Systems and processes for continuous addition of a cryoprotective agent

Abstract

An integrated system and method for preparing sperm cells to improve their survival rate during cryopreservation is described herein. The system has a container, a controlled dispenser, and a dispensing tube. The dispensing tube has a first end fluidly connected to the dispenser and a second end disposed within the container. The second end can be submerged in a sperm cell fluid. The controlled dispenser can be a syringe pump including a syringe for containing a cryoprotectant and a pushing mechanism for displacing the syringe. The syringe pump is configured to expel the cryoprotectant through the dispensing tube into the container, thereby dispensing the cryoprotectant into the sperm cell fluid. Mixing of the fluids is achieved using agitation with a vibration table.

Description

[0001] This application is a divisional application of the following parent application:

[0002] International application data: PCT / US2020 / 028850 2020.04.17;

[0003] Entering the national phase: October 12, 2021;

[0004] Application date: April 17, 2020;

[0005] Application number: 202080028183.1;

[0006] Invention title: System and process for continuously adding cryoprotectant.

[0007] Cross-reference content

[0008] This application claims the benefit of U.S. Provisional Application No. 62 / 835,676, filed April 18, 2019, the entire contents of which are incorporated herein by reference. Technical Field

[0009] This invention generally relates to the processing and cryopreservation of semen. In particular, this invention discloses systems and methods in which fluid is added with high precision and continuously under agitation to optimize the cryopreservation of sperm cells. Background Technology

[0010] Cryopreservation of human sperm involves the stability of sperm cells at low temperatures. This technique is commonly used in artificial insemination procedures or to preserve male fertility. During cryopreservation, a cryoprotectant is added to the sperm sample to protect the sperm from freezing damage. A drawback of this method is that the absorption of the cryoprotectant can stress the sperm cells, leading to cell death. It is believed that a large mismatch between the cryoprotectant concentration and the sperm cell concentration only increases this applied stress. Previously known solutions have included adding the cryoprotectant medium dropwise by means of droplets detached from a suspended dispensing tube, or a gradual addition comprising multiple discrete additions spaced several minutes apart.

[0011] In dropwise addition, a setup similar to burette titration is used. The cryoprotectant medium (cryoprotectant) is added dropwise over an extended period. In stepwise or batch addition, the cryoprotectant is suspended in a container above the sperm sample. A stopcock valve connected to the container is used to start / stop the flow. There is no deliberate control of the flow rate; instead, mass balance is used to estimate the volume to be delivered. The addition of the cryoprotectant is completed within minutes to read the total volume to be added. For example, the cryoprotectant can be added in three discontinuous batches at 15-minute intervals. These processes still result in cell death; therefore, it is necessary to reduce the rate of cell death during the addition of cryoprotectant medium to sperm cells. Summary of the Invention

[0012] The object of this invention is to provide systems and methods capable of mitigating the harmful effects of cryopreservation on sperm integrity, as described in the independent claims. Embodiments of the invention are given in the dependent claims. Where embodiments of the invention are not mutually exclusive, they may be freely combined with each other.

[0013] In some aspects, the present invention features an integrated cryopreservation system that can improve sperm cell viability and processing yield. In exemplary embodiments, the invention features a dispensing system comprising a container, a controlled dispenser, and a dispensing tube. The controlled dispenser can be configured to dispense fluid through the dispensing tube and dispense fluid into the container. A non-limiting example of the controlled dispenser is an injection pump comprising a syringe for containing fluid (e.g., a cryoprotectant) and a push mechanism for moving the syringe plunger. The injection pump can be configured to dispense fluid into the container through the dispensing tube as the syringe is moved by the push mechanism. In some embodiments, the dispensing tube has a first end fluidly connected to the outlet of the controlled dispenser and a second end disposed within the container. In one embodiment, the second end may contact material contained within the container. The material contained within the container may be cells, such as sperm cells, in a sample medium (i.e., a liquid medium containing, for example, salts, sugars, buffers, proteins, antibiotics, lipids, antioxidants, etc.). In the case of sperm cells, the sperm cells can be sexed (i.e., post-processed) or non-sexed (i.e., “routine”). The system can also be used for other cell types, such as cell cultures, oocytes, and embryos. Alternatively, the second end can contact the inner surface of the container.

[0014] According to some embodiments, the actuation mechanism may include a movable pusher, a motor operatively coupled to the pusher, and a motor controller for controlling the motor. The pusher and syringe may be arranged on a track and aligned such that the pusher can press against the plunger of the syringe. The motor is configured to move the pusher along the track in a direction that pushes the plunger into the barrel of the syringe, thereby displacing the syringe. Displacement of the syringe ejects fluid contained in the barrel through an outlet into a dispensing tube. The fluid exits the dispensing tube through a second end and enters a container.

[0015] In other aspects, the dispensing system may also include a mixer for mixing the contents of the container. In another embodiment, the dispensing system may also include one or more additional controlled dispensers, and a dispensing tube that fluidly connects each controlled dispenser to the container. When the actuating mechanism moves the syringe, the additional dispensers can dispense the same or different fluids, such as cryoprotectants or gases, into the container through their respective dispensing tubes.

[0016] In one embodiment, the controlled dispenser can continuously dispense fluid. In a non-limiting example, the actuation mechanism can move the syringe plunger such that fluid is continuously dispensed from a second end of a dispensing tube immersed in sperm cells. In another embodiment, the controlled dispenser can continuously dispense fluid, thereby preventing droplet formation, and the fluid is dispensed without dripping into the container. In yet another embodiment, the controlled dispenser can dispense at a flow rate less than or equal to the maximum flow rate f. max The flow rate distributes the fluid into the container, the maximum flow rate f max Determined by the following formula: f max (ml / min)=0.0045 * V, where V = material volume in ml. In alternative embodiments (e.g., embodiments with a shorter dispensing time of approximately 45 minutes), the maximum flow rate f max It can be determined by the following formula: f max (ml / min)=0.0056 * V, where V = material volume, in ml. The maximum flow rate is determined by the volume of cryoprotectant (which in turn is determined by the sample size being measured), which is added over a set time period. Typically, the addition time is about 60 minutes, but in some cases it can be about 45 minutes. For larger samples, a larger volume of cryoprotectant will be required, and therefore a higher maximum flow rate will be required. Therefore, the addition time can be approximately constant, independent of the sample size and the volume of cryoprotectant added, because the total flow rate can be linearly proportional to the volume.

