyte solution can include a single oocyte or multiple oocytes in a medium such as water, culture medium (e.g., KSOMaa Evolve, HEPES medium, Human Tubal Fluid, MOPS medium, tissue culture medium 199, serum substitute supplement, human serum albumin, fetal bovine serum), carbon dioxide, or combinations thereof. For cry opreservation of a fertilized egg, embryo, or blastocyst, the inlet 118 is configured to receive a solution including a fertilized species (e.g., a fertilized egg, embryo, or blastocyst). The received oocyte (or fertilized egg, embryo, or blastocyst) flows into the trap 114, which is a U- shaped structure that is sized to receive and retain the oocyte, fertilized egg, embryo, or blastocyst. An inlet valve 104 is positioned between the inlet 118 and the trap 114 and operates to regulate fluid flow between the inlet 118 and the trap 114 in the cryopreservation module 102.

[0082] The trap 114 is in fluid communication with the waste outlet 126 through a neck microchannel 117. Two elements being in fluid communication generally indicates that a fluid may pass substantially unrestricted between the two elements. During use, when the oocyte solution (or solution including a fertilized egg, embryo, or blastocyst) is flowed into the cry opreservation module 102 through the inlet 118, a portion of the solution flows from the inlet 118 to the trap 114 and through the neck microchannel 117 to the waste outlet 126. Fluid flow through the waste outlet 126 is controlled by a waste outlet valve 110. Because the neck microchannel 117 is dimensioned to offer a lower fluidic resistance than alternative paths in the cry opreservation module 102, the oocyte 116 (or fertilized egg, embryo, or blastocyst) becomes trapped within the trap 114.

[0083] The media solution inlet group 120 includes an equilibrium solution inlet 120a configured to receive equilibrium solution from an equilibrium solution source 130. In some embodiments, the equilibrium solution includes a CPAs. Suitable CPAs can include, but are not limited to, ethylene glycol and dimethyl sulfoxide. For example, the equilibrium solution can include from 1% to 15% (v / v) of a first CPA (e.g., ethylene glycol) and from 1% to 15% (v / v) of a second CPA (e.g., dimethyl sulfoxide). Exposing the oocyte 116 to an equilibrium solution allows the oocyte to equilibrate (partially dehydrate and permit permeating CPAs to enter) before it is exposed to higher concentrations of a vitrification solution, minimizing osmotic shock and toxicity.

[0084] The media solution inlet group 120 also includes a media inlet 120b configured to receive a culture medium from a culture medium source 132. Suitable culture mediums can include, but are not limited to, water, KSOMaa Evolve, HEPES medium, Human Tubal Fluid, Attorney Docket No. 29539-0851 WO1 / MGH2025-035

[0085] MOPS medium, tissue culture medium 199, serum substitute supplement, human serum albumin, fetal bovine serum, or combinations thereof. Flow through the equilibrium solution inlet 120a and the media inlet 120b is controlled by an equilibrium valve 106.

[0086] The equilibrium solution provided through the equilibrium solution inlet 120a and the culture medium provided through the media inlet 120b are mixed with prior to entering the trap 114. For example, as shown in FIG. 3, media solution inlet group 120 includes include a serpentine micromixer 134 that is positioned to receive the culture media from the media inlet 120b and the equilibrium solution from the equilibrium solution inlet 120a. The serpentine micromixer 134 includes multiple turns such that the equilibrium solution is mixed with the culture media as they flow through the serpentine micromixer 134. The serpentine micromixer 134 includes a sufficient number of turns to ensure uniform mixing of the equilibrium solution and the culture media, e.g., between 10 and 200 turns, e.g., between 40 and 80 turns. The mixing of the culture medium and the equilibrium solution dilutes the CPA concentration in the solution that is delivered to the trap 114. In some examples, the mixing can be implemented to create a gradually increasing (e.g., linearly increasing) concentration of CPA during delivery of equilibrium solution to the oocyte or fertilized egg, embryo, or blastocyst, as discussed below with reference to FIG. 6.

[0087] The vitrification solution inlet 122 is configured to receive vitrification solution from a vitrification solution source 136. The vitrification solution can include one or more types of CPAs, such as ethylene glycol and dimethyl sulfoxide. In some embodiments, the vitrification solution includes from 1% (v / v) to 15% (v / v) of a first CPA (e.g., ethylene glycol) and from 1% (v / v) to 15% (v / v) of a second CPA (e.g., dimethyl sulfoxide). A vitrification valve 108 is configured to regulate fluid flow along the vitrification solution inlet 122 and along a collection outlet 124, through which the CPA-loaded oocyte or fertilized egg, embryo, or blastocyst is recovered from the cry opreservation module 102.

[0088] Referring specifically to FIG. 3, in some embodiments, a serpentine channel 129 is positioned between the trap 114 and the inlet 118. The serpentine channel 129 includes multiple turns (e.g., at least two turns, at least three turns, or at least four turns). In some embodiments, the plurality of turns are about 180° bends within the serpentine channel 129. In some embodiments, the serpentine channel 129 includes a siphoning channel 131 that allows a portion of fluid flow in the serpentine channel 129 to pass to the waste outlet 126. In some embodiments, the serpentine channel 129 includes multiple siphoning channels 131 (e.g., from 1 to 50, from 5 to 50, from 10 to 45, from 15 to 45, from 20 to 43 siphoning channels 131). Oocytes 116 or fertilized species may be prone to deformation (“blebbing”) within the neck Attorney Docket No. 29539-0851 WO1 / MGH2025-035 microchannel 117 if the velocity of fluid flow to the trap 114 is too high. Incorporating turns in the serpentine channel 129 and / or inclusion of siphoning channels 131 with the serpentine channel 129 reduces fluid flow velocity within the serpentine channel 129, which can reduce deformation. The turns in the serpentine channel 129 and the siphoning channels 131 also can promote the oocyte 116 or fertilized species to travel along the walls of the serpentine channel 129 during delivery, which can facilitate trapping the oocyte 116 or fertilized species in the trap 114.

[0089] Referring specifically to FIG. 2B, operation of the valves of the cryopreservation module 102 is controlled to achieve a desired sequence of fluid flow, e.g., in order to expose the oocyte or fertilized species to a gradually increasing CPA concentration, and then to expel the oocyte under exposure to vitrification solution. In a first phase of operation (“Step #1”), an oocyte 116 or fertilized species is loaded into the cryopreservation module 102 through the inlet 118 and trapped in the trap 114. Oocyte or fertilized species loading and trapping is achieved by controlling the inlet valve 104 to open and the equilibrium valve 106 and vitrification valve 108 to remain closed. In addition, the waste outlet valve 110 is opened to allow excess solution to flush out of the cry opreservation module 102. Oocyte or fertilized species loading and trapping is continued for a predetermined duration, e.g., 200 seconds in the illustrated example. Other durations are possible, e.g., durations of 50 seconds to 500 seconds.

