TWI778747B - A sperm sorting device - Google Patents

Abstract

This invention provides a sperm sorting device, which includes a micro-heater and a microfluidic sorting chip. The micro-heater is distributed with a gradient temperature field. The microfluidic sorting chip is disposed on the micro-heater, and comprises a fluid inlet, a semen inlet, a selected sperm outlet, a waste outlet and a flow channel unit. The direction from the fluid inlet to the waste outlet is a flow field direction, the gradient temperature field of the micro-heater gradually decreases along the flow field direction.

Description

Sperm sorting device

The present invention relates to a sperm worm sorting device, in particular to a sperm worm sorting device which can improve the sperm worm recovery rate through the design of gradient temperature.

There are many causes of infertility, which can generally be divided into male factors, female factors, both factors, or unknown reasons. Among them, male factors account for about 40% of infertility, and most of them are due to poor quality of male sperm. Therefore, more and more attention has been paid to sperm screening in assisted reproductive technology.

Among the existing sperm sorting methods, the common ones are for example the upper swimming method or the density gradient centrifugation method. However, it takes a long time to carry out sperm sorting with these methods, and the longer the sorting time is, the easier it is to cause the sperm motility to decline. , In addition, when the sperm is sorted by centrifugation, the centrifugal force will easily break the sperm DNA, thereby causing the failure of artificial insemination.

Therefore, the object of the present invention is to provide a sperm sorting device.

Therefore, the sperm sorting device of the present invention includes a micro heating platform and a microfluidic sorting chip.

The micro heating platform is distributed with a gradient temperature field. The microfluidic sorting wafer is set on the micro heating platform and located on the gradient temperature field, and includes a sperm extraction port and a waste liquid outlet located at two opposite ends, and a sperm extraction port located at the same end and spaced apart The provided fluid injection port, a semen injection port arranged between the sperm worm extraction port and the fluid injection port, and a flow connecting the fluid injection port, the semen injection port, the sperm worm removal port, and the waste fluid outlet. A channel unit, the direction from the fluid injection port to the waste liquid outlet is the direction of the flow field, and the temperature of the gradient temperature field of the micro heating platform gradually decreases along the direction of the flow field.

The effect of the present invention lies in that, by utilizing the characteristics of reverse swimming of sperms, the direction of the flow field is opposite to the swimming direction of sperms, so that sperms with poor quality cannot compete with the flow field generated by the fluid in the flow channel, and are flushed to the rear end of the flow field. In the waste liquid outlet, the fine sperm and the fluid speed are balanced and can be taken out through the sperm extraction port. The sperm extraction port and the waste liquid outlet are designed at different outlets, which can ensure that the recovered sperm are all sorted high-quality sperm, and then Improve the recovery quality, and use the thermotaxis of sperm to cooperate with the micro heating platform, so that the high-motility sperm in the area with insufficient flow velocity at the rear end swim toward the sperm outlet, which greatly improves the sperm recovery rate.

Before the present invention is described in detail, it should be noted that in the following description, similar elements are designated by the same reference numerals.

Referring to FIGS. 1 to 3 , the sperm sorting device of the present invention comprises a micro-heating platform 2, a micro-fluidic sorting wafer 3 disposed on the micro-heating platform 2, and an injection extraction device communicated with the micro-fluidic sorting wafer unit 4.

The microfluidic sorting wafer 3 includes a body structure 30 , a fluid injection port 31 formed on the body structure 30 , a semen injection port 32 downstream of the fluid injection port 31 , and a semen injection port 32 . The downstream sperm extraction port 33, the waste liquid outlet 34 located at the two opposite ends of the sperm extraction port 33 and located downstream of the sperm extraction port 33, and the fluid injection port 31, the sperm injection port 32, the The sperm extraction port 33, and the flow channel unit 35 of the waste liquid outlet 34, that is, the sperm extraction port 33 and the waste liquid outlet 34 are located at two opposite ends, respectively, and the fluid injection port 31 and the sperm extraction port 33. At the same end and spaced apart, the semen injection port 32 is located between the sperm extraction port and the fluid injection port.

The flow channel unit 35 has a main channel 351 that communicates with the sperm extraction port 33 and the waste liquid outlet 34, and a connecting branch 352 that communicates with the fluid injection port 31, the semen injection port 32, and the sperm extraction port 33; Wherein, the direction from the fluid injection port 31 to the waste liquid outlet 34 is the direction F of the flow field. Preferably, in this embodiment, the size of the flow channel of the flow channel unit 35 is in the order of millimeters, so the flow channel unit 35 has a large throughput, so that a large number of specimens can be processed at the same time.