[0017] According to other embodiments, the cryopreservation system of the present invention can be used in methods for preparing cryopreserved biological samples. When a syringe pump is used as a controlled dispenser, the method may include adding a cryoprotectant to a syringe, adding a biological sample to a container, connecting the syringe to the container via a dispensing tube, and dispensing the cryoprotectant into the container at a desired flow rate, thereby adding the cryoprotectant to the biological sample. In one embodiment, one end of the dispensing tube may contact the biological sample or the inner surface of the container. In some embodiments, the cryoprotectant is continuously dispensed into the container. In a preferred embodiment, the syringe is dispensed to prevent droplet formation, and the cryoprotectant is dispensed without dripping into the container. In other embodiments, the cryoprotectant is dispensed at a flow rate less than or equal to a maximum flow rate f. max The flow rate is distributed into the container, the maximum flow rate f max Determined by the aforementioned equation.

[0018] A unique technical feature of this invention, compared to batch or dropwise addition, is the continuous addition of cryoprotectant to the sample. The same volume of cryoprotectant medium, such as a glycerol-based cryoprotectant, can be added at a slow and continuous rate over a specific time period. Another technical feature of this invention is that the dispensing tube is immersed in or in contact with the inner surface of the container, allowing the cryoprotectant to be added to the sperm cell solution with low or soft impact. This feature allows for the gradual introduction of the cryoprotectant into the sperm cell solution in a manner that reduces cellular stress. Another technical feature of this invention is the dilution addition of the cryoprotectant, wherein the cryoprotectant enters the sperm cell fluid in a relatively low volume to reduce cytotoxicity to local cells at or near the point of entry. Without wishing to be limited by a particular theory or mechanism, these technical features are believed to reduce process risk and cell death, thereby improving sperm cell survival and process yield. No existing references or works currently known possess the unique inventive features of this invention.

[0019] Furthermore, the inventive technical features of this invention have led to surprising results. Following extensive experimentation, it was unexpectedly found that, by using a continuous immersion process, cell viability increased by an average of 5% compared to a stepwise addition process with three batches added at 15-minute intervals. In both processes, the ratio of sample volume to dispensed cryoprotectant volume was the same. Moreover, it was surprisingly found that cells prepared using the cryopreservation preparation process of this invention exhibited an 8% increase in viability after the freeze / thaw process. Therefore, this invention is surprisingly capable of increasing the processing yield of sperm cells, a result that would not be easily predicted or conceived by one of ordinary skill in the art. Any feature or combination of features described herein is included within the scope of this invention, provided that the features included in any such combination are not contradictory, as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art. Further advantages and aspects of the invention will become apparent from the following detailed description and claims. Attached Figure Description

[0020] The features and advantages of the invention will become apparent from the following detailed description given in conjunction with the accompanying drawings, in which:

[0021] Figure 1 A non-limiting embodiment of the dispensing or cryopreservation system of the present invention is shown.

[0022] Figure 2A-2B A non-limiting example of a controlled allocator in an allocation system is shown. Figure 2A A partially displaced syringe pump was described. Figure 2B The syringe pump in a fully displaced position is depicted.

[0023] Figure 3 Another non-limiting embodiment of the cryopreservation system is shown.

[0024] Figure 4A This is another non-limiting implementation of the cryopreservation system. Figure 4B An implementation scheme that can process multiple samples simultaneously is shown.

[0025] Figure 5A The initial position of the syringe clamp is shown. Figure 5B The displacement position of the syringe clamp holding the syringe is shown.

[0026] Figure 6A The initial position of the inductive position sensor connected to the syringe clamp is shown; the position change is proportional to the voltage change. Figure 6B The sensor's displacement position is shown when the syringe holder holds the syringe.

[0027] Figure 7This is a non-limiting schematic diagram of the pumping mechanism of the present invention.

[0028] Figures 8A-8C This is a non-limiting example of a process flow diagram for dispensing cryoprotectants according to the present invention.

[0029] Figure 9 A non-limiting example of a feed stream for distributing cryoprotectant is shown. Detailed Implementation

[0030] The following is a list of components corresponding to the specific components mentioned in this article:

[0031] 100 System

[0032] 102 Fluids or cryoprotectants

[0033] 104 biological samples

[0034] 110 Container

[0035] 112 The inner surface of the container

[0036] 115 Controlled dispensers, such as syringe pumps

[0037] 120 syringes

[0038] 122 plunger

[0039] 124 tubes

[0040] 126 Discharge outlet

[0041] 127 Syringe clamp

[0042] 129 Position Sensor

[0043] 130 Promoting Organizations

[0044] 132 Push Blocks

[0045] 134 motor

[0046] 136 Motor Controller

[0047] 138 orbits

[0048] 140 Distribution Pipe

[0049] 142 First end of the distribution pipe

[0050] 144 The second end of the distribution pipe

[0051] 150 Mixer

[0052] As used herein, “cryoprotectant” refers to a chemical substance added to a biological sample to protect the sample from freezing damage during cryopreservation. The term “cryoprotectant” may be used interchangeably with the terms “cryoprotectant medium” or “cryoprotectant medium.” Examples of cryoprotectants include, but are not limited to, glycerol, ethylene glycol, dimethyl sulfoxide (DMSO), sucrose, trehalose, glucose, fructose, tris(hydroxymethyl)aminomethane (TRIS), TRIS B, tri-fructose-egg yolk-glycerol (TFEG), citric acid, and 1,2-propanediol.