[0090] Following the loading and trapping of the oocyte or fertilized species, in a second phase of operation (“Step #2”), equilibrium solution (“ES”) is loaded into the cryopreservation module 102 with gradually increasing CPA concentration. ES loading is achieved by controlling the inlet valve 104 to close and the vitrification valve 108 to open. The equilibrium valve 106 remains closed and the waste outlet valve 110 remains open. ES loading is continued for a predetermined duration, e.g., 900 seconds in the illustrated example. Other durations are possible, e.g., durations of 500 seconds to 1500 seconds. During the flow of ES solution, the concentration of CPA is gradually (e.g., linearly) increased, using a mixing approach discussed below with reference to FIG. 6. The gradual increase of CPA concentration helps to reduce osmotic stress.

[0091] Following ES loading, in a third phase of operation (“Step #3”), vitrification solution (“VS”) is loaded into the cry opreservation module 102 and the oocyte or fertilized species, now loaded with CPA, is collected. VS loading and oocyte or fertilized species collection is achieved by controlling the vitrification valve 108 to open and the equilibrium valve 106 and waste outlet valve 110 to close. The inlet valve 104 remains closed. This configuration of valves exposes the oocyte or fertilized species to VS and flushes the oocyte or fertilized species Attorney Docket No. 29539-0851 WO1 / MGH2025-035 out of the cry opreservation module through the collection outlet 124. VS loading and oocyte or fertilized species collection is continued for a predetermined duration, e.g., 75 seconds in the illustrated example. Other durations are possible, e.g., durations of 30 seconds to 500 seconds.

[0092] Referring generally to FIG. 6, during the ES solution loading phase, the culture medium is mixed with the equilibrium solution to create a ramping linear concentration profile of CPA in the ES that is flowed past the oocyte or fertilized species. Referring specifically to FIG. 6(B), a resistor-capacitor (RC) charging circuit 133 is connected between the equilibrium solution source 130 and the culture medium source 132. The RC charging circuit 133 enables generation of a linear CPA concentration profile in the ES supplied to the oocyte or fertilized species from a constant pressure supply of ES and culture media. The RC charging circuit 133 includes three resistors R3, R4, and Rs and a capacitor C4 that together implement a low-pass filter (equilibrium solution source - R4 - C4 - R3) between the equilibrium solution source 130 and the serpentine micromixer 134. Another resistor Rs is positioned between the culture medium source 132 and the serpentine micromixer 134. In some embodiments, the capacitor C4 is a rigid container containing gas with an adjustable gas volume and the resistors R3, R4, and Rs are tubing or microchannels of smaller diameter that impart fluidic resistance to fluid flow within the flow path. The RC charging circuit 133 creates a linearly increasing flow rate of the equilibrium solution, thereby achieving a linearly increasing CPA concentration profile in the equilibrium solution flowing through the cry opreservation module 102. In a specific example, the RC charging circuit 133 can be configured to increase the flow rate of the equilibrium solution over time to achieve a ratio of equilibrium solution to culture medium of 1 : 1 at the end of the equilibrium loading state of the cry opreservation module 102.

[0093] Figs. 6E and 6F illustrate the flow rates of ES and culture media, as mediated by the RCE charging circuit 133, in an example experimental implementation. As illustrated, the flow rate of equilibrium solution gradually and linearly increases, while the flow rate of culture media decreases less quickly, thereby resulting in a combined equilibrium solution having a gradually increasing concentration of CPA.

[0094] FIG. 8 shows example dimensions of a cryopreservation module. The neck microchannel 117 has a width W117 from about 5 pm to 30 pm, which prevents the highly deformable oocyte 116 or fertilized species from passing through the neck microchannel 117. In some embodiments, the neck microchannel 117 has a width W117 from about 5 pm to about 20 pm, from about 5 pm to about 15 pm, or about 10 pm. In some embodiments, a width W128 of the inlet microchannel 128 is from about 100 pm to about 150 pm, from about 100 pm to Attorney Docket No. 29539-0851 WO1 / MGH2025-035 about 140 pm, from about 110 pm to about 130 pm, or about 120 pm. In some embodiments, a width W124 of the waste outlet 124 is from about 100 pm to about 500 pm, from about 100 pm to about 400 pm, from about 100 pm to about 300 pm, from about 100 pm to about 200 pm, from about 100 pm to about 150 pm, from about 100 pm to about 140 pm, from about 110 pm to about 130 pm, or about 120 pm. In some embodiments, a width W124 of the vitrification microchannel 124 is from about 100 pm to about 500 pm, from about 150 pm to about 400 pm, from about 150 to about 300 pm, from about 150 pm to about 200 pm, from about 150 pm to about 180 pm, from about 155 pm to about 165 pm, or about 160 pm. In some embodiments, the trap 116 includes a container region having a width from about 100 pm to about 300 pm, from about 100 pm to about 200 pm, from about 100 pm to about 150 pm, from about 100 pm to about 140 pm, from about 110 pm to about 130 pm, or about 125 pm. In some embodiments, the container region of the trap 116 has a length of about 80 pm to about 300 pm, from about 80 pm to about 200 pm, from about 80 pm to about 120 pm, from about 90 pm to about 115 pm, from about 100 pm to about 110 pm, or about 105 pm. The depth of the microchannels is from about 100 pm to about 400 pm, from about 100 pm to about 300 pm, from about 100 pm to about 200 pm, or from 100 pm to about 150 pm.

[0095] As used herein, the term “about” means + / — 10% of any recited value.

[0096] FIG. 1, FIGS. 4(A-C), and FIGS. 5(B-E) illustrate the microfluidic transistor module 115. The microfluidic transistor module 115 includes multiple (e.g., three) fluidic timer circuits 138 and fluidic logic gate circuit 140 include multiple (e.g., four) fluidic logic gates. Referring particularly to FIG. 5(D), the microfluidic transistor module 115 may be formed from a multilayer structure. In the example of FIG. 5(D), the microfluidic transistor module 115 includes a four-layer structure (e.g., Layer 0, Layer 1, Layer 2, and Layer 3) that forms the fluidic timer circuits 138 and the fluidic logic gate circuit 140.

[0097] In general, each fluidic timer circuits 138 generate a time-dependent pressure outputs Pout. Specifically, referring to the truth table of FIG. 5(A), the three time-dependent pressure outputs from the three fluidic timer circuits 138 are referred to as Pi, P2, P3. The timedependent pressure outputs Pout from the fluidic timer circuits 138 are communicated as inputs to the fluidic logic gate circuit 140 through flow of a fluid, such as water, a buffer solution (e.g., PBS), or combinations thereof. For example, the microfluidic transistor module 115 includes shared ports between Layer 1 and Layer 2 and a channel in Layer 3 that allows the fluid exiting the fluidic timer circuits 138 to be in fluid communication with the fluidic logic gate circuit 140. Responsive to the three time-dependent pressure outputs, the fluidic logic gate circuit 140 generates four logic gate outputs A, B, C, D (see FIGS. 5(B) and 5(C)). Each Attorney Docket No. 29539-0851 WO1 / MGH2025-035 logic gate output controls the opening and closing of a corresponding valve 104-110 of the cry opreservation module 102.