In this embodiment, the main flow channel 351 of the microfluidic sorting chip 3 is located between the sperm extraction port 33 and the waste liquid outlet 34 , and the main flow channel 351 goes from the sperm extraction port 33 to the waste liquid outlet 34 direction gradually expands to form a sperm sorting area 36, and from the sperm sorting area 36 equal width to the direction of the waste liquid outlet 34 to form an enhanced recovery area 37, and then from the enhanced recovery area 37 to the waste liquid outlet 34 The tapered extension forms a waste liquid region 38 .

The material for making the body structure 30 is not particularly limited, and can be selected from materials such as glass, quartz, poly(methyl methacrylate), or polydimethylsiloxane (PDMS), etc. In this embodiment, the body structure 30 is made of polydimethylsiloxane (PDMS) as an example for illustration.

The micro heating platform 2 includes a base 21 , a plurality of temperature plates 22 arranged on the base 21 and arranged in the same direction, and a control unit 23 connected to the temperature plates 22 . Specifically, the temperature plates 22 are arranged on the base 21 along the direction F of the flow field. The control unit 23 is used to connect the temperature plates 22 and adjust the temperature of the temperature plates 22 respectively. .

In detail, the control unit 23 includes a plurality of connection regulators 231 and a controller 232, the connection regulators 231 are respectively disposed at one end of the temperature plates 22, and the controller 232 is connected to the connection regulators The component 231 is used to control the respective connection control components 231 through the controller 232 to change the temperature of the corresponding temperature plate 22 , so that the micro heating platform 2 distributes a gradient temperature field.

Preferably, in this embodiment, the temperature of the gradient temperature field of the micro heating platform 2 is between 30°C and 40°C, and the temperature of the gradient temperature field gradually decreases along the flow field direction F. In the embodiment, the minimum temperature of the temperature plate 22 is 30°C as an example, and the temperature difference of each temperature plate 22 is maintained at 2°C, and the temperature plate 22 is composed of five pieces, so that the micro heating platform 2 The gradient temperature field is between 30°C and 38°C, but not limited to this.

The temperature plates 22 suitable for this embodiment are selected from, for example, indium tin oxide (ITO) conductive substrates, the connection control elements 231 are selected from variable resistors, and the controller 232 is selected from power supplies.

In detail, the micro heating stage 2 is generally fabricated in the following manner. First, the indium tin oxide (ITO) conductive glass substrate is cut into a plurality of elongated conductive plates of appropriate size, and then rinsed and dried to form the temperature plates 22 . Next, the temperature plates 22 are arranged in an array on the base 21 which has been designed to accommodate the temperature plates 22, and preferably the temperature plates 22 are further fixed on the base 21 through copper foil tape, for example. , and its fixing method is not limited to this.

Wherein, before using the copper foil tape to fix the temperature plates 22, a plurality of wires 20 are respectively welded to one end of the temperature plates 22, so that the wires 20 can be electrically connected to the temperature plates 22 respectively, and then Then, the two opposite ends of the temperature plate 22 are fixed on the base 21 through copper foil tape, and the other ends of the wires 20 are respectively connected with variable resistors (ie, the connection control member 231), and finally the power supply A supplier (ie, the controller 232 ) is electrically connected with the variable resistors to complete the setting of the micro heating platform 2 .

It can be seen from this that the micro heating platform 2 of the present invention can adjust the resistance values of the variable resistors, so that when the power supply provides power to the variable resistors, due to the different resistance values, the corresponding variable resistors can be adjusted accordingly. The isothermal plates 22 have different temperatures.

Referring to FIG. 4 , it is worth mentioning that the temperature distribution of the micro heating platform 2 is confirmed through a thermal imager to adjust the variable resistance and then correct the temperature of the respective temperature plates 22 to realize a gradient temperature field, and further The temperature stability experiment was carried out on the micro-heating platform 2, as shown in Figure 4, for the position of one point of the micro-heating platform 2, after a 3-hour temperature test, the experimental results showed that the temperature of the micro-heating platform was not affected by the ambient temperature. , has good temperature stability.

The injection extraction unit 4 includes a fluid supply 41 connected to the fluid injection port 31 of the microfluidic sorting wafer 3 , a syringe pump 42 connected to the semen injection port 32 , and a sperm extraction port 33 The extraction pump 43; wherein, the fluid supplier 41 can be used to accommodate a culture medium, and is arranged at a height difference with the microfluidic sorting wafer 3 and is located at a relatively high position in the direction of gravity.

