Transport, separation and storage container for use in the preservation of motile cells

Abstract

The invention relates to a transport, separation, and storage container for use in the preservation of motile cells.

Description

[0001] DESCRIPTION TRANSPORT, SEPARATION AND STORAGE CONTAINER FOR USE IN THE PRESERVATION OF MOTILE CELLS

[0002] Technical Field of the Invention

[0003] The invention relates to a transport, separation, and storage container for use in the preservation of motile cells. In the container subject to the invention, the motile cells in the samples are forced to move to a certain location by means of the medium and channels used. In this way, healthy and motile cells can move away from other dead cells, and thus can be transported and stored with less exposure to oxidative stress and with less loss. In addition, the decomposition process with said container is carried out in real time by examination under a microscope.

[0004] State of the Art

[0005] The preservation of motile cells is of great importance, especially in biological research, medical applications, and agriculture field. Firstly, from a reproductive health perspective, for couples, especially those undergoing fertility treatment, the storage of healthy cells increases the chances of having a child. Cancer patients may have the chance to preserve their fertility by saving sperm or egg cells before treatment. For animal and plant species, storing motile cells is important to preserve cells of threatened species and genetic diversity, and to support future populations. Furthermore, in contingency planning, the storage of specific cells provides a resource for reproduction in case of emergency.

[0006] Sperm cells in particular are used in genetic research and biomedical studies. Storage allows for long-term analysis of these cells. In sperm storage and transportation, centrifugation is primarily used to separate sperm cells and other liquid components. This process is done to improve sperm quality and make the sperm samples more concentrated. Centrifugation separates the sperm cells from other, heavier components, resulting in more healthy sperm. It is also useful for increasing sperm motility and vitality. For this reason, it is frequently used, especially in assisted reproductive techniques such as in vitro fertilization. Containers designed for separating sperm, usually having different compartments, are used. This allows sperm cells to be separated according to their density. By using these containers during the centrifugation process, sperm cells and other components are effectively separated. Specially designed containers for freezing are used for freezing and long-term storage of sperm cells. Said containers are usually designed to be stored in liquid nitrogen and have special formulations to prevent damage to the cells during freezing.

[0007] In the state of the art, many methods for long-term storage of semen from animals have been developed in the field of reproductive biotechnology. Comparing these methods between each other reveals their advantages and disadvantages. It was realized that there are still shortcomings in the cryobiological aspect, for this reason, experiments were carried out on current methods for freezing semen. Volumetrically large amounts of semen (2-10 ml) can be stored at once by directional freezing. The encapsulation method involves a gel-like structure surrounding the cells. The storage method called lyophilization is a drying process and organisms, cells, tissues, and even all biological products can find a place in this group [1]. Directional freezing is a method of freezing sperm cells in a specific direction and speed during freezing. This process is used to preserve the health of sperm cells and prevent damage during the freezing process. Sperm cells are frozen at a certain speed. Freezing too fast can cause ice crystals to form inside the cells, which can damage the cells. Directional freezing ensures controlled freezing. The freezing process is harmonized with the direction in which the cells are frozen. This helps to control the crystallization process of the water inside the cells. Thus, the formation of ice crystals in intracellular and extracellular areas is minimized. Cryoprotectants (e.g. DMSO or glycol) are used in the freezing process, these materials preserve the water content of the cells and reduce the damage that can occur during freezing. While directional freezing is beneficial in terms of orienting the freezing, there is still a risk that some cells may be affected by ice crystals. In addition, it may not have the same effect on all sperm cells, and some cells may be more damaged. Another method, encapsulation, is a technique used to ensure the protection of sperm cells, this method traps the sperm cells in a protective environment and thus increases their resistance to various negative influences. Materials used for encapsulation include natural polymers such as alginate, chitosan, and agarose. These substances interact with sperm cells to form a protective structure. However, the effects of the polymers used on sperm cells should be carefully evaluated, as some materials negatively affect the functionality of the cells. Lyophilization, on the other hand, is a technique used to ensure long-term storage of sperm cells, this method preserves them by removing the water content of the cells. In said method, sperm cells are frozen at a specific temperature. This stage ensures that the water inside the cells freezes. The frozen cells are kept at a certain temperature for water vapor removal under vacuum. In this process, water is removed by direct evaporation. In the final stage, the cells are allowed to dry such that the cells are in a