[0098] In the example truth table illustrated in FIG. 5A, the inputs to the logic gates are the outputs of three timer circuits, denoted as Pi, P2, and P3, which change states from 1 to 0 (i.e., high pressure to low pressure) at 1100, 200, and 1175 seconds, respectively. The logic gate outputs control the open / close status of the corresponding valve A, B, C, and D, where an output value of 1 represents high pressure to close the valve and an output value of 0 represents low pressure to open the valve.

[0099] Referring to FIG. 4(A) and FIGS. 5(C-E), each fluidic timer circuit 138 includes a resistor-capacitor (RC) low-pass filter 142 and an inverter 144. Each of the RC low-pass filters 142 includes a fluidic resistor and a respective fluidic capacitor. Each fluidic resistors is implemented as a serpentine channel, where the number of turns and / or the dimensions of the channel defines the resistance of the resistor. Each fluidic capacitor is implemented as a rigid container containing gas with an adjustable gas volume, where the capacitance of the capacitor depends on with the initial gas volume. In the example of FIG. 4(C), the capacitor is implemented as a syringe; in some implementations, the capacitor can be a microfluidic device. Further details of modeling of the behavior and modeling of the capacitor are provided below as an Example. The time constant of each RC low-pass filter 142 is defined by the resistance R of its resistor and the capacitance C of its capacitor, e.g., with the duration of the timer increasing linearly with the gas volume inside the rigid container capacitor.

[0100] As illustrated in Fig. 4A (see also Figs. 5C-D), in some embodiments, the low-pass filter 142 receives fluid from a first fluid source having a fluidic pressure PSUpi and the inverter 144 receives fluid from a second fluid source having a fluidic pressure Psup2. The fluidic pressure PSUpi of the first fluid source is a constant pressure that is greater than the constant pressure of the fluidic pressure Psup2 of the second fluid source. In some embodiments, the fluidic pressure PSUpi of the first fluid source is a constant pressure from 190 to 210 kPa, from 195 to 205 kPa, or at about 200 kPa; and the fluidic pressure Psup2 of the second fluid source is a constant pressure from 170 to 190 kPa, from 175 to 185 kPa, or at about 180 kPa. Specifically, fluid is fed from the first fluid source to Layer 0 in the microfluidic transistor module 115, from where it communicates with the fluidic resistor in each fluidic timer circuit 138 by a port shared between Layer 0 and Layer 1 of the microfluidic transistor module 115. Fluid is also fed from the second fluid source to Layer 0 in the microfluidic transistor module 115, from where it communicates with the inverter 144 in each fluidic circuit 138 by a port shared between Layer 0 and Layer 2 of the microfluidic transistor module 115. Attorney Docket No. 29539-0851 WO1 / MGH2025-035

[0101] Each inverter 144 is includes a first microfluidic transistor Ti and a second microfluidic transistor T2. The output of each RC low-pass filter 142 acts as a gating signal for the inverter 144. Fluidic transistors are described in more detail in U.S. Patent Application No. 18 / 287,948, filed April 22, 2022,, the contents of which are incorporated here by reference in their entirety. In some embodiments, the first microfluidic transistor Ti and the second microfluidic transistor T2 each includes an elastic member (e.g., silicone membrane) that is sandwiched between Layer 1 and Layer 2 of the microfluidic transistor module 114. The first microfluidic transistor Ti is positioned at an intersection between Layer 1 and Layer 2. For example, the intersection can be located where a first channel and a second channel intersect, where the first channel is defined by a channel length in Layer 1 between the respective capacitor Ci, C2, and C3 and a dead-end channel Pcdownstream of the respective capacitor Ci, C2, and C3. The second channel is defined by a channel length in Layer 2 between the second fluidic timer circuit source Psup2 and the ground port outlet Gnd of Layer 2. Depending on the applied pressure, the first microfluidic transistor T 1 can mitigate the flow in the first channel in Layer 1 or the second channel in Layer 2. For example, the first microfluidic transistor T 1 can be a Quake-style valve that induces a flow-limitation to block fluid flow in the respective channel.

[0102] The second microfluidic transistor T2 is positioned at an intersection between Layer 1 and Layer 2, where the intersection is located along a third channel and the second channel. In some embodiments, the third channel is defined by a channel length in Layer 1 between a deadend channel Pa and a respective shared port between Layer 1 and Layer 2. Depending on the applied pressure, the second microfluidic transistor T2 can mitigate the flow in the third channel in Layer 1 or the second channel in Layer 2. For example, the second microfluidic transistor T2 can be a Quake-style valve that induces a flow-limitation to block fluid flow in the respective channel.

[0103] During operation, the low-pass filter 142 of each fluidic timer 138 creates a respective fixed time delay in the rise of capacitor pressure Pc, which triggers the inverter 144 and causes the respective time-dependent output pressure Pout, Pi, P2, P3 to drop. Increasing pressure at the respective capacitor Ci, C2, and C3 causes the second liquid to enter the rigid container of the respective capacitor Ci, C2, and C3, which compresses the air inside and raises the air pressure until equilibrium is reached. As Pcexceeds the supply pressure for the inverter (e.g., PsuP2, about 180 kPa), the time-dependent pressure at the shared port Pout, Pi, P2, P3 between Layer 1 and Layer 2 decreases. The time-dependent pressure output of each fluidic timer circuit 138 can be tailored to a pre-determined time point based on the air volume in the respective Attorney Docket No. 29539-0851 WO1 / MGH2025-035 capacitor Ci, C2, and C3. For example, air volumes of 0.6, 3.7, and 3.9 mL achieves the predetermined time points of 200s, 1,100s, and 1,175 s for the example operation of FIG. 2B.

[0104] In some embodiments, an air volume of the first capacitor Ci is from 0.01 to 0.6 mL, from 0.1 to 0.6 mL, or from 0.2 to 0.6 mL. In some embodiments, the pre-determined time point for the first fluidic timer circuit having the first capacitor Ci is from 0.1 seconds to 200 seconds, from 5 seconds to 200 seconds, from 10 seconds to 200 seconds, from 50 seconds to 200 seconds, or from 100 to 200 seconds.

[0105] In some embodiments, an air volume of the second capacitor C2 is from 0.6 to 3.7 mL, from 1 to 3.7 mL, from 1.5 to 3.7 mL, or from 2 to 3.7 mL. In some embodiments, the predetermined time point for the second fluidic timer circuit having the second capacitor C2 is from 200 seconds to 1,100 seconds, from 300 seconds to 1,100 seconds, from 400 seconds to 1,100 seconds, from 500 seconds to 1,100 seconds, or from 600 to 1,100 seconds.

