A microfluidic cryoculture device suitable for single seminiferous tubules

Abstract

The present invention relates to a microfluidic freezing culture device suitable for a single seminiferous tubule, comprising a dual-channel microfluidic chip, wherein the dual-channel microfluidic chip can be divided into an upper cavity chip and a lower cavity chip, and a semipermeable membrane is arranged between the upper cavity chip and the lower cavity chip, and two upper liquid inlets and outlets are arranged on the upper surface of the upper cavity, and a plurality of identical independent units are arranged in the lower cavity chip, and the independent units are long grooves, and a plurality of tooth grooves are arranged evenly in the long groove, and each tooth groove is arranged at intervals; microinjection fixing needles are also arranged on both sides of the long groove; the advantages are as follows: the present invention realizes differential perfusion inside and outside the lumen (including cryoprotectant and culture fluid) and microperfusion regulation of intervention factors in the lumen through a microfluidic chip and a microperfusion channel, thereby realizing long-term culture of seminiferous tubules and regulation of the spermatogenic microenvironment, and improving spermatogenic function.

Description

Technical Field

[0001] The invention relates to the technical field of medical experimental research, in particular to a microfluidic freezing culture device suitable for a single seminiferous tubule. Background Art

[0002] The current seminiferous tubule microfluidic culture chip contains two layers of polydimethylsiloxane structure (PDMS); the upper chip contains an injection port and a liquid channel for perfusion of culture fluid; the lower layer includes a sample chamber connected to the injection port for placing the culture sample; a porous polycarbonate membrane is placed on the upper and lower chips as a semipermeable membrane, and the two layers of chips are bonded by a plasma cleaning machine to complete the production. The use of the chip is to add the seminiferous tubule tissue from the upper injection port to the lower sample chamber, and then close the injection port after adding. Two steel needles are inserted into the liquid channels on both sides. The culture medium is pumped from the steel needles through the microfluidic chip by a microfluidic pump, and penetrates into the lower layer through the porous polycarbonate membrane to achieve microfluidic culture of the seminiferous tubules. At present, testicular tissue is frozen in block tissue, and relevant protective agents are added during the cryopreservation process and then frozen and thawed. After thawing, the testicular tissue is washed to remove the protective agent and then placed in the microfluidic for tissue culture.

[0003] At present, freezing of seminiferous tubules is mainly based on freezing of block seminiferous tubules. During the freezing process, the penetration of seminiferous tubules is poor, and the cryoprotectant cannot quickly penetrate into the lumen of the seminiferous tubules, resulting in the formation of ice crystals near the lumen during freezing, which leads to adverse results such as cell apoptosis. At the same time, since spermatogenic cells and spermatids are mainly distributed on the lumen side of the seminiferous tubules, and supporting cells and interstitial cells are mainly distributed on the wall side of the seminiferous tubules, different cell types have different tolerance and effects on the same protective agent. However, the current freezing can only choose one protective agent to penetrate, which is easy to cause the differentiation of cells in the lumen and affect the overall recovery effect of the seminiferous tubules, thereby causing damage to the sperm microenvironment after recovery, and finally affecting the outcome of seminiferous tubule culture. After the seminiferous tubules are recovered, a gradient washing solution is needed to remove the cryoprotectant in the tissue. In the process of removing the protective agent, the same problems as freezing are faced, namely the effects of liquid penetration and differentiation of luminal cells. These problems will affect the recovery rate of freezing and thawing and the subsequent culture effect.

