Method for integrating cell layer culture and common observation of cell layer and sperms
By forming a chamber on a microscope slide, the culture of the cell layer and the two-dimensional and three-dimensional observation of sperm are achieved, solving the problem of difficulty in simultaneously performing cell layer culture and sperm observation in the prior art, reducing the complexity of operation and the consumption of sperm solution.
Patent Information
- Application Number
- CN202510278031.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-10
- Publication Date
- 2025-06-06
AI Technical Summary
The prior art is difficult to meet the needs of cell layer culture and observation of cell layer and sperm at the same time, especially when conducting two-dimensional and three-dimensional observations.
By tightly fitting the single-well gasket on the microscope slide, an open chamber is formed, and the cell layer is cultured and sperm solution is added in the chamber to achieve joint observation of the cell layer and sperm.
Efficient culture of the cell layer and two-dimensional and three-dimensional observation of sperm are achieved, which solves the problem of observing sperm motor behavior in the prior art, and reduces the complexity of the operation steps and the consumption of sperm solution.
Smart Images
Figure CN120098899A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for integrating cell layer culture and co-observation of cell layers and sperm. Background Art
[0002] For mammals, sperm reaches the egg and completes fertilization by passing through the female reproductive tract. In this process, the interaction with the cell layer in the reproductive tract determines the migration behavior and movement response of sperm above it, which is crucial to its fertilization outcome. However, the migration and fertilization process of sperm is difficult to study in situ. Therefore, it is particularly important to develop in vitro cell models and methods that can simulate the reproductive tract cell environment to observe sperm migration behavior. The difficulty lies in the successful cultivation of the reproductive tract cell layer, and how to introduce sperm without disturbance and achieve co-observation with the cell layer under a microscope. At present, the existing cell climbing technology needs to be transferred after the cell layer is formed. The operation steps are cumbersome and may affect the cell state. Although there are chamber slides on the market that can directly culture cell layers, the chamber volume and bottom area of such cell culture devices are currently too large. The sperm solution usually configured is difficult to fill the entire chamber and seal the top to solve the problem of liquid surface shaking affecting trajectory tracking. It can only be used for cell layer culture, and it is difficult to meet the needs of cell layer culture and co-observation of cell layers and sperm at the same time.
[0003] At the same time, limited by the principles of traditional microscopic imaging, the observation of sperm movement in the past was mainly carried out on the two-dimensional focal plane of the imaging, so the thickness of the observation chamber used was only at the micron level. In recent years, with the development of three-dimensional microscopic observation methods such as digital holographic microscopes, optical coherence tomography, and spinning disk confocal microscopes, in order to observe and study the movement behavior of sperm in a three-dimensional space that is closer to the real environment, we need an observation chamber that is more convenient for sperm to swim in three dimensions without disturbance and with a greater depth of field. However, there is currently no relevant chamber design on the market that can well meet the needs of two-dimensional and three-dimensional observation of sperm at the same time. Summary of the invention
[0004] The object of the present invention is to provide a method for integrating cell layer culture and joint observation of cell layers and sperm, which can perform cell layer culture and directly perform real-time two-dimensional and three-dimensional observation of the movement behavior of sperm on the cell layer under a microscope, thereby solving the problem that existing cell culture devices are difficult to simultaneously meet the needs of cell layer culture and joint observation of cell layers and sperm, and cannot well meet the needs of two-dimensional and three-dimensional observation of sperm.
[0005] The purpose of the present invention is achieved through the following technical solutions:
[0006] A method for integrating cell layer culture and co-observation of cell layer and sperm comprises the following steps: a single-hole gasket is tightly attached to a sterile microscope slide to obtain an open chamber, a cell suspension is added to the chamber, a complete culture medium is supplemented, and a CO 2 The cells are incubated in an incubator, and after the cell layer is formed, the complete culture medium in the chamber is aspirated, and after the chamber is filled with a sperm solution pre-diluted with culture medium, a cover slip is attached to the single-hole gasket to seal the chamber, and then the sealed chamber is placed under a microscope to observe the cell layer and the sperm together.