[0106] In some embodiments, an air volume of the third capacitor C3 is from 3.7 to 4 mL, from 3.8 to 4 mL, or about 3.9 mL. In some embodiments, the pre-determined time point for the third fluidic timer circuit having the third capacitor C3 is from 1,100 seconds to 1,240 seconds, from 1,100 seconds to 1,200 seconds, from 1,100 seconds to 1,175 seconds, from 1,100 seconds to 1,100 seconds, or from 1,100 to 1,150 seconds.

[0107] The fluidic logic gate circuit 140 receive the time-dependent pressures produced from the plurality of fluidic timer circuits 138 via the transfer of fluid from the fluidic timer circuits 138 to the fluidic logic gate circuit 140. Specifically, the inputs to the fluidic logic gate circuit 140 are the outputs from the fluidic timer circuits 138. Responsive to the time-dependent pressures Pi, P2, P3 produced from the fluidic timer circuits 138, the fluidic logic gate circuit 140 generates a pressure outputs A, B, C, and D, each of which controls opening and closing of a respective valve 104-110 in the cryopreservation module 102.

[0108] Referring to FIG. 5B, the fluidic logic gate circuit 140 is composed of two NOT gates and two NAND gates that control opening and closing the plurality of deformable valves 102- 112 to control the sequential state change process of the cryopreservation module 102. To achieve the desired outputs, Karnaugh mapping and NAND simplification, resulting in a circuit with four logic gates including two NOT and two NAND gates. The NOT and NAND gates are fluidically connected such that combinations of inputs (time-dependent pressures Pi, P2, P3 produced from the plurality of fluidic timer circuits 138) produce outputs A-D as shown in the truth table of FIG. 5 A.

[0109] The pressure outputs A, B, C, and D from the logic gate circuit 140 autonomously cause the cry opreservation module 102 to undergo the sequential state change process of FIG. 2B by Attorney Docket No. 29539-0851 WO1 / MGH2025-035 controlling the valves 104-110 to open and close. Specifically, with reference to both FIGS. 2B and 5A, in Step #1, the “0” signals output through channels A and D open valves 104 and 110, and the “1” signals output through channels B and C cause vales 106 and 108 to remain closed. In Step #2, channel A is changed to a “1” signal, closing valve 104; and channel B is changed to a “0” signal, opening valve 106. In Step #3, channels B and D are changed to “1” signals, closing valves 106 and 110; and channel C is changed to a “1” signal, opening valve 108. Notably, the sequential state change process occurs using only constant fluidic pressure supplies to the cryopreservation module and the microfluidic transistor module and without external electronic control.

[0110] Referring to FIG. 7, in an example method for CPA loading of an oocyte or cryopreservation of a fertilized species, a time-dependent fluid pressure signal is produced by each of multiple fluidic timer circuits of a microfluidic device (700). By a fluidic logic gate circuit of the microfluidic device, multiple second time-dependent fluid pressure signals are generated responsive to receiving the first time-dependent fluid pressure signals as inputs (702). Operation of each of multiple valves of the microfluidic device is controlled by a corresponding one of the second time-dependent fluid pressure signals (704). Each valve is configured to control fluid flow through a corresponding one of multiple microchannels of the microfluidic device, where the microchannels are in fluid communication with an trap of the microfluidic device that is configured to retain an oocyte or fertilized species. Specifically, in a first phase of operation, a first valve is opened (706) to introduce an oocyte or fertilized species into the microfluidic device through an inlet microchannel, wherein the introduced oocyte or fertilized species is retained in the trap. In a second phase of operation, the first valve is closed and a second valve is opened (708) to flow an equilibrium solution through a media channel group, wherein the equilibrium solution is flowed past the oocyte or fertilized species retained in the trap. In a third phase of operation, the second valve is closed, a third valve is opened, and a waste outlet valve is closed (710) to flow a vitrification solution through a vitrification microchannel to remove the oocyte or fertilized species from the microfluidic device through the vitrification microchannel.

[0111] Additional features can be implemented to facilitate clinical translation. For instance, instead of passively waiting for 200 seconds to trap the oocyte or fertilized species, actively detecting the trapping of an oocyte or fertilized species through backpressure can be implemented to facilitate seamless operations. Second, multiplexing the design to handle more oocytes or fertilized species can greatly enhance efficiency. Third, with the ability to autonomously sense and actuate complex sequences of fluidic events in a scalable and low- Attorney Docket No. 29539-0851 WO1 / MGH2025-035 cost manner can be implemented in the context of automating other IVF-on-a-chip steps such as oocyte denudation, ICSI, embryo culture and cryopreservation.

[0112] Attorney Docket No. 29539-0851 WO1 / MGH2025-035

[0113] Examples:

[0114] Device Fabrication:

[0115] A cryopreservation chip and a microfluidic transistor chip were fabricated using polydimethylsiloxane (PDMS) soft lithography techniques. SU-8 negative photoresist (Microchem) was spinned onto a silicon wafer and patterned using UV photolithography to define the master for the chip. The feature dimensions were measured using a surface profilometer (Veeco Instruments Inc). Then PDMS (Sylgard 184) was poured, degassed, and cured using SU-8 as the master mold. For the cryopreservation chip, the cured PDMS was peeled from SU-8, followed by hole punching, oxygen plasma treatment and then bonded to the oxygen plasma treated glass slide. A separate valve device was made to control different flows in and out of the cryopreservation chip. Specifically, a silicone membrane (20 pm thickness, Wacker Chemie) was sandwiched between two PDMS layers as the valve. The upper PDMS layer (i.e., above the membrane) is connected to the pressure outputs (i.e., A, B, C, D in FIGS. 5(A-F)) from the transistor chip. The lower PDMS layer is connected to different inlets and outlets of the cry opreservation chip.

[0116] To fabricate a 4-layer microfluidic transistor chip (e.g., see FIG. 5(D)), a 1 ,2-mm biopsy punch was first used to punch out appropriate holes in layer 0, 1, and 2 PDMS. Then layer 1 PDMS was bonded to the silicone membrane (20 pm thickness, Wacker Chemie) using oxygen plasma treatment. A 1.2-mm biopsy punch was then used to punch through the silicone membrane to create shared ports (in FIGS. 4(A-D) and FIGS. 5(A-F)) with layer 2 PDMS. Next, the layer 1 PDMS - silicone membrane assembly was bonded to layer 2 PDMS following oxygen plasma treatment. Finally, layer 0 and layer 3 PDMS were similarly bonded to the layer 1 PDMS - silicone membrane - layer 2 PDMS assembly.