[0004] At the same time, the current single-channel microfluidic tissue culture system can only successfully culture mouse seminiferous tubules. There is still no effective method for microfluidic in vitro culture of human seminiferous tubules. Although the single-channel microfluidic testis culture chip can preliminarily simulate the in vivo conditions and the continuous culture time of the tissue can be maintained for 3-6 months, the culture of human seminiferous tubules not only needs to simulate the capillary circulation system in the body, but also needs to maintain the survival of spermatogenic cells and maintain the homeostasis of the spermatogenic microenvironment. The single-channel chip can only penetrate the culture medium into the surface of the seminiferous tubules, and the penetration effect on the wall of the seminiferous tubules is poor. At the same time, since the seminiferous tubules are mainly distributed on the lumen side, spermatogenic cells and spermatids, and the seminiferous tubules are mainly distributed on the wall side, different cells need different specific culture media for culture. The single-channel microfluidic chip can only pass through one culture medium, and cannot achieve the differentiated culture of different cells inside and outside the lumen. Therefore, the current single-channel microfluidic chip can only maintain the survival of seminiferous tubules for a certain period of time, and cannot perform good cryopreservation and cell tissue culture regulation.

[0005] Therefore, in summary, there is an urgent need for a microfluidic freezing and culture device suitable for a single seminiferous tubule that is easy to operate, has strong adaptability, can complete the microfluidic perfusion of differentiated culture medium inside and outside the lumen of a single seminiferous tubule, and can also complete the freezing and thawing, culture and cell growth regulation of a single seminiferous tubule. However, there is no report on this new type of microfluidic freezing and culture device suitable for a single seminiferous tubule. Summary of the invention

[0006] The purpose of the present invention is to solve the above-mentioned problems, and a microfluidic freezing culture device suitable for a single seminiferous tubule is provided.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] A microfluidic freezing culture device suitable for a single seminiferous tubule comprises a dual-channel microfluidic chip, wherein the dual-channel microfluidic chip is formed by bonding a double layer of PDMS and an intermediate porous polycarbonate membrane; the dual-channel microfluidic chip can be divided into an upper cavity chip and a lower cavity chip, and a semi-permeable membrane is arranged between the upper cavity chip and the lower cavity chip, two upper liquid inlets and outlets are arranged on the upper surface of the upper cavity chip, and three identical independent units are arranged in the lower cavity chip, and the independent units are long grooves, and a plurality of evenly arranged tooth grooves are arranged in the long grooves, and each tooth groove is arranged at intervals; microinjection fixing needles are also arranged on both sides of the long grooves.

[0009] In the above-mentioned microfluidic freezing culture device applicable to a single seminiferous tubule, as a preferred embodiment, the upper cavity chip is used for microfluidic culture fluid flow outside the seminiferous tubule.

[0010] In the above-mentioned microfluidic freezing culture device suitable for a single seminiferous tubule, as a preferred embodiment, the width of the long groove is 300 μm and the length is 1 cm.

[0011] In the above-mentioned microfluidic cryoculture device suitable for a single seminiferous tubule, as a preferred embodiment, the microinjection fixing needle is in a vertical structure and passes through the upper cavity chip and leaves a portion thereof.

[0012] The advantages of the present invention are:

[0013] 1. The present invention has reasonable structural design, convenient operation, firm fixation, safety and stability, simple manufacture, easy cleaning and disinfection, can meet the needs of freezing and thawing of a single seminiferous tubule and in vitro culture, has strong practicality, provides great convenience for medical experimental research on in vitro culture of seminiferous tubules, and improves work efficiency.

[0014] 2. The present invention adds an intraluminal culture channel for seminiferous tubules on the basis of the culture survival of a single-channel microfluidic chip, which can not only realize the differentiated loading and removal of protective agents during tissue freezing and thawing, but also carry out differentiated culture inside and outside the tube on this basis, and can also realize long-term culture of seminiferous tubules and regulation of the spermatogenic microenvironment, so as to better complete the research on the regulation of human spermatogenic microenvironment, in vitro seminiferous tubule culture and in vitro spermatogenesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Attached Figure 1 It is a structural schematic diagram of the microfluidic freezing culture device suitable for a single seminiferous tubule described in the present invention.

[0016] Attached Figure 2 It is a partial structural schematic diagram of the upper cavity and the lower cavity in the microfluidic freezing culture device suitable for a single seminiferous tubule described in the present invention.