[0007] Furthermore, the material of the single-hole gasket is PDMS or PU; the material of the microscope slide is Permanox plastic or polystyrene. In a preferred embodiment of the present invention, in addition to PDMS or PU, the material of the single-hole gasket can also be other materials that can achieve good adhesion with plastic.
[0008] Furthermore, the length of the single-hole gasket is 22mm-26mm, the width is 22mm-26mm, the height is 3mm-5mm, the diameter of the chamber is 8mm-15mm, and the chamber is arranged at the central position of the single-hole gasket. The advantage is that the single-hole gasket can form a stable adhesion with the microscope slide, which is convenient to replace and easy to customize. The volume of the central chamber formed not only meets the needs of cell culture, but also takes into account the needs of large depth of field three-dimensional observation of sperm, so as to study the three-dimensional movement and interaction of sperm above the cell layer.
[0009] Furthermore, the single-hole gasket is closely attached to the microscope slide after pretreatment, and the pretreatment process includes: sterilizing the single-hole gasket in an autoclave, then soaking it in an ethanol solution for standby use, and taking it out of the ethanol solution and drying it under an ultraviolet lamp before use. The advantage is that the single-hole gasket can be reused after only disinfection.
[0010] Furthermore, the preparation method of the cell suspension is as follows: take out the cell freezing tube, transfer 1 mL of cells therein into a centrifuge tube, add 3 mL-5 mL of complete culture medium and centrifuge it in a centrifuge, discard the supernatant after centrifugation, add 5 mL of complete culture medium to resuspend the cells to obtain a resuspended liquid, and then transfer the resuspended liquid into a cell culture dish with a diameter of 6 cm, and after the cells are evenly distributed, place the cell culture dish in a 37°C CO 2The cells were statically cultured in an incubator, and when the cell confluence reached 80%, the complete medium in the cell culture dish was discarded, 3 mL-5 mL PBS buffer was added to wash the cells, the PBS buffer was discarded, 1 mL-2 mL preheated trypsin was added for digestion, and 3 mL-5 mL complete medium was added after digestion to terminate digestion to obtain a single cell suspension, which was transferred to a centrifuge tube, the supernatant was discarded after centrifugation, and 3 mL complete medium was added to resuspend the cells to obtain a cell suspension.
[0011] Furthermore, the cell is a primary cell or a cultured cell of the reproductive tract or non-reproductive tract of a mammal; or, a primary cancer cell or a cultured cancer cell of the reproductive tract or non-reproductive tract of a mammal.
[0012] Furthermore, the added volume of the cell suspension is 20 μL-60 μL.
[0013] Furthermore, the step of supplementing the complete medium includes adding a volume of complete medium exceeding the volume of the chamber to the chamber, and maintaining the complete medium in the chamber without overflowing by utilizing the surface tension of the liquid; the supplemented complete medium is the same as the complete medium used to prepare the cell suspension. The advantage is that the chamber can accommodate a sufficient volume of the complete medium without the need for repeated addition during the cell culture process.
[0014] Furthermore, the method for determining the formation of the cell layer is that if the cell confluence reaches 80% or more, the cell layer is determined to be formed. The advantage is that when the cell confluence reaches 80% or more, it means that the cells now occupy most of the space in the chamber, there are fewer blank areas, and the cell layer area is large enough, which is conducive to the subsequent joint observation of the cell layer and the sperm.
[0015] Furthermore, the concentration of sperm in the sperm solution is not higher than 10 6 The volume of the sperm solution added is just enough to fill the chamber. 6 The sperm concentration of 100 / mL can reduce the influence of the distance between sperms on three-dimensional tracking, and the amount of the sperm solution used is small, avoiding excessive consumption of the sperm solution, thereby effectively reducing the experimental cost.
[0016] Furthermore, before the cover glass is attached to the single-hole gasket, it is first cleaned with a plasma cleaner, then immersed in a piranha solution, and then stored in a 75% ethanol solution using ultrapure water ultrasound. 2 The advantage is that the cover glass can effectively seal the chamber to prevent the liquid surface from shaking during observation.
[0017] Furthermore, the ratio of the piranha solution is 98% H 2 SO 4 : 30% H 2 O 2 =7:3(v / v).