[0117] Fluidic timer circuit modelling

[0118] To model a fluidic capacitor, a rigid container filled with air was considered with initial pressure and volume as Po and Vo, respectively. As shown in FIG. 4A, the capacitor is connected to pressure supply of PSUpi through a resistor with resistance of Ri. To solve the Pc (the air pressure inside the rigid container) as a function of time, the following governing equations were considered to account for the varying capacitance over time: Attorney Docket No. 29539-0851 WO1 / MGH2025-035

[0119] W c(t) = V0* P0(4)

[0120] Plugging Eq 4 into Eq3 yields: where Q(t) is the flow through the resistor into the capacitor, C is the capacitance, Vci / t) is the liquid volume inside the rigid container, Vc(t) is the air volume inside the rigid container, Pc(t) is the pressure inside the rigid container. MATLAB was used to solve the above equations.

[0121] For the constant capacitance model in FIG. 4 A, a constant capacitance value is calculated from Eq. 5 using the final equilibrium value of Pc, which is PSUpi.

[0122] Fluidic timer circuit and logic gate circuit measurements

[0123] To measure Pc (FIG. 4 A), a 40 cm-long peek tubing with 0.01 -inch inner diameter (IDEX-HS) was used as the resistor. To create a rigid sealed container as the capacitor, the plunger of a 1 mL syringe was pulled to the position of 0.7 mL and then glued to the syringe wall. A 200 kPa pressure supply was provided using a computer-controlled pressure source (FlowEZ, Fluigent). The pressure at Pc was measured using a pressure sensor (ABPDRRV015PDAA5, Honeywell).

[0124] To measure Pout (FIG. 4A and 4C), two pressure supplies were set to 200 kPa (PSUpi) and 180 kPa (Psup2), respectively. Different air volumes were used as the capacitor. A MATLAB script was used to ramp up / down the pressure supplies and record Pout. Unless otherwise specified, all reservoirs for the pressure sources (P-CAP, Fluigent) were filled with l x phosphate-buffered saline (Fisher Scientific).

[0125] For the measurement of time-dependent digital pressure outputs (FIG. 5F), three pressure supplies were set to 200 kPa (Psupi), 180 kPa (Psup2) and 170 kPa (Psup3), respectively. A 15 cm-long 0.02-inch inner diameter Tygon tubing served as R2 (FIG. 5C). Regarding capacitors, syringes with 0.6, 3.7, and 3.9 mL air were used for C2, Ci, and C3, respectively. The 4-layerPDMS device was primed with PBS. AMATLAB script was used to ramp up / down the constant pressure supplies and record the pressure of ports A, B, C, and D.

[0126] Linear CPA concentration

[0127] To create a linear CPA concentration profile from a constant pressure supply, the RC charging circuit and serpentine mixer were employed as shown in FIG. 6B. The resistors R3, R4, and R5 were 6 cm, 10 cm and 50 cm long 0.01 inch inner diameter tubing, respectively. A Attorney Docket No. 29539-0851 WO1 / MGH2025-035 syringe with 4 mL air was used as the capacitor (C4 in FIG. 6B). The ES is 15% EG + 15% DMSO prepared in KSOMaa Evolve w / HEPES medium (LifeGlobal Group). The pressure supply (PSUp4) was set to 30 kPa. To visualize the mixing of ES and media in the serpentine mixer, 0.1 mg / mL rhodamine B was added to the media for fluorescent imaging using an upright microscope (Nikon Eclipse 90i). The flow rates of each flow into and out of the cry opreservation chip were measured using Sensirion flow meters (SLI-1000).

[0128] LTspice was used to simulate the mixer circuit (FIG. 6B). Fluidic to electric domain conversions are as follows: Pressure: 1 kPa = 1 Volt, Resistance: 1 kPa*s / uL = 1 ohm, Flow rate: 1 uL / s = 1 amp, Capacitance: 1 uL / kPa = 1 farad. The resistance of the mixer channel is estimated to increase linearly with the flow rate ratio of ES to media. A small nonzero source pressure for t < 200 s, which is the pressure resultant of imperfect valve seals, results in the capacitor having an initial charge and less reverse flow across R3 at the beginning of step 2. A 3D laminar flow model (Comsol 4.4) was built to simulate the laminar flow fields of the cryopreservation chip.

[0129] Autonomous CPA loading into the mouse oocyte

[0130] Frozen mouse Mil oocytes were purchased from Embryotech laboratories (Haverhill, MA) and thawed as instructed by the vendor. To load the oocyte into the cry opreservation chip, an individual oocyte was first transferred into a pipette tip with 135 microns inner diameter (EZ-Tip, Cooper Surgical) under the microscope. The pipette tip was then inserted into a 1 / 32 inch inner diameter Tygon tubing connected to a Tee tubing junction connector (McMaster- Carr). The other two ports of the Tee connector were connected to a tubing from the media reservoir and a tubing to the inlet of the cry opreservation chip. After injecting 10 pL suspension containing the selected oocyte from the pipette to the tubing leading to the inlet of the cryopreservation chip, the pipette tip was clamped at the other end. The oocyte was pushed into the cryopreservation chip by the flow from the media reservoir when pressurized. A MATLAB script was used to ramp up / down the pressure supplies. The sequence of different actions including oocyte loading & trapping, linear loading of 7.5% EG + 7.5% DMSO in 15 min, subsequent loading of VS and oocyte collection were performed automatically. Videos of the oocyte were recorded with an upright microscope under lOx objective (Nikon Eclipse 90i).

[0131] Long duration fluidic timer with the microfluidic transistor

[0132] A fluidic timer was constructed by combining a RC low-pass filter and an inverter (FIG. 4A). As the supply pressure (PSUpi) increases from 0 to 200 kPa, the low pass filter creates a Attorney Docket No. 29539-0851 WO1 / MGH2025-035 fixed time of delay in the rise of capacitor pressure (Pc), which triggers the inverter and causes the output pressure (Pout) to drop. The microfluidic channel layout consistent with the illustration in FIG. 4A, with a 20 pm thickness silicone membrane (not shown) sandwiched between two PDMS layers. Two microfluidic transistors were used in this timer circuit. Air inside a rigid container (in this example, a syringe with the plunger glued to the syringe wall; FIG. 4C) was used as the fluidic capacitor. When the capacitor syringe was connected to the microfluidic circuit, increasing pressure at the connection point drove liquid into the syringe, which compresses the air inside and raises the air pressure until equilibrium is reached. The capacitance C can be calculated as C = dV / dP, where V and P are the liquid volume and pressure inside the capacitor syringe, respectively. As presented in Fig. 4B, the model that accounts for the time-dependent capacitance demonstrates a better match with experimental measurement of Pc, compared to the constant capacitance model.

[0133] As Pc exceeds the supply pressure for the inverter (Psup2, 180 kPa), the output pressure (Pout) decreases from 175 kPa to 11 kPa. By increasing the air volume in the capacitor syringe from 0.1 mL to 2 mL and 4 mL, timer durations of 63, 584 and 1237 seconds, respectively, can be achieved, as shown in FIG. 4C. Additionally, with constant pressure supplies (PSUpi and PsuP2), the model indicates that the timer duration is proportional to the initial air volume in capacitor syringe and the resistor Ri. Indeed, as air volume was varied from 0.1 mL to 4 mL, a linear increase of the timer was observed from 63 to 1237 seconds.