[0017] Attached Figure 3 It is a partial structural schematic diagram of the lower cavity in the microfluidic freezing culture device suitable for a single seminiferous tubule described in the present invention.

[0018] Attached Figure 4 It is a partial flow diagram of the microfluidic freezing culture device suitable for a single seminiferous tubule described in the present invention. DETAILED DESCRIPTION

[0019] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0020] The reference numerals and components involved in the drawings are as follows:

[0021] 1. Dual-channel microfluidic chip 2. Upper cavity chip 3. Lower cavity chip

[0022] 4. Semipermeable membrane 5. Inlet and outlet of upper liquid 6. Long groove

[0023] 7. Tooth socket 8. Microinjection fixation needle

[0024] Example 1 A microfluidic cryo-culture device suitable for a single seminiferous tubule

[0025] Please see attached Figure 1-4 As shown, attached Figure 1 Schematic diagram of the structure of the microfluidic cryo-culture device for a single seminiferous tubule according to the present invention. Figure 2 Schematic diagram of the partial structure of the upper cavity and the lower cavity in the microfluidic freezing culture device for a single seminiferous tubule described in the present invention. Figure 3 Schematic diagram of the partial structure of the lower cavity in the microfluidic cryo-culture device for a single seminiferous tubule according to the present invention. Figure 4 It is a partial flow diagram of the microfluidic freezing culture device suitable for a single seminiferous tubule described in the present invention.

[0026] This device mainly solves the technologies related to freezing, thawing and culturing of a single seminiferous tubule. Specifically, according to the lumen structure of the seminiferous tubule and the difference in cell distribution inside and outside the seminiferous tubule, a second channel is added to the traditional single-channel microfluidic chip to complete the microfluidic perfusion of differentiated culture medium inside and outside the lumen of a single seminiferous tubule, complete the freezing, thawing, culturing and cell growth regulation of a single seminiferous tubule, and provide an experimental platform for the cryopreservation and culture of testicular tissue in vitro; it specifically provides a microfluidic freezing and culturing device suitable for a single seminiferous tubule, the device mainly includes a dual-channel microfluidic chip, the dual-channel microfluidic chip is bonded together by a double-layer PDMS and an intermediate porous polycarbonate membrane; the dual-channel microfluidic chip 1 can be divided into an upper cavity chip 2 and a lower cavity chip 3, the upper cavity chip 2 is mainly used as a flow channel for liquid, and the lower cavity chip 3 is mainly used for As tissue cell culture of seminiferous tubules; and a semipermeable membrane 4 is arranged between the upper cavity chip 2 and the lower cavity chip 3, and the semipermeable membrane 4 is used to prevent the tissue cells in the lower cavity chip from drifting into the upper cavity chip, and two upper liquid inlets and outlets 5 are arranged on the upper surface of the upper cavity chip 2, and a plurality of identical independent units are arranged in the lower cavity chip 3 (preferably 3 in this embodiment, and this arrangement can meet the culture needs in many cases as needed, thereby improving the practicality of the device), and the independent unit is a long groove 6, and a plurality of evenly arranged tooth grooves 7 are arranged in the long groove 6, and each tooth groove 7 is arranged at intervals, and the tooth groove 7 is used to fix the seminiferous tubules and allow a certain amount of liquid to contact the tubules for nutrient exchange; microinjection fixing needles 8 are also arranged on both sides of the long groove 6.

[0027] In this embodiment, the upper cavity chip 2 is preferably used for microfluidic culture fluid flow outside the seminiferous tubules.

[0028] In this embodiment, the width of the long groove 6 is 300 μm and the length is 1 cm, and a single seminiferous tubule of about 1 cm can be placed in each groove.

[0029] In this embodiment, the microinjection fixing needle 8 is preferably in a vertical structure and passes through the upper cavity chip 2 with a portion reserved. This arrangement can facilitate the culture injection of tissues in the lower cavity chip.