[0053] As used herein, a “controlled dispenser” refers to any device capable of delivering fluids (e.g., cryoprotectants or gases) at a controlled flow rate. In some preferred embodiments, the dispenser is automated. In other embodiments, the dispenser may be non-automated. Non-limiting examples of controlled dispensers that can be used according to the invention include syringe pumps, mechanical pumps, peristaltic pumps, gravity pumps, direct lift pumps, microfluidic pumps, centrifugal pumps, compressors, gas regulators, or any positive displacement pump. In some preferred embodiments, the dispenser may be any flow controller capable of delivering fluids with a precision similar to that of a syringe pump. In another example, the controlled dispenser may include a container having a controlled pressure above the fluid and a downstream flow rate measurement. A simple feedback loop controls the pressure to a setpoint based on the flow rate error. Alternatively, a throttle valve may be used to control the flow rate rather than the pressure above the fluid.

[0054] As used herein, the term "syringe" refers to a positive displacement pump comprising a barrel and a movable (e.g., slidable) plunger into the barrel. The plunger may include a seal that allows the plunger to generate suction when the plunger is pulled away from the barrel, thereby drawing fluid in and retaining it within the barrel. Pushing the plunger into the barrel forces, for example, the retained fluid out of the barrel. As used herein, when used in conjunction with a syringe, the term "displacement" and its derivatives refer to the pushing of the plunger into the barrel.

[0055] Now for reference Figure 1 In one embodiment, the invention is characterized by a dispensing system (100) comprising a container (110), a controlled dispenser (115), and a dispensing tube. In some embodiments, the dispensing tube (140) has a first end (142) in fluid connection with an outlet (126) of the controlled dispenser and a second end (144) disposed within the container (110). In one embodiment, the second end (144) may contact material (104) contained within the container. In another embodiment, the second end (144) may contact the inner surface (112) of the container, such as... Figure 3As shown. In the following text, the controlled dispenser may be an example of a syringe pump; however, it should be understood that the controlled dispenser is not limited to a syringe pump, and it may be any device suitable for delivering fluid and controlling its flow rate. In some embodiments, the syringe pump (115) may include a syringe (120) for containing fluid (102) and a push mechanism (130) for moving said syringe (120). The syringe pump (115) may be configured to dispense fluid into the container (110) through a dispensing tube (140) when the syringe (120) is moved by the push mechanism (130).

[0056] According to some embodiments, the dispensing system (100) of the present invention can be used in cryopreservation processes. For example, in one embodiment, the present invention is characterized by a cryopreservation system (100) for dispensing a cryoprotectant (102) to a biological sample (104). In some embodiments, the cryoprotectant (102) may include glycerol, ethylene glycol, dimethyl sulfoxide (DMSO), 1,2-propanediol, or any other suitable substance that can prevent damage to the biological sample during freezing.

[0057] In one embodiment, the biological sample (104) may include sperm cells. Sperm cells may include sex-classified or sex-biased cells, wherein they primarily contain either the X or Y chromosome. For example, sperm cells may be sex-classified to primarily contain the X chromosome to increase the likelihood that the resulting fetus will be female. In another embodiment, sperm cells may be unclassified. In other embodiments, the invention is not limited to sperm cells. According to the invention, other types of samples can be cryopreserved. For example, biological samples may include tissues, stem cells, bone marrow, embryos, eggs, oocytes, ovum cells, amniotic fluid and umbilical cord, liver cells, blood cells, neurons, organs, teeth, plant seeds, and microorganisms.

[0058] In one embodiment, the cryopreservation system (100) may include a container (110) for containing a biological sample (104), an injection pump (115) including a syringe (120) for containing cryoprotectant (102), a push mechanism (130) for moving the syringe (120), and a dispensing tube (140) having a first end (142) in fluid connection with an outlet (126) of the syringe and a second end (144) disposed within the container (110). When the syringe (120) is moved by the push mechanism (130), the injection pump (115) may dispense the cryoprotectant (102) through the dispensing tube (140) and dispense it into the container (110).

[0059] In some embodiments, the second end (144) may contact the inner surface (112) of the container. For example, the second end (144) may be positioned directly in contact with or adjacent to the inner surface (112) of the container. In other embodiments, the second end (144) may contact the biological sample (104) when it is contained in the container (110). For example, the second end (144) of the tube may be immersed in the biological sample (104). In a preferred embodiment, by bringing the second end (144) of the tube into contact with the biological sample (104) or the inner surface (112) of the container, the dispensed cryoprotectant (102) is prevented from dripping into the container (110). As used herein, the term “droplet” and its derivatives refer to a droplet detached from the suspended dispensing tube.

[0060] In one embodiment, the inner surface (112) of the container may include the inner side surface or the inner bottom surface of the container. In some embodiments, the container (110) may be a flask, a tube, or any container with sufficient volumetric capacity to contain the cryoprotectant (102) and the biological sample (104). For example, the container (110) may have a volumetric capacity of about 20 ml to about 100 ml. In another embodiment, the capacity of the container (110) may be about 100 ml to about 250 ml or about 250 ml to about 500 ml. In yet another embodiment, the capacity of the container (110) may be greater than 500 ml. In other embodiments, the container (110) may be a closed or sealed container. Figure 4A As shown, the container may include a lid having an inlet for receiving the dispensing tube (140), thus allowing the container to be sealed while still providing access to the interior of the container. In some embodiments, the container (110) may be made of a rigid or semi-rigid material, such as glass or plastic. In other embodiments, if the sample is photosensitive, the material may be opaque or substantially transparent to visibility into the container.