[0134] Generating time-dependent digital pressure signal using fluidic logic gates

[0135] A microfluidic transistor chip was implemented in experiment according to the integrated electrical schematic diagram of FIG. 4A). Specifically, two pressure supplies were employed to drive three fluidic timers and one pressure supply to drive four fluidic logic gates, all connected to a common ground. A total of 18 microfluidic transistors, along with 4 fluidic resistors and 3 fluidic capacitors, work together to compute 4 digital pressure outputs, entirely without electronics. To translate the electrical diagram to the fluidic domain, a 4-layer PDMS device was fabricated. As illustrated in FIG. 4D, in addition to the layer 1 and layer 2 microchannels which create microfluidic transistors, two routing layers were included to prevent undesired microchannel crossings. The layer 0, positioned above layer 1, bridges ports with shared pressure supplies and ground, while layer 3 (gray), beneath layer 2, feeds the timer circuit outputs to the logic gates.

[0136] The fully assembled microfluidic transistor chip filled with green dye is presented in FIG. 5E, with an overall size of 4.5 cm by 3.5 cm. Once connected to pressure supplies and Attorney Docket No. 29539-0851 WO1 / MGH2025-035 external capacitor syringes, the device automatically produces time dependent digital pressure outputs as demonstrated in FIG. 5F. For instance, pressure A stays low (i.e., < 20 kPa) for the first 200 seconds and rises to high (i.e., > 140 kPa) at 200 seconds and remains high afterwards. In the cry opreservation chip, this indicates that valve A is open for the first 200 seconds to allow oocyte loading and trapping, and remains closed after 200 seconds. Moreover, the chip has the ability to alternate output pressure state multiple times. In FIG. 5F, pressure B starts high for the first 200 seconds, drops to low at 200 seconds and remains low for the next 900 seconds, and eventually switches back to high after 1100 seconds. This sequence commands valve B of the cry opreservation chip to open only between 200 seconds to 1100 seconds for linear ES loading into the oocyte, while remain closed during oocyte loading (< 200 seconds) and VS loading & oocyte extraction (1100 - 1175 seconds). After 1175 seconds, all output pressures stay high, closing all the valves to stop any flow within the cryopreservation chip. Note that the first 25 seconds of the pressure curves in Fig. 5F reflects the transition phase as the pressure supplies gradually ramp up.

[0137] Optimizing the trap and linear CPA concentration profile

[0138] Two key functionalities of the cry opreservation module are 1) trapping the oocyte or fertilized species securely within the device and 2) generating a linear CPA concentration profile. The distribution of flow streamline from simulation confirmed the preferrable flow path through the trap (FIG. 9). In addition, the narrow microchannel (e.g., dimensioned as indicated in FIG. 8) prevents the highly deformable oocyte or fertilized species from squeezing through the trap. To reduce the oocyte loading time, an oocyte was transferred directly into tubing (0.03-inch inner diameter) connected to the inlet port of the cryopreservation chip. By adjusting the inlet flow rate to 90 pL / min, the oocyte can be loaded and trapped within 2 minutes. However, the high flow rate in the trap pushes a portion of the oocyte through the narrow channel, causing severe deformation (FIG. 10). To address this, a parallel siphoning channel was connected to the oocyte inlet pathway (i.e., prior to the trap) through 43 small channels (15 pm wide and 175 pm long; FIG. 11), which slows down the flow by 25-fold as indicated by the numerical simulation.

[0139] Referring specifically to FIG. 6A, a mouse oocyte is held in the trap, with flow directions from and to various inlets and outlet labeled. The ES and VS are composed of 15% EG + 15% DMSO, and 15% EG + 15% DMSO + 0.5 M sucrose, respectively. Referring to FIG. 6B, to generate a linear CPA concentration profile that flows over the oocyte for 15 minutes, an RC charging circuit was employed to achieve linear increase of QES. The ES is Attorney Docket No. 29539-0851 WO1 / MGH2025-035 then mixed with media via a serpentine micromixer, targeting a 1 : 1 mixing ratio at 15 minutes, resulting in a final concentration of 7.5% EG + 7.5% DMSO over the oocyte.

[0140] Specifically, a RC low pass filter was utilized to create a linearly increasing flow rate of ES, as the result of capacitor charging. The resistor R4 and capacitor C4 were tuned to ensure that the 900-second linear ES loading occurred within the linear region of the varying capacitance model of the RC circuit (FIG. 6B). In the cryopreservation chip, ES and media meet at a T-junction and enter the serpentine mixing channel with 80 turns, before flowing over the oocyte for CPA loading (FIG. 6A-B). Rhodamine B was added to the media to confirm uniform mixing of ES and media due to Dean flow after 49 turns, as indicated by the fluorescent intensity (FIG. 6C).

[0141] The CPA concentration flowing over the oocyte is determined by the flow rate ratio of ES to media. Notably, the resistance of the serpentine mixer is determined by this flow rate ratio, which changes over time and, in turn, affects the flow rate of both ES and media. To better estimate these flow rates, this complex circuit was analyzed using an analog circuit simulator (details in Method). As suggested by the model, when valve B opens at 200 seconds, media flows through the T-junction to the capacitor syringe, resulting in negative ES flow rates, as confirmed by experimental measurements shown in FIG. 6E. This negative ES flow rate delays the onset of linear ES loading and reduces the actual CPA loading time. In addition, as the pressure increases at the T-junction, the model indicates a slight decrease in media flow rate, which is supported by experimental measurement in FIG. 5F. Resistances and capacitance were tuned to achieve a linear increase in ES flow rate over time and a final flow ES to media rate ratio of 1 : 1 at 15 minutes (FIGS. 6E-F).

[0142] Autonomous CPA loading of the mouse oocyte

[0143] The cryopreservation chip was integrated with the microfluidic transistor chip to achieve fully autonomous oocyte trapping, linear ES loading, oocyte dehydration with VS, and oocyte extraction from the device. By connecting the outputs of the microfluidic transistor chip to automatically control the valves A, B, C and D, the flow rates of each inlet and outlet of the oocyte chip were measured. For the cry opreservation chip, solely one pressure supply is used to drive the complex flow of four distinct solutions. As shown in FIG. 6E, the inlet flow rate remains at 90 pL / min only for the first 200 seconds, allowing sufficient time to load and trap the oocyte. Subsequently, the flow from ES and media inlets is activated to enable linear CPA loading between 200 seconds and 1100 seconds (FIG. 6E-F). Afterward, the flow to the waste outlet is stopped, and the flow from VS inlet is turned on for dehydration and Attorney Docket No. 29539-0851 WO1 / MGH2025-035 oocyte collection from 1100 seconds to 1175 seconds. Finally, all valves are closed, with no active flow in the cryopreservation chip. Overall, successful control of each valve was demonstrated and desired flow conditions were achieved throughout the multi-step protocol.