[0018] Furthermore, the sealed chamber is placed on a sample stage of a microscope for observation, which is beneficial in that it can be used directly to track and study the movement of sperm on the cell layer.
[0019] Compared with the prior art, the present invention has the following advantages and beneficial effects:
[0020] The present invention can culture the cell layer by adding a cell suspension in the chamber, and can directly add a sperm solution in situ on the basis of the formed cell layer for joint observation, without the need for cumbersome steps such as transferring the cell slide and can be reused, and the consumption of sperm solution is reduced. The chamber with a millimeter depth can simultaneously meet the needs of two-dimensional observation and three-dimensional observation of sperm, and realizes the observation of two-dimensional or three-dimensional movement and interaction of sperm above the cell layer. The method is compatible with various commercial microscopes, easy to operate and install, and reusable. It provides a convenient method for studying the movement and interaction of sperm above the cell layer using various commercial microscopes, and is suitable for promotion and use. The method can provide a powerful tool for analyzing the dynamic characteristics of sperm and its interaction mechanism with the reproductive microenvironment, and is expected to be widely used in the fields of assisted reproductive technology, diagnosis and treatment of male infertility, and animal husbandry. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 It is an exploded schematic diagram of a single-hole gasket, a microscope slide and a cover glass in a method of integrating cell layer culture and co-observation of cell layer and sperm in one embodiment of the present invention;
[0022] Figure 2 It is a front view of a single-hole gasket, a microscope slide and a cover glass for a method of integrating cell layer culture and co-observation of cell layer and sperm according to an embodiment of the present invention;
[0023] Figure 3 is a light microscopic image of a layer of African green monkey kidney cells (Vero cells) cultured in a chamber in Example 1 of the present invention;
[0024] Figure 4 It is a hologram of human sperm on top of a layer of African green monkey kidney cells (Vero cells) in Example 1 of the present invention;
[0025] Figure 5 is a fluorescent staining image of live cells and dead cells in the cell layer in Example 1 of the present invention;
[0026] Figure 6is a light microscopic image of a layer of human endometrial cancer cells (Ishikawa cells) cultured in the chamber in Example 2 of the present invention;
[0027] Figure 7 is a hologram of human sperm on top of a layer of human endometrial cancer cells (Ishikawa cells) in Example 2 of the present invention;
[0028] Figure 8 is a fluorescent staining image of live cells and dead cells in the cell layer in Example 2 of the present invention;
[0029] Fig. 9 is a schematic diagram of the two-dimensional trajectory of sperm above the cell layer in Example 2 of the present invention;
[0030] Fig.10 It is a schematic diagram of the three-dimensional trajectory of sperm near the cell layer in Example 2 of the present invention.
[0031] Among them: 1. Single-hole gasket; 2. Microscope slide; 3. Chamber; 4. Cover glass. DETAILED DESCRIPTION
[0032] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.
[0033] Example 1
[0034] The material of the single-hole gasket 1 is polydimethylsiloxane (PDMS), and the material of the microscope slide 2 is Permanox plastic. The overall size of the single-hole gasket 1 is 22.5 mm × 22.5 mm × 4.0 mm, and the diameter of the chamber 3 is Φ = 14.0 mm. The pretreatment process includes: placing the single-hole gasket 1 in an autoclave for sterilization, soaking the sterilized single-hole gasket 1 in a 75% ethanol solution for standby use, and taking out the single-hole gasket 1 from the 75% ethanol solution before use and placing it under a UV lamp to dry.