[0061] In one embodiment, if the second end (144) of the dispensing tube is in contact with the biological sample (104), the cryoprotectant (102) is added directly to the biological sample (104). For example, when the cryoprotectant (102) is added to the container, the second end (144) is immersed in both the biological sample (104) and the cryoprotectant (102). In another embodiment, if the second end (144) of the dispensing tube is in contact with the inner surface of the container but not with the biological sample (104) (e.g., the container sidewall), the cryoprotectant (102) can be added indirectly to the biological sample (104). Figure 3As shown, if the second end (144) contacts the container sidewall, the cryoprotectant (102) flows along the container sidewall as it is gradually added to the sample. In either embodiment, droplet formation is disrupted, and the dispensed cryoprotectant (102) does not drip into the container (110).

[0062] To avoid limiting the invention to a specific device, the syringe pump can control the delivery rate of the cryoprotectant via precise displacement of the syringe plunger. In one embodiment, the addition rate is programmably set as a function of cell volume. Since the batch size of sperm cells can vary considerably depending on the number of cells obtained from semen, a wide range of addition volumes must be accommodated. In some embodiments, the volume of the sperm cell fluid can range from about 1 ml to about 100 ml per semen sample. In a preferred embodiment, the total volume of the added cryoprotectant can be from about 10% to about 25% of the sperm cell sample volume. For example, a 25 ml semen sample might require about 20% cryoprotectant, thus the cryoprotectant volume would be about 5 ml.

[0063] In some embodiments, the total cryogenic medium addition for each batch lasts approximately 60 minutes. In other embodiments, the addition time can be approximately 45 to 90 minutes. Since the total volume of the cryogenic medium varies with the sperm cell volume, the flow rate is set based on the target volume divided by the addition time. In one embodiment, the flow rate of the dispensing fluid (also referred to as the addition rate) can be in the range of approximately 0.01 ml / min to approximately 1 ml / min. For example, the flow rate could be 0.017–0.333 ml / min. In another embodiment, the flow rate could be approximately 0.01 ml / min to approximately 0.05 ml / min or approximately 0.05 ml / min to approximately 0.1 ml / min. In yet another embodiment, the flow rate could be approximately 0.1 ml / min to approximately 1 ml / min or greater than 1 ml / min. Continuing with the previous example, 5 ml of cryogenic medium can be continuously added to a 25 ml semen sample over a time span of approximately 60 minutes, thus the cryogenic medium flow rate is approximately 0.083 ml / min.

[0064] like Figure 2A-2B As shown, the actuating mechanism (130) may include a movable pusher (132) disposed on a track (138). The pusher (132) and the syringe (120) may be arranged on the track (138) such that the pusher (132) is aligned with the syringe (120). The pusher (132) may be moved along the track (138) to press against the plunger (122) of the syringe. Figure 5A-7As shown, the actuation mechanism (130) also includes a motor (134) operatively coupled to the pusher (132), and a motor controller (136) for controlling the motor (134). The motor (134) can be configured to move the pusher (132) along a track (138) in a direction that pushes the plunger (122) into the barrel (124) of the syringe, thereby displacing the syringe (120) to spray fluid (102) (e.g., a cryoprotectant) contained in the barrel (124) through the outlet (126) into the dispensing tube (102). The fluid (102) then exits the dispensing tube (140) through a second end (144) and enters a container.

[0065] like Figure 7 As shown, in some implementations, an external processor, such as a computer, is used to control the entire system. The computer can execute LabVIEW software to operate the system. For example, upon receiving user input that can be entered via a peripheral device such as a mouse or keyboard, the computer transmits a signal to a motor controller that can be integrated into the syringe pump. In one implementation, the motor controller (136) may include a processor and a memory operatively coupled to the processor. The memory may store a set of instructions that, when executed by the processor upon receiving a signal from the computer, cause the motor (134) to move the pusher (132) along a track in the direction of the dispensing syringe (120) with desired step sizes. Thus, cryoprotectant (102) is added to the biological sample (104) at a desired flow rate.

[0066] In some implementations, continuous flow can refer to smooth or non-discrete flow, steady flow, or uninterrupted flow. In one implementation, the pusher (132) displaces the syringe (120) such that cryoprotectant (102) is added to the biological sample (104) at a continuous flow rate. For example, the motor moves the pusher (132) in specific steps such that the cryoprotectant flow rate is approximately 0.083 ml / min, to continuously dispense 5 ml of cryoprotectant into a 25 ml semen sample over approximately 60 minutes, such as... Figure 9 As shown, feed A.

[0067] In other embodiments, the pusher (132) can displace the syringe (120) such that the cryoprotectant (102) is added to the biological sample (104) at a pulsed or intermittent flow rate. A pulsed or intermittent flow rate refers to a flow rate with discrete interruptions in the flow; however, the flow is not dropwise. For example, 25% of the cryoprotectant volume is slowly and continuously added to the sample over a 10-minute time span, then paused for 5 minutes, and then repeated until the entire volume of cryoprotectant has been dispensed. As another example, 10% of the cryoprotectant volume is slowly and continuously added to the sample over a 3-4 minute time span, then paused for 2-3 minutes, and then repeated until the entire volume of cryoprotectant has been dispensed. Various non-limiting examples of pulsed or intermittent flow are described in [the following text is missing from the original extract]. Figure 9 The diagram shows feed BG. In some embodiments, the pulse flow rate can be a stable or constant flow rate, for example, in feeds BD and FG. Alternatively, the cryoprotectant can be fed at varying pulse flow rates (e.g., non-constant flow rates) during the flow process shown in feed E. In a preferred embodiment, the cryoprotectant is dispensed in a manner and / or at a flow rate that inhibits droplet formation.