[0144] As the final measure, the integrated device was validated with mouse oocyte. After transferring the mouse oocyte to the inlet tubing, the entire process proceeded automatically without human intervention and delivered vitrification-ready oocytes. Step loading and linear loading of ES (7.5% EG + 7.5% DMSO) were performed separately and the volume change of mouse oocytes was compared by recording the entire process using an upright microscope. During the 15 min loading of ES, fluorescent image confirmed that the ES flowed over the oocyte and was not blocked by the trapped oocyte, ensuring sufficient mass transport (FIG. 12). In addition, these results demonstrate that traditional step loading induced sudden and substantial oocyte shrinkage due to osmotic shock, while the linear loading produced a much more gradual and milder volume change (FIG. 6H). The rate of shrinkage (% of initial volume per second) during linear loading is 0.077 ± 0.009 % / s, which is 44-fold lower than that of step loading, 3.4 ± 0.7 % / s. Additionally, the minimal oocyte volume (% of initial volume) with step loading and linear loading was 62.3 ± 1.7% and 88.1 ± 3.1% respectively (FIG. 61). As presented in FIG. 61, plasma membrane of oocyte underwent collapsing / folding during step loading, which can potentially cause structural damage. Indeed, the blebbing of oocyte (i.e., 2 out of 5 oocytes) was observed during step loading (FIG. 13). In contrast, no blebbing occurred during linear loading (n=5). Further, to demonstrate the versatility of the device, a 10-min linear loading of ES was performed, resulting in a shrinkage rate of 0.13 ± 0.04 % / s, minimal volume of 74.2 ± 1.5 % and no blebbing (n=5, FIG. 14). The vitrification-ready oocytes were successfully collected from the device (n=15).

[0145] Various embodiments of the features of this disclosure are described herein. However, it should be understood that such embodiments are provided merely by way of example, and numerous variations, changes, and substitutions can occur according to those skilled in the art without departing from the scope of this disclosure. It should also be understood that various alternatives to the specific embodiments described herein are also within the scope of this disclosure.

[0146] Other features and advantages of the present disclosure will be apparent from the following detailed description, the figures, and the claims.

Claims

Attorney Docket No. 29539-0851 WO1 / MGH2025-035What is claimed is:

1. A microfluidic device comprising: a trap configured to retain an oocyte or fertilized species; a plurality of microchannels in fluid communication with the trap; a plurality of valves, wherein each valve is configured to control fluid flow through a corresponding set of one or more of the plurality of microchannels; a plurality of fluidic timer circuits each configured to produce a corresponding first time-dependent fluid pressure signal; and a fluidic logic gate circuit configured to receive the first time-dependent pressure signals and to produce a plurality of second time-dependent fluid pressure signals, wherein each second time-dependent fluid pressure signal controls operation of a corresponding one of the valves.

2. The microfluidic device of claim 1, wherein the plurality of microfluidic channels comprise: an inlet microchannel configured to receive an oocyte or fertilized species; an media channel group configured to receive an equilibrium solution; a vitrification microchannel configured to receive a vitrification solution and configured for output of the oocyte or fertilized species; and a waste outlet microchannel.

3. The microfluidic device of claim 2, comprising a serpentine microchannel positioned between the trap and the inlet channel, wherein the serpentine microchannel includes at least two turns.

4. The microfluidic device of claim 3, comprising one or more siphoning microchannels fluidically connecting the serpentine microchannel to the waste outlet microchannel.

5. The microfluidic device of any of claims 2 to 4, wherein a width of the vitrification microchannel is between about 100 pm and about 500 pm.

6. The microfluidic device of claim 5, wherein a width of the vitrification microchannel is between about 150 pm and about 200 pm.Attorney Docket No. 29539-0851 WO1 / MGH2025-0357. The microfluidic device of any of claims 2 to 6, wherein a width of the waste outlet microchannel is between about 100 m and about 500 pm.

8. The microfluidic device of claim 7, wherein a width of the waste outlet microchannel is between about 100 pm and about 150 pm.

9. The microfluidic device of any of claims 2 to 8, wherein the media channel group comprises: a media inlet microchannel; an equilibrium solution inlet microchannel; and a serpentine micromixer microchannel in fluid communication with the media inlet microchannel and the equilibrium solution inlet microchannel and disposed between the equilibrium solution inlet microchannel and the trap and between the media inlet microchannel and the trap.

10. The microfluidic device of claim 9, wherein the serpentine micromixer microchannel includes between 10 to 200 turns.

11. The microfluidic device of claim 10, wherein the serpentine micromixer microchannel includes between 40 to 80 turns.

12. The microfluidic device of any of the preceding claims, wherein a channel depth of the plurality of microchannels is between about 100 pm and about 400 pm.

13. The microfluidic device of claim 12, wherein a channel depth of the plurality of microchannels is between about 100 pm and about 150 pm.

14. The microfluidic device of any of the preceding claims, wherein the trap comprises: a container region having a width of between about 100 pm and about 300 pm and a length of about 80 pm to about 300 pm; and a neck region having a width of between about 5 pm and about 30 pm.

15. The microfluidic device of claim 14, wherein the container region has a width of between about 100 pm and about 150 pm and a length of about 80 pm to about 120 pm.Attorney Docket No. 29539-0851 WO1 / MGH2025-03516. The microfluidic device of any of the preceding claims, wherein each fluidic timer circuit is a microfluidic circuit comprising: a fluidic transistor; and a low-pass filter circuit, wherein an output pressure of the low-pass filter circuit is a gate signal for the fluidic transistor.

17. The microfluidic device of claim 16, wherein an output channel from each fluidic transistor is connected to an input of the fluidic logic gate circuit.

18. The microfluidic device of claim 16 or 17, wherein the low-pass filter circuit comprises: a fluidic resistor; and a fluidic capacitor, wherein a resistance of the fluidic resistor and a capacitance of the fluidic capacitor define a time constant of the low-pass filter circuit.

19. The microfluidic device of claim 18, wherein the fluidic capacitor comprises a rigid container containing a gas with an adjustable gas volume, wherein the capacitance of the fluidic capacitor is based on the volume of gas in the rigid container.

20. The microfluidic device of claim 18 or 19, wherein the fluidic resistor comprises a serpentine channel, wherein the resistance of the fluidic resistor is based on a length of the serpentine channel.