Embodiments of application of the sperm sorting device of the present invention will be described in detail below.

First, let the control unit 23 heat the temperature plates 22 respectively to make them exhibit different temperatures, thereby creating a gradient temperature field on the micro heating platform 2; then, the microfluidic sorting wafer 3 is directly placed on the On the micro heating platform 2 , the fluid injection port 31 is located at a relatively high temperature of the micro heating platform 2 , and the waste liquid outlet 34 is correspondingly located at a relatively low temperature of the micro heating platform 2 .

Then, the fluid supplier 41 , the syringe pump 42 , and the extraction pump 43 are respectively connected to the fluid injection port 31 , the semen injection port 32 , and the sperm extraction port 33 . Since the fluid supplier 41 is arranged at a high position relative to the microfluidic sorting wafer 3 , the culture fluid in the fluid supplier 41 flows into the flow channel unit 35 through the fluid injection port 31 by gravity and flows into the flow channel unit 35 therein. A flow field is generated, and the flow field direction F from the fluid injection port 31 to the waste liquid outlet 34 is formed; wherein, the culture can be further controlled by the height difference between the fluid supplier 41 and the microfluidic sorting wafer 3 The flow rate of the liquid can be adjusted to adjust the velocity of the flow field, thereby controlling the grade of the semen for subsequent sorting.

The culture medium suitable for this embodiment is a fluid that can flow to establish the required flow field, and is a buffer fluid that does not harm spermatozoa. In this embodiment, the culture medium is modified Human Tubal Fluid (mHTF). ) as an example to illustrate, but not limited to.

The main flow channel 351 in the flow channel unit 35 has a gradually expanding design from the sperm extraction port 33 to the waste liquid outlet 34, so that a slow gradient flow field can be generated. The semen injection port 32 is pushed into and flows into the main channel 351 through the connecting branch 352 in the flow channel unit 35. When the sperm swimming speed and the flow field speed reach a balance, the flow tendency of the sperm is used, and the motility is good. The flow direction of the spermatozoa is opposite to the flow field direction F of the culture solution, and can flow in the direction of the spermatozoa extraction port 33; The direction F is flushed to the waste region 38 at the rear end.

However, the sperm injected through the semen injection port 32 at the beginning will generate an excessively fast flow rate and many high-motility sperm will be flushed to the area where the flow rate is insufficient at the rear end of the microfluidic sorting wafer 3. Therefore, with the micro-heating The gradient temperature field generated by the platform 2 utilizes the thermotaxis of spermatozoa, and the active spermatozoa will swim from low temperature to high temperature (that is, swim in the direction F of the reverse flow field), and then enter the spermatozoa through the enhanced recovery zone 37 again. Selecting 36 gradient flow fields for sorting, the recovery rate is greatly increased (~60%) to meet clinical needs.

The sperms swimming back from the main channel 351 are extracted from the sperm extraction port 33 through the suction pump 43 . Using the extraction method to take out the sorted sperm from the sperm extraction port 33 at the front end to ensure that it is not mixed with the waste liquid. This method can collect a large number of sperm with fast swimming speed, strong motility and normal shape.

Compared with the traditional microfluidic sperm sorting chip that only sorts sperm motility, the sperm sorting device of the present invention allows sperm with poor motility, or sperm with high motility but deformity, by means of reverse swimming of sperm, which cannot resist the flow field. be washed away, so that the sperms with linearity, poor motility, and high motility but deformed (big head, tilted head...) can be sorted, and the sperm extraction port 33 and the waste liquid outlet 34 are designed at different outlets , which can ensure that the sperms are all sorted high-quality sperm after recovery, thereby improving the quality of recovery, and using the thermotaxis of sperm to cooperate with the micro heating platform 2, so that the sperm with high motility in the area with insufficient flow velocity at the back end swim to the front gradient. The flow field area greatly improves the recovery rate.

To sum up, by using the characteristics of reverse swimming of spermatozoa, the swimming direction is opposite to the flow field direction F of the culture solution, so that the spermatozoa with poor quality cannot compete with the flow field of the culture solution in the flow channel, and are flushed to the surface. The rear end outlet, and the fine sperm can reach equilibrium with the fluid velocity and flow toward the sperm extraction port 33, and the area with insufficient rear end flow velocity can pass through the thermotaxis of the sperm, and use the gradient temperature field formed by the micro heating platform 2 to make The active sperm can swim to the front-end gradient flow field area, which greatly improves the recovery rate. This device can be used in artificial assisted reproductive technology as the pretreatment of sperm samples, and its high recovery rate is more in line with clinical needs. Therefore, It can indeed achieve the purpose of the present invention.