[0035] The preparation method of cell suspension is as follows: take out the cell cryopreservation tube (the cells are Vero cells) and put it into a 37°C constant temperature water bath to thaw quickly. Transfer 1mL of cells into a 15mL centrifuge tube, add 5mL of complete culture medium and centrifuge it at 1000rpm for 5min. After centrifugation, discard the supernatant, add 5mL of complete culture medium to resuspend the cells to obtain a resuspension, and then transfer the resuspension into a cell culture dish with a diameter of 6cm, repeatedly blow and shake crosswise to make the cells evenly distributed. After the cells are evenly distributed, place the cell culture dish at 37°C and 5% CO 2Cultivate statically in an incubator. When the cell confluence reaches 80%, discard the complete medium in the cell culture dish, add 3mL PBS buffer to wash the cells, repeat three times and discard the PBS buffer in the cell culture dish, add 1mL preheated trypsin digestion for 3min. After the timing is over, take out the cell culture dish, add 5mL complete medium to terminate the digestion, and obtain a single cell suspension, which is then transferred to a centrifuge tube and centrifuged at 1000rpm for 5min. After centrifugation, discard the supernatant, add 3mL complete medium to resuspend the cells to obtain a cell suspension. In this embodiment, the formula of the complete medium used to prepare the cell suspension is EMEM + 10% fetal bovine serum (FBS) + 1% penicillin-streptomycin solution (P / S).
[0036] like Figure 1 and 2 As shown, a method for integrating cell layer culture and co-observation of cell layer and sperm comprises the following steps: a single-hole gasket 1 is tightly attached to a sterile microscope slide 2 to obtain a device having a chamber 3, 60 μL of cell suspension is added to the chamber 3 and a complete culture medium is supplemented (the step of supplementing the complete culture medium comprises adding a complete culture medium with a volume exceeding the volume of the chamber 3 to the chamber 3, and maintaining the complete culture medium in the chamber 3 without overflowing by utilizing the surface tension of the liquid; the supplemented complete culture medium is the same as the complete culture medium used to prepare the cell suspension), and after the cells are evenly distributed, the device is placed in a 37°C CO 2 Incubate in an incubator until a cell layer is formed (e.g. Figure 3 After the cell layer confluence reaches more than 80%, the complete culture medium in chamber 3 is aspirated, and 650 μL of sperm solution diluted with culture medium in advance is added to chamber 3 (the sperm concentration in the sperm solution is 10 5 pcs / mL, sperm is human sperm), a cover glass 4 is attached to the single-hole gasket 1 to seal the chamber 3, and then the sealed device is placed under a microscope to observe the cell layer and sperm together. In this embodiment, the sealed device is placed on the sample stage of a digital holographic microscope that can perform three-dimensional sperm movement tracking and photographed, as shown in FIG. Figure 4 Shown is a hologram of human sperm on top of a layer of Vero cells.
[0037] In this embodiment, the culture medium is sperm washing fertilization solution (G-IVF PLUS); before the cover glass 4 is attached to the single-hole gasket 1, it is first cleaned with a plasma cleaner, then immersed in piranha solution, and then stored in a 75% ethanol solution with ultrapure water ultrasound. Before use, it is washed with N 2 Blow dry; the ratio of piranha solution is 98% H 2 SO 4 : 30% H 2 O 2 =7:3 (v / v), where 98% H2 SO 4 and 30% H 2 O 2 98% and 30% are mass percentage concentrations respectively.
[0038] Calcein-AM and propidium iodide (PI) were used to fluorescently stain live and dead cells in the Vero cell layer, respectively. Figure 5 (a) shows viable Vero cells. Figure 5 (b) shows dead Vero cells. Figure 5 (c) is a schematic diagram of the combined state of living Vero cells and dead Vero cells. The Vero cell layer cultured in the present invention has good activity.
[0039] Example 2
[0040] The material of the single-hole gasket 1 is polydimethylsiloxane (PDMS), and the material of the microscope slide 2 is Permanox plastic. The overall size of the single-hole gasket 1 is 22.5 mm × 22.5 mm × 4.0 mm, and the diameter of the chamber 3 is Φ = 14.0 mm. The pretreatment process includes: placing the single-hole gasket 1 in an autoclave for sterilization, soaking the sterilized single-hole gasket 1 in a 75% ethanol solution for standby use, and taking out the single-hole gasket 1 from the 75% ethanol solution before use and placing it under a UV lamp to dry.