[0068] In one implementation, if the linear step size of the injection pump is fixed, the added accuracy can be a function of the syringe bore diameter. To achieve the same accuracy over a range of added volumes, syringes of various sizes can be used as a percentage of the delivered volume to the target volume. In some implementations, the control software specifies the syringe size based on the volume of sperm cells. If an incorrect syringe is placed on the system, the amount of cryoprotectant medium delivered may be significantly greater or less than the specified amount. This error corresponds to a significant loss of cells due to initial cell death (overdelivery) during the process or increased cell death (underdelivery) measured after thawing of cryopreserved cells. This risk can be mitigated by using a position sensor (128) to determine the outer diameter of the syringe placed on the system. Figures 5A-6B As shown, a position sensor (128) can be connected to a syringe holder (127). When the syringe holder holds the syringe, the sensor is displaced, and this change in sensor position is proportional to a change in voltage, which is related to the outer diameter of the syringe.

[0069] In a preferred embodiment, the syringe used according to the invention should be biocompatible with the sample. The syringe should be free of lubricants or other substances that may be harmful to sperm cells. A non-limiting example of a syringe suitable for use in the invention is the NORM-JECT luer locking syringe. In some embodiments, the syringe volume capacity can range from about 2 ml to about 50 ml. In one embodiment, the syringe volume capacity can be from about 2 ml to about 5 ml. In another embodiment, the syringe volume capacity can be from about 5 ml to about 20 ml. In yet another embodiment, the syringe volume capacity can be from about 20 ml to about 50 ml. In a further embodiment, the syringe volume capacity can be greater than 50 ml.

[0070] In other embodiments, the dispensing tube may be made of plastic, rubber, or silicone materials. For example, the dispensing tube may be made of PVC, DEHP-free PVC, etc. The tubing is made of high-density polyethylene, platinum-cured silicone, or peroxide-cured silicone. Furthermore, the tubing material can be of medical-grade quality. In one embodiment, the dispensing tube may be made of PEEK with an OD of 1 / 16” and an ID of 0.020”. The inner diameter of the tube can vary from about 0.01” to about 0.1”. For example, the inner diameter may be 0.020”. The dispensing tube may be of a predetermined length or optionally cut to the desired length to adequately cover the distance between the syringe and the container. In some embodiments, the tubing material may be opaque. Alternatively, the tubing material may be substantially transparent to allow visibility within the tube, for example, to observe any blockages within the tube. In other embodiments, the dispensing tube may be flexible, semi-rigid, or rigid. Tubing expansion under pressure can increase errors in the target delivery volume. To ensure accuracy, the tube may have a high OD / ID ratio and be made of a rigid material, thereby reducing the risk of tubing expansion.

[0071] In the prior art, suspended containers with stopcocks or burettes for dispensing have physical limitations that prohibit the immersion of the dispensing tip. For example, the typical opening size of the container or the size of the burette tip limits the ability to immerse the tip. Furthermore, by using a burette typically made of brittle glass, physical contact with the container wall applies bending forces to the glass, putting it at risk of breakage. Radial agitation of the container only increases the risk of breakage. In contrast, the dispensing tube of the present invention allows for both immersion of the dispensing tip and agitation of the container.

[0072] Once sperm cells are packaged and sold, each batch undergoes bacterial testing. If bacterial levels exceed a threshold, the batch is not sold because it may pose a health risk. Furthermore, preventing cross-contamination between batches is paramount. Therefore, it is necessary to prevent the freezing medium from being contaminated by bacteria and / or other cells. This invention addresses these problems by using a reusable or single-use fluid path. Without wishing to be limited to a particular theory or mechanism, the entire fluid path eliminates the risk of contamination from bacteria and cross-contamination between different samples (e.g., male-to-male).

[0073] In some embodiments, the dispensing tube and container can be reused through a washing and autoclaving process. In other embodiments, the syringe can be discarded after use. In a preferred embodiment, the dispensing tube is immersed in a fluid containing sperm cells. By immersing the end of the dispensing tube rather than suspending it, the dosing rate is controlled by an injection pump, rather than by the adhesive force of droplets suspended from the end of the dispensing tube.

[0074] In a further embodiment, the system (100) may also include a mixer (150) for mixing the cryoprotectant (102) and the biological sample (104) contained in the container (110). Without limiting the invention, the mixer (150) can prevent the biological sample (104) from adhering to the inner surface (112) of the container. In one embodiment, the mixer (150) may be a vibration table. As an example, the vibration table may include a fixed base and a movable platform that supports the container. Figure 4A As shown, a container (110) can be placed on a vibrating table that agitates the fluid in the container, thereby mixing the cryoprotectant medium into the fluid containing sperm cells and reducing the local concentration at the end of the dispensing tube. If the radius of rotation of the device is fixed, the agitation rate of the vibrating table can be programmed to be set by the rotation speed. While the vibrating table can reduce contamination by eliminating contact with the contents of the container, other types of mixing devices can also be used with the system, such as magnetic stirring rods with agitation plates or overhead mixers with impellers, blades, or paddles. However, acoustic mixing can be implemented according to the invention because the sperm cells may be physically damaged by the submerged rotating object, as shear forces on them need to be minimized. For example, acoustic mixing may involve acoustic treatment, which is agitation using acoustic energy.

[0075] like Figure 4BAs shown, in some embodiments, the dispensing system (100) can be a multi-dispensing system that implements multiple controlled dispensers and containers to process multiple samples simultaneously. In some embodiments, each container may contain its own sample, which may be the same as or different from the samples in other containers. Each container may be coupled to its own controlled dispenser, such as an infusion pump, to dispense its own fluid. The fluid in each dispenser may be the same as or different from the fluid in other dispensers. For example, one dispensing system may be used to dispense glycerol into a semen sample, while a second dispensing system may be used to dispense glycerol into another semen sample from the same or different sources. As another example, one dispensing system may be used to dispense glycerol into a tissue sample, while a second dispensing system may be used to dispense glycerol into a semen sample. In another embodiment, one dispensing system may be used to dispense glycerol into an embryo sample, while a second dispensing system may be used to dispense DMSO into a semen sample. Containers may be placed on a single vibration table, provided that the vibration table is capable of supporting and mixing multiple containers. Alternatively, each container may have its own mixer.