21. The microfluidic device of any of claims 16 to 20, wherein each fluidic timer circuit comprises: a first microfluidic channel and constituting at least a portion of the low-pass filter circuit; a second microfluidic channel crossing the first microfluidic channel at an intersection; and a deformable membrane separating the first microfluidic channel and the second microfluidic channel at the intersection, wherein the fluidic transistor is defined at the intersection.Attorney Docket No. 29539-0851 WO1 / MGH2025-03522. The microfluidic device of claim 21, wherein the deformable membrane comprises a PDMS membrane.

23. The microfluidic device of claim 21 or 22, wherein the first microfluidic channel is defined in a first plane and the second microfluidic channel is defined in a second plane.

24. The microfluidic device of any of the preceding claims, wherein the fluidic logic gate circuit comprise two NOT gates and two NAND gates.

25. The microfluidic device of any of the preceding claims, wherein the plurality of second time-dependent fluid pressure signals are configured to sequentially: open a first valve of the plurality of valves to introduce the oocyte or fertilized species into the microfluidic device through an inlet microchannel, wherein the introduced oocyte or fertilized species is retained in the trap; close the first valve and open a second valve of the plurality of valves to flow an equilibrium solution through a media channel group, wherein the equilibrium solution is flowed past the oocyte or fertilized species retained in the trap; and close the second valve and open a third valve of the plurality of valves to flow a vitrification solution through a vitrification microchannel and to remove the oocyte or fertilized speciesfrom the microfluidic device through the vitrification microchannel.

26. The microfluidic device of any of the preceding claims, wherein the plurality of second time-dependent fluid pressure signals are configured to close a waste outlet valve concurrently with opening the third valve.

27. The microfluidic device of any of the preceding claims, comprising: a first microfluidic chip, wherein the trap and the plurality of microchannels are defined in the first microfluidic chip; a second microfluidic chip, wherein the plurality of fluidic timer circuits and the fluidic logic gate circuit are defined in the second microfluidic chip; and fluid flow passages connecting the first microfluidic chip and the second microfluidic chip.Attorney Docket No. 29539-0851 WO1 / MGH2025-03528. A method comprising: producing, by each of a plurality of fluidic timer circuits of a microfluidic device, a respective first time-dependent fluid pressure signal; generating, by a fluidic logic gate circuit of the microfluidic device, a plurality of second time-dependent fluid pressure signals responsive to receiving the first time-dependent fluid pressure signals as inputs; and by each second time-dependent fluid pressure signal, controlling operation of a corresponding valve of a plurality of valves of the microfluidic device, wherein each valve is configured to control fluid flow through a corresponding one of a plurality of microchannels of the microfluidic device, and wherein the plurality of microchannels are in fluid communication with an trap of the microfluidic device that is configured to retain an oocyte or fertilized species.

29. The method of claim 28, wherein controlling operation of the valves of the microfluidic device comprises controlling an opening and closing sequence of the valves.

30. The method of claim 28 or 29, wherein controlling operation of the valves of the microfluidic device comprises controlling a timing of opening and closing of each of the valves.

31. The method of any of claims 28 to 30, wherein controlling operation of the valves of the microfluidic device comprises sequentially: opening a first valve to introduce an oocyte or fertilized species into the microfluidic device through an inlet microchannel, wherein the introduced oocyte or fertilized species is retained in the trap; closing the first valve and opening a second valve to flow an equilibrium solution through a media channel group, wherein the equilibrium solution is flowed past the oocyte or fertilized species retained in the trap; and closing the second valve and opening a third valve to flow a vitrification solution through a vitrification microchannel to remove the oocyte or fertilized species from the microfluidic device through a vitrification microchannel.

32. The method of claim 31, wherein controlling operation of the valves of the microfluidic device comprises closing a waste outlet valve concurrently with opening the third valve.Attorney Docket No. 29539-0851 WO1 / MGH2025-03533. The method of claim 31 or 32, comprising, when the first valve is opened, flowing a fluid containing the oocyte or fertilized species through a serpentine microchannel connecting the inlet microchannel and the trap.

34. The method of claim 33, comprising flowing at least some of the fluid from the serpentine microchannel to a waste outlet microchannel.

35. The method of any of claims 31 to 34, comprising: closing the first valve and opening the second valve about 200 seconds after opening the first valve; and closing the second valve and opening the third valve about 900 seconds after opening the second valve.

36. The method of any of claims 31 to 35, comprising, when the first valve is closed and the second valve is open, flowing a fluid through the media channel group, comprising: flowing media through a media inlet microchannel; flowing equilibrium solution through an equilibrium solution inlet microchannel; and mixing the media and the equilibrium solution in a serpentine micromixer microchannel in fluid communication with the media inlet microchannel and the equilibrium solution inlet microchannel and disposed between the equilibrium solution inlet microchannel and the trap and between the media inlet microchannel and the trap.

37. The method of claim 36, comprising by mixing the media and the equilibrium solution in the serpentine micromixer microchannel, gradually increasing a concentration of the equilibrium solution in fluid that flows past the trap.

38. The method of any of claims 28 to 37, wherein controlling operation of the valves occurs without external electronic control.

39. The method of any of claims 28 to 38, wherein producing a time-dependent fluid pressure signal by a fluidic timer circuit comprises: gating a fluidic transistor with an output pressure of a low-pass filter circuit.Attorney Docket No. 29539-0851 WO1 / MGH2025-03540. The method of claim 39, wherein an output channel from each fluidic transistor is connected to an input of the fluidic logic gate circuit.

41. The method of claim 39 or 40, comprising operating the low-pass filter circuit, comprising: supplying the output pressure of the low-pass filter circuit for a time defined by a resistance of a fluidic resistor of the low-pass filter circuit and a capacitance of a fluidic capacitor of the low-pass filter circuit.

42. The method of any of claims 28 to 41, wherein the fluidic logic gate circuit comprises two NOT gates and two NAND gates configured to output the plurality of second timedependent fluid pressure signals.

43. The method of any of claims 28 to 42, wherein the microfluidic device further comprises a serpentine channel positioned between the trap and the inlet, wherein the serpentine channel includes at least two turns.

44. The method of claim 43, wherein the serpentine channel includes a plurality of siphoning channels that allows a portion of fluid flow in the serpentine channel to pass to the waste outlet, wherein the plurality of siphoning channels are configured to reduce flow velocity through the trap.

45. The method of any of claims 28 to 44, wherein the microfluidic device further comprises: a media inlet in fluid communication with the trap; and a serpentine micromixer in fluid communication with the media inlet and the equilibrium solution inlet, wherein the serpentine micromixer is positioned between the equilibrium solution inlet and the trap and between the media inlet and the trap, and wherein the serpentine micromixer is configured to mix an equilibrium solution and a media solution.

46. The method of claim 45, wherein the serpentine micromixer includes from 10 to 200 turns to achieve mixing of the equilibrium solution and the media solution.Attorney Docket No. 29539-0851 WO1 / MGH2025-03547. The method of claim 46, wherein the serpentine micromixer includes from 40 to 80 turns to achieve mixing of the equilibrium solution and the media solution.

Images