[0041] The preparation method of the cell suspension is as follows: take out the cell cryopreservation tube (the cells are Ishikawa cells) and put it in a 37°C constant temperature water bath to thaw quickly. Transfer 1mL of cells to a 15mL centrifuge tube, add 5mL of complete culture medium and centrifuge it at 1000rpm for 5min. After centrifugation, discard the supernatant, add 5mL of complete culture medium to resuspend the cells to obtain a resuspension, and then transfer the resuspension to a cell culture dish with a diameter of 6cm, repeatedly blow and cross-shake to make the cells evenly distributed. After the cells are evenly distributed, place the cell culture dish at 37°C and 5% CO 2The cells were cultured in an incubator. When the cell confluence reached 80%, the complete medium in the cell culture dish was discarded, 3 mL of PBS buffer was added to wash the cells, and the PBS buffer in the cell culture dish was discarded after three times. 1 mL of preheated trypsin digestion was added for 3 min. After the timing was over, the cell culture dish was taken out, 5 mL of complete medium was added to terminate the digestion, and a single cell suspension was obtained. The single cell suspension was then transferred to a centrifuge tube and centrifuged at 1000 rpm for 5 min. After centrifugation, the supernatant was discarded, and 3 mL of complete medium was added to resuspend the cells to obtain a cell suspension. In this embodiment, the formula of the complete medium used to prepare the cell suspension is MEM + 15% fetal bovine serum (FBS) + 1% non-essential amino acids (NEAA) + 1% glutamine dipeptide (GlutaMAX) + 1% penicillin-streptomycin solution (P / S).
[0042] A method for integrated cell layer culture and joint observation of cell layer and sperm comprises the following steps: a single-hole gasket 1 is tightly attached to a sterile microscope slide 2 to obtain a device having a chamber 3, 60 μL of cell suspension is added to the chamber 3 and a complete culture medium is supplemented (the step of supplementing the complete culture medium comprises adding a complete culture medium with a volume exceeding the volume of the chamber 3 to the chamber 3, and maintaining the complete culture medium in the chamber 3 without overflowing by utilizing the surface tension of the liquid; the supplemented complete culture medium is the same as the complete culture medium used to prepare the cell suspension), and after the cells are evenly distributed, the device is placed in a 37°C CO 2 Incubate in an incubator until a cell layer is formed (e.g. Figure 6 After the cell layer confluence reaches more than 80%, the complete culture medium in chamber 3 is aspirated, and 650 μL of sperm solution diluted with culture medium in advance is added to chamber 3 (the sperm concentration in the sperm solution is 10 5 pcs / mL, sperm is human sperm), a cover glass 4 is attached to the single-hole gasket 1 to seal the chamber 3, and then the sealed device is placed under a microscope to observe the cell layer and sperm together. In this embodiment, the sealed device is placed on the sample stage of a digital holographic microscope that can perform three-dimensional sperm movement tracking and photographed, as shown in FIG. Figure 7 Shown is a hologram of human spermatozoa above a layer of Ishikawa cells.
[0043] In this embodiment, the culture medium is a sperm washing and fertilization solution (G-IVF PLUS); before the cover glass 4 is attached to the single-hole gasket 1, it is first cleaned with a plasma cleaner, then immersed in a piranha solution, and then stored in a 75% ethanol solution with ultrapure water ultrasound, and blown dry with N2 before use; wherein the ratio of the piranha solution is 98% H 2 SO 4 : 30% H 2 O 2 =7:3 (v / v), where 98% H2 SO 4 and 30% H 2 O 2 98% and 30% are mass percentage concentrations respectively.
[0044] Calcein-AM and propidium iodide (PI) were used to fluorescently stain live and dead cells in the Ishikawa cell layer, respectively. Figure 8 (a) shows viable Ishikawa cells. Figure 8 (b) shows dead Ishikawa cells. Figure 8 (c) is a schematic diagram showing the combined state of living Ishikawa cells and dead Ishikawa cells. The activity of the Ishikawa cell layer cultured in the present invention is good.
[0045] The trajectory of spermatozoa above the cell layer obtained by defocusing the digital holographic microscope is shown in the following figure. Fig. 9 As shown, the two-dimensional trajectory tracking software is ImageJ.
[0046] The trajectory results of the three-dimensional tracking of sperm above the cell layer obtained by defocusing the digital holographic microscope are as follows: Fig.10 As shown, the 3D tracking software is Matlab and Python.