[0076] In other embodiments, the multi-dispensing system can implement multiple controlled dispensers, such as syringe pumps, fluidly coupled to a single container. This allows for shorter processing times by dispensing cryoprotectant fluids from multiple dispensers. Alternatively, the dispensers can dispense various fluids (e.g., cryoprotectants or gases) into a single container simultaneously or sequentially. For example, the dispensing system may include multiple syringe pumps that dispense various fluids into a single container for vitrification of sperm cells. Each fluid can be dispensed at its own independent flow rate and timing. In one embodiment, the dispensing system may include a syringe pump dispensing cryoprotectant and a second controlled dispenser dispensing gas into a single container. Each fluid can be dispensed at its own independent flow rate and timing. In one embodiment, the multi-dispensing system can be configured to dispense two or more different media added to the cells, thereby defining the delivery similar to a recipe. The flow rate of each medium can be varied independently, and / or the delivery between media can be coordinated, for example, by increasing the flow rate.

[0077] In some embodiments, the container may be equipped with a cap that creates a gas seal, allowing gas to be added and contained within the container. The cap may include a port for receiving gas and other ports for receiving liquid. For example, the preparation of samples for in vitro fertilization may require a lower oxygen concentration and / or some type of gas coverage in the container to improve viability. In one embodiment, the container may be sealed and purged with argon, CO2, and / or nitrogen, which may be added via one or more ports, each connected to its own pump or gas regulator. A separate outlet may be used to maintain atmospheric conditions and prevent pressure build-up in the container. In another embodiment, the process may be operated under reduced pressure by connecting a port to a vacuum pump. In some embodiments, the cryogenic medium may be added along with the gas via a separate inlet and pump. In conjunction with or as an alternative to a syringe pump, other types of pumps, such as microfluidic pumps, centrifugal pumps, compressors, gas regulators, or other positive displacement pumps, may be used according to the invention to dispense the cryogenic medium, gas, or other fluids.

[0078] According to some embodiments, the dispensing system of the present invention can achieve a temperature ramp during cryopreservation. When preparing biological samples for cryopreservation, the samples and cryoprotectants are preferably in a temperature-controlled environment. In a preferred embodiment, the samples and cryoprotectants are maintained at a temperature of about 5°C. Any temperature deviation should not exceed ±2°C. For example, a temperature deviation can be ±1°C. In some embodiments, the entire system can be located in a temperature-controlled room. If a dedicated cold storage room is not available, at least the syringe pump and container can be contained in a temperature-controlled room, such as a cold box, to maintain a constant temperature for the samples and cryoprotectants. In another embodiment, the container and / or syringe can be jacketed with a heating / cooling device. Temperature feedback can be achieved by thermocouples or thermistors, which can be immersed in the sample solution or attached to the container.

[0079] According to some aspects, the system described herein can be used in methods for dispensing cryoprotectants into biological samples, such as... Figures 8A-8C As shown. As a crucial initial step, based on the sample quantity, the mass of the sample is determined by pre-weighing the container, adding the sample, weighing the combined container and sample, and subtracting the mass to determine the sample's mass. The appropriate volume of cryoprotectant can then be calculated, thus determining the volume of cryoprotectant to be added. In... Figure 8AIn one embodiment shown, the method may include providing any of the cryopreservation systems (100) described herein, fluidly connecting cryoprotectant (102) to a controlled dispenser (115), adding a biological sample (104) to a container (110), and activating the controlled dispenser (115) to continuously dispense the cryoprotectant (102) through a dispensing tube (140) and dispense it into the container (110). In one embodiment, the controlled dispenser (115) includes an injection pump having a syringe and pumping mechanism as described above. Preferably, a push mechanism (130) moves the syringe (120) such that the cryoprotectant (102) is continuously added to the biological sample (104) at a desired flow rate. This prevents droplet formation, allowing the cryoprotectant (102) to be dispensed without dripping into the container (110).

[0080] In such Figure 8B In another embodiment shown, the method may include providing any of the cryopreservation systems (100) described herein, adding a desired volume of cryoprotectant (102) to a syringe (120), adding a biological sample (104) to a container (110), and activating a push mechanism (130) to move the syringe (120). By moving the syringe, the cryoprotectant (102) is dispensed into the container (110) through a dispensing tube (140). Preferably, the push mechanism (130) moves the syringe (120) such that it is at a flow rate less than or equal to the maximum flow rate f. max The cryoprotectant (102) is added to the biological sample (104) at a flow rate of f, wherein the maximum flow rate f max Determined by the following formula:

[0081] f max (ml / min)=0.0045 * V, where V = volume of the biological sample, in ml.

[0082] Returning to the previous example, 5 ml of cryoprotectant was dispensed into a 25 ml semen sample. max The calculated flow rate is 0.11 ml / min. Therefore, the flow rate of the cryoprotectant should be less than or equal to 0.11 ml / min, with a minimum dispensing time of approximately 45 minutes. The previous example flow rate of 0.083 ml / min and dispensing time of 60 minutes fall within these parameters.