However, the above are only examples of the present invention, and should not limit the scope of implementation of the present invention. Any simple equivalent changes and modifications made according to the scope of the patent application of the present invention and the contents of the patent specification are still included in the scope of the present invention. within the scope of the invention patent.

2: Micro heating platform 20: Wire 21: Base 22: Temperature plate 23: Control unit 231: Connection tuning controls 232: Controller 3: Microfluidic sorting wafers 30: Ontology structure 31: Fluid injection port 32: Sperm Injector 33: Sperm extraction port 34: Waste liquid outlet 35: runner unit 351: Main Street 352: Connection branch 36: Sperm sorting area 37: Enhanced recycling area 38: Waste area 4: Injection extraction unit 41: Fluid Supply 42: Syringe Pump 43: Extraction pump F: flow field direction

Other features and effects of the present invention will be clearly presented in the embodiments with reference to the drawings, wherein: 1 is a schematic perspective view illustrating an embodiment in which a control unit is omitted from the sperm sorting device of the present invention; 2 is a schematic diagram illustrating a microfluidic sorting wafer of the sperm sorting device of the present invention; 3 is a schematic diagram illustrating a micro-heating platform in the sperm sorting device of the present invention; and Figure 4 is a temperature versus time graph illustrating the temperature stability of the micro heating platform of the present invention.

2: Micro heating platform

21: Base

22: Temperature plate

3: Microfluidic sorting wafers

4: Injection extraction unit

41: Fluid Supply

42: Syringe Pump

43: Extraction pump

F: flow field direction

Claims (7)

A sperm sorting device, comprising: a micro heating platform, distributed with a gradient temperature field, including a base, a plurality of temperature plates arranged along the direction of the flow field, and a control unit connected to the temperature plates, the control unit includes a plurality of connection regulators, and a controller, the connection regulators are respectively connected to the temperature plates, the controller is connected to the connection regulators, and can respectively regulate the temperature of the respective temperature boards; and a microfluidic separator The selection wafer is arranged on the micro heating platform and located on the gradient temperature field, and includes a sperm extraction port and a waste liquid outlet located at two opposite ends, and a fluid that is located at the same end and spaced apart from the sperm extraction port An injection port, a semen injection port disposed between the sperm worm extraction port and the fluid injection port, and a flow channel unit communicating with the fluid injection port, the semen injection port, the sperm worm extraction port, and the waste liquid outlet, The direction from the fluid injection port to the waste liquid outlet is the direction of the flow field, and the temperature of the gradient temperature field of the micro heating platform gradually decreases along the direction of the flow field. The sperm sorting device according to claim 1, wherein the temperature plates are selected from conductive substrates, the connection control elements are selected from variable resistors, and the controller is selected from power supplies. The sperm sorting device according to claim 1, wherein the temperature of the gradient temperature field of the micro heating platform is between 30°C and 40°C. The sperm sorting device according to claim 1, wherein the flow channel unit has a main channel between the sperm removal port and the waste liquid outlet that communicates the sperm removal port and the waste liquid outlet, and the The main channel is taken out by the sperm It gradually expands towards the waste liquid outlet to form a sperm sorting area, and extends from the sperm sorting area equal width to the waste liquid outlet direction to form an enhanced recovery zone, and then tapers from the enhanced recovery zone to the waste liquid outlet It extends to form a waste liquid area. The sperm sorting device according to claim 1, further comprising a fluid supplier communicating with the fluid injection port of the microfluidic sorting wafer, the fluid supplier can be used to contain a culture medium, and is connected to the microfluidic sorting chip. The fluid-separating wafer is arranged at a height difference and is located at a relatively high position in the direction of gravity, so that the culture fluid flows into the flow channel unit through the fluid injection port in a gravity manner and generates a flow field therein. The sperm sorting device according to claim 1, further comprising a syringe pump and an extraction pump respectively communicating with the semen injection port and the sperm extraction port, and the injection pump can transfer the semen with sperm from the semen injection port It is pushed into the main channel through the flow channel unit, and the extraction pump extracts the sperms swimming back from the main channel through the sperm extraction port. The sperm sorting device according to claim 1, wherein the size of the flow channel of the flow channel unit is in the order of millimeters.

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