[0047] The above embodiments are preferred implementation modes for fully illustrating the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement modes and shall be included in the protection scope of the present invention.
Claims
1. A method for integrating cell layer culture and co-observation of cell layer and sperm, characterized in that: The method comprises the following steps: closely fitting a single-hole gasket to a sterile microscope slide to obtain an open chamber, adding a cell suspension into the chamber and supplementing the chamber with a complete culture medium, and then placing the chamber in a CO2 incubator for incubation, aspirating the complete culture medium in the chamber after a cell layer is formed, adding a sperm solution diluted with a culture medium in advance to fill the chamber, fitting a cover glass onto the single-hole gasket to seal the chamber, and then placing the sealed chamber under a microscope to observe the cell layer and the sperm together.
2. A method for integrated cell layer culture and joint observation of cell layers and sperm according to claim 1, characterized in that: The material of the single-hole gasket is PDMS or PU; the material of the microscope slide is Permanox plastic or polystyrene.
3. A method for integrated cell layer culture and joint observation of cell layer and sperm according to claim 2, characterized in that: The length of the single-hole gasket is 22mm-26mm, the width is 22mm-26mm, the height is 3mm-5mm, the diameter of the cavity is 8mm-15mm, and the cavity is arranged at the central position of the single-hole gasket.
4. A method for integrated cell layer culture and co-observation of cell layers and sperms according to claim 3, characterized in that: The single-hole gasket is tightly fitted with the microscope slide after pretreatment, and the pretreatment process includes: placing the single-hole gasket in a high-pressure sterilizer for sterilization, then soaking it in an ethanol solution for standby use, taking it out of the ethanol solution before use and placing it under an ultraviolet lamp to dry.
5. The method for integrated cell layer culture and co-observation of cell layer and sperm according to claim 1, characterized in that: The preparation method of the cell suspension is as follows: take out the cell freezing tube, transfer 1mL of cells therein to a centrifuge tube, add 3mL-5mL of complete culture medium and then place it in a centrifuge for centrifugation, discard the supernatant after centrifugation, add 5mL of complete culture medium to resuspend the cells to obtain a resuspension, then transfer the resuspension to a cell culture dish with a diameter of 6cm, and after the cells are evenly distributed, place the cell culture dish in a 37°C CO2 incubator for static culture, when the cell confluence reaches 80%, discard the complete culture medium in the cell culture dish, add 3mL-5mL PBS buffer to wash the cells, discard the PBS buffer, add 1mL-2mL of preheated trypsin for digestion, add 3mL-5mL of complete culture medium after digestion to terminate digestion, and obtain a single cell suspension, transfer the single cell suspension to a centrifuge tube, discard the supernatant after centrifugation, add 3mL of complete culture medium to resuspend the cells to obtain a cell suspension.
6. A method for integrated cell layer culture and co-observation of cell layers and sperms according to claim 5, characterized in that: The cells are primary cells or passaged cells of the reproductive tract or non-reproductive tract of a mammal; or primary cancer cells or passaged cancer cells of the reproductive tract or non-reproductive tract of a mammal.
7. The method for integrated cell layer culture and co-observation of cell layer and sperm according to claim 1, characterized in that: The added volume of the cell suspension is 20 μL-60 μL.
8. The method for integrated cell layer culture and co-observation of cell layer and sperm according to claim 1, characterized in that: The step of supplementing the complete culture medium comprises adding a complete culture medium having a volume exceeding the volume of the chamber into the chamber, and maintaining the complete culture medium in the chamber without overflowing by utilizing the surface tension of the liquid; the supplemented complete culture medium is the same as the complete culture medium used to prepare the cell suspension.
9. The method for integrated cell layer culture and co-observation of cell layer and sperm according to claim 1, characterized in that: The method for determining the formation of the cell layer is that if the cell confluence reaches 80% or more, the cell layer is determined to be formed.
10. A method for integrated cell layer culture and co-observation of cell layers and sperms according to claim 9, characterized in that: The concentration of sperm in the sperm solution is not higher than 10 6 / mL, and the volume of the sperm solution added is just enough to fill the chamber.