[0083] Now for reference Figure 8CAnother embodiment of the method may include providing any of the cryopreservation systems (100) described herein, adding a desired volume of cryoprotectant (102) to a syringe (120), adding a biological sample (104) to a container (110), adjusting a second end (144) of a dispensing tube such that the second end (144) contacts the biological sample (104) or the inner surface (112) of the container, and actuating a pushing mechanism (130) to move the syringe (120). By moving the syringe, the cryoprotectant (102) is dispensed into the container (110) through the dispensing tube (140). Preferably, the pushing mechanism (130) moves the syringe (120) such that the cryoprotectant (102) is added to the biological sample (104) at a desired flow rate. More preferably, by bringing the second end (144) of the dispensing tube into contact with the inner surface (112) of the container or the biological sample (104), the dispensed cryoprotectant (102) is prevented from dripping into the container (110).

[0084] Consistent with the preceding embodiments, the method described herein may further include continuously or intermittently mixing the cryoprotectant (102) and the biological sample (104) contained in the container (110). The cryoprotectant (102) and the biological sample (104) may be mixed using any of the mixing devices described herein. In a further embodiment, the method may further include adding one or more additional fluids to the container by activating one or more additional controlled dispensers fluidly connected to the container, each dispenser containing one additional fluid. The method may utilize a multi-dispensing system as described above. In some embodiments, the one or more additional fluids may be a cryoprotectant or a gas. For example, a second fluid may be added to the container by activating a second injection pump having a syringe containing the second fluid and fluidly connected to the container.

[0085] Consistent with the preceding implementation, the method described herein may further include weighing the container at the end of the addition process to verify that the correct mass of cryoprotectant has been added. At this stage, if it is determined that the mass is lower than it should be, additional cryoprotectant may be added manually or automatically to bring the total amount of cryoprotectant to the calculated amount. If the volume added manually or in stages is large compared to the total calculated volume, it may be added in multiple sub-volume additions with waiting time intervals. As a non-limiting example, if the manually added volume is as high as 32% of the total volume, the remaining volume may be delivered in a single addition. If the manually added volume is 33-65% of the total volume, it may be divided into two equal volumes with a 15-minute rest period between additions. If the manually added volume is equal to or greater than 66% of the total volume, it may be divided into three equal volumes with two 15-minute rest periods between additions.

[0086] In some respects, high concentrations of cryoprotectants can be toxic to biological samples, such as sperm cells. Distributing cryoprotectants by adding them in large quantities or dropwise can deliver high concentrations to cells closest to the cryoprotectant entry point before the cryoprotectant diffuses into the cell solution, resulting in localized cytotoxicity to the cells. In other respects, dropwise addition of cryoprotectants can cause stress or physical damage due to droplet impact on cells. Without intending to limit the invention to a particular theory or mechanism, the invention allows for the addition of cryoprotectants in small volumes via slow, continuous flow, low-impact entry, and / or determined flow rates, which reduces local concentrations and thus minimizes cytotoxicity and cellular stress.

[0087] In some embodiments, the dispensing system of the present invention can be used in a cold storage compartment or a large refrigeration cabinet to maintain sample viability. In alternative embodiments, the system can be installed inside a refrigerator or mini-fridge. Some components of the system, such as the frame, may need to be reduced in size so that the frame, main injection pump, and vibration table are all suitable for installation inside the refrigerator or mini-fridge. In some embodiments, a secondary frame may be located on the exterior of the refrigerator or mini-fridge (e.g., on top of the refrigerator or mini-fridge), and components such as a computer, power supply, and monitor may be mounted to the secondary frame.

[0088] In some implementations, the pump plunger may also be designed to function as a spring-loaded clamp holding the syringe within the pump and block. Certain plungers and clamps can lead to inconsistent measurement results. As a non-limiting example, the distance from the unit's sensor to the tag can be altered, which in turn can cause the system to incorrectly identify which syringe is loaded into the pump. Therefore, a dedicated plunger may be advantageous. Additionally, a dedicated injection pump cap may be advantageous to provide safer cable management and easier maintenance removal.

[0089] Example

[0090] The following are non-limiting examples of the invention. It should be understood that these examples are not intended to limit the invention in any way. Equivalents or alternatives are within the scope of the invention.

[0091] A cryopreservation system was implemented and compared with existing cryopreservation procedures. This system adds cryoprotectant at a continuous flow rate, while the existing process involves three discontinuous additions at 15-minute intervals. In both processes, the ratio of sample volume to dispensed cryoprotectant volume was the same. The present invention resulted in a 5% cell recovery, thus providing an average of 5% more cells than the existing process. It is not intended to limit the invention to a specific theory or mechanism; the cryopreservation system reduces cell death by cryopreserving from freezing to thawing. Furthermore, the present invention increases the fertilization dose in the pipette by 8% compared to the existing process.

[0092] As used in this article, the term “about” means plus or minus 10% of the cited figure.

[0093] Although preferred embodiments of the invention have been shown and described, it will be apparent to those skilled in the art that modifications can be made thereto without departing from the scope of the appended claims. Therefore, the scope of the invention is defined only by the appended claims. In some embodiments, the figures presented in this patent application are drawn to scale, including angles, size ratios, etc. In some embodiments, the figures are merely representative, and the claims are not limited by the size of the figures. In some embodiments, the description of the invention using the phrase "comprising" herein includes embodiments that can be described as "consistently consisting of" or "consisting of," and thus satisfies the requirement of a written description claiming one or more embodiments of the invention using the phrase "consistently consisting of" or "consisting of."

[0094] The reference numerals used in the following claims are merely for the convenience of examination of this patent application and are exemplary, and are not intended to limit the scope of the claims in any way to specific features having the corresponding reference numerals in the drawings.