[0030] The method of using the present invention is as follows: first, a single seminiferous tubule is placed in a long groove in a lower cavity, and a single seminiferous tubule of about 1 cm in length can be placed in each groove. After placement, two microinjection fixation needles with a diameter of 50 μm can be inserted into the seminiferous tubule cavity on both sides and fixed. After tissue loading is completed, the upper cavity and the lower cavity and the semipermeable membrane between the upper cavity and the lower cavity are bonded, and then microinfusion can be started after a liquid channel is established by a steel needle and a capillary.

[0031] It should be noted that: the present invention can add a culture channel in the lumen of the seminiferous tubules on the basis of the culture survival of the single-channel microfluidic chip, which can not only realize the differentiated loading and removal of the protective agent during the freezing and thawing of the tissue, but also can carry out differentiated culture inside and outside the tube on this basis, and can also realize the microenvironment regulation in the lumen of the seminiferous tubules, so as to better complete the research on the regulation of the human spermatogenic microenvironment and the realization of in vitro seminiferous tubule culture and in vitro spermatogenesis; secondly, since the seminiferous tubules need to be divided into single tubes and placed in the device when freezing the seminiferous tubules, and the two sides are fixed with steel needles respectively, at this time, the cryoprotectant can be loaded and perfused according to the required protective agent type, temperature, flow rate, concentration, and perfusion time, and differentiated protection loading inside and outside the tube can be realized; and after the seminiferous tubules are revived, they can be placed in the dual-channel microfluidic control chip again, and the two sides are fixed with steel needles respectively. At this time, the washing solution type, temperature, flow rate, concentration, and perfusion time can be required. The chip can be perfused at the right time to remove the cryoprotectant in the best way inside and outside the tube, so as to achieve the removal of the differentiated protective agent inside and outside the tube. At the same time, anti-oxidative stress and anti-apoptotic factors can be added in the removal process as needed, and the precise flow rate and concentration can be controlled through the microfluidic channel to complete the rapid penetration and reduce the freeze-thaw damage. After the recovery is completed, the seminiferous tubules can be kept on the chip, and the double-channel microfluidic culture process of the seminiferous tubules can be directly started through the microperfusion channel. Different culture media can be used for perfusion culture according to the differences in cell types inside and outside the seminiferous tubules. Cell regulatory factors can be added as needed to achieve differentiated culture inside and outside the lumen. The overall structure of the device is reasonably designed, easy to operate, firmly fixed, safe and stable, simple to make, easy to clean and disinfect, and can meet the needs of freezing and thawing and in vitro culture of a single seminiferous tubule. It is highly practical and provides great convenience for medical experimental research on in vitro culture of seminiferous tubules, thereby improving work efficiency.

[0032] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the principle of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. A microfluidic freezing culture device suitable for a single seminiferous tubule, characterized in that: The invention comprises a dual-channel microfluidic chip, wherein the dual-channel microfluidic chip is formed by bonding a double layer of PDMS and an intermediate porous polycarbonate membrane together; the dual-channel microfluidic chip can be divided into an upper cavity chip and a lower cavity chip, and a semi-permeable membrane is arranged between the upper cavity chip and the lower cavity chip, two upper liquid inlets and outlets are arranged on the upper surface of the upper cavity chip, and a plurality of identical independent units are arranged in the lower cavity chip, and the independent units are long grooves, and a plurality of evenly arranged tooth grooves are arranged in the long grooves, and each tooth groove is arranged at intervals; microinjection fixing needles are also arranged on both sides of the long grooves.

2. The microfluidic cryoculture device for a single seminiferous tubule according to claim 1, characterized in that: The upper cavity chip is used for microfluidic culture fluid flow outside the seminiferous tubules.

3. The microfluidic freezing culture device for a single seminiferous tubule according to claim 1, characterized in that: The width of the long groove is 300 μm and the length is 1 cm.

4. The microfluidic cryoculture device for a single seminiferous tubule according to claim 1, characterized in that: The microinjection fixing needle is in a vertical structure and passes through the upper cavity chip and leaves a portion in reserve.

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