Claims

1. A cryoprotectant dispensing system (100), comprising: a) A container (110) for containing semen; b) The controlled distributor (115) is configured to operate at a flow rate less than or equal to the maximum flow rate f. max The flow rate dispenses the cryoprotectant (102) fluid into the container, the maximum flow rate being determined by the volume of cryoprotectant added within a set time, wherein the volume of cryoprotectant is determined by the amount of sperm cell fluid; c) A dispensing tube (140) having a first end (142) fluidly connected to an outlet (126) of the dispenser and a second end (144) disposed within the container (110), the second end (144) contacting the inner surface (112) of the container or being immersed in the semen contained within the container; The controlled dispenser (115) is configured to dispense the cryoprotectant (102) fluid through the dispensing tube (140) and distribute it into the container (110) to prevent the formation of droplets, and the cryoprotectant (102) is dispensed without dripping into the container (110); The system (100) also includes: d) A mixer (150) for mixing the cryoprotectant (102) and the semen contained in the container (110), the mixer (150) being a vibration table on which the container (110) is placed, the vibration of which agitates the cryoprotectant (102) and the semen, the mixer (150) preventing the semen from adhering to the inner surface (112) of the container.

2. The system as claimed in claim 1, wherein, The controlled dispenser (115) includes an injection pump comprising a syringe (120) for receiving the fluid and a push mechanism (130) for moving the syringe (120), wherein the push mechanism (130) includes a movable push block (132), a motor (134) operably coupled to the push block (132), and a motor controller (136) for controlling the motor (134), wherein the injection pump is configured to dispense the fluid when the syringe (120) is moved by the push mechanism (130).

3. The system as described in claim 2, wherein, The pusher (132) and the syringe (120) are arranged on a track (138), wherein the pusher (132) is aligned with the syringe (120) such that the pusher (132) can press against the plunger (122) of the syringe, wherein the motor (134) is configured to move the pusher (132) along the track (138) in a direction that pushes the plunger (122) into the barrel (124) of the syringe, thereby displacing the syringe (120), wherein the displacing of the syringe (120) will spray the fluid contained in the barrel (124) through the outlet (126) into the dispensing tube (140), wherein the fluid exits the dispensing tube (140) through the second end (144) and enters the container (110).

4. The system as claimed in claim 1, wherein, The controlled dispenser (115) is configured to continuously dispense the fluid into the container (110).

5. The system of claim 1, further comprising one or more additional controlled dispensers (115), and a dispensing pipe (140) fluidly connecting each controlled dispenser (115) to the container (110), wherein, The one or more additional controlled dispensers (115) are configured to dispense the same or different fluids into the container (110) through the dispensing tube (140).

6. A cryopreservation system (100) for dispensing a cryoprotectant (102) into a biological sample (104), the system (100) comprising: a) Container (110) for containing the biological sample (104); b) An injection pump (115) comprising a syringe (120) for receiving the cryoprotectant (102) and a push mechanism (130) for moving the syringe (120). c) A dispensing tube (140) having a first end (142) and a second end (144), the first end (142) being fluidly connected to the outlet (126) of the syringe, and the second end (144) being disposed within the container (110) such that when the biological sample (104) is contained in the container (110), the second end (144) contacts the inner surface (112) of the container or the biological sample (104); The syringe pump (115) is configured to dispense the cryoprotectant (102) through the dispensing tube (140) and into the container (110) when the syringe (120) is moved by the push mechanism (130), the syringe pump (115) dispensing at a flow rate less than or equal to the maximum flow rate f. max The flow rate dispenses the cryoprotectant (102) into the container (110), the maximum flow rate being determined by the volume of cryoprotectant added within a set time, wherein the volume of cryoprotectant is determined by the amount of sperm cell fluid; In this way, droplet formation is prevented by bringing the second end (144) of the tube into contact with the inner surface (112) of the container or the biological sample (104), and the cryoprotectant (102) is dispensed without dripping into the container (110). d) A mixer (150) for mixing the cryoprotectant (102) and the biological sample (104) contained in the container (110), the mixer (150) being a vibration table on which the container (110) is placed, the vibration of the vibration table agitating the cryoprotectant (102) and the biological sample (104), the mixer (150) preventing the biological sample (104) from adhering to the inner surface (112) of the container.

7. The system of claim 6, wherein, The second end (144) of the tube is immersed in the biological sample (104).

8. The system of claim 6, wherein, The pushing mechanism (130) includes a movable push block (132), a motor (134) operably coupled to the push block (132), and a motor controller (136) for controlling the motor (134).

9. The system of claim 8, wherein, The push block (132) and the syringe (120) are arranged on a track (138), wherein the push block (132) is aligned with the syringe (120) such that the push block (132) can press against the plunger (122) of the syringe, wherein the motor (134) is configured to move the push block (132) along the track (138) in a direction that pushes the plunger (122) into the barrel (124) of the syringe, thereby moving the syringe (120). The displacement of the syringe (120) sprays the cryoprotectant (102) contained in the cylinder (124) into the dispensing tube (140) through the outlet (126), wherein the cryoprotectant (102) leaves the dispensing tube (140) through the second end (144) and enters the container (110).

10. The system of claim 8, wherein, The motor controller (136) includes a processor and a memory operatively coupled to the processor and storing a set of instructions that, when executed by the processor, cause the motor (134) to move the pusher (132) in a desired step size in the direction of moving the syringe (120), thereby adding the cryoprotectant (102) to the biological sample (104) at a desired flow rate.

11. The system of claim 10, wherein, The pusher (132) moves the syringe (120) so that the cryoprotectant (102) is added to the biological sample (104) at a continuous flow rate.

12. The system of claim 10, wherein, The pusher (132) moves the syringe (120) so that the cryoprotectant (102) is added to the biological sample (104) at a pulsed flow rate.

13. The system of claim 6, further comprising one or more additional injection pumps (115), each additional injection pump comprising a syringe (120) containing another fluid and a push mechanism (130) for moving the syringe (120), and a dispensing tube (140) fluidly connecting each syringe (120) to the container (110), wherein, One or more additional injection pumps (115) are configured to dispense the other fluid into the container (110) via the dispensing tube (140) when the syringe (120) is moved by the push mechanism (130).

14. The system of claim 13, wherein, The other fluid is a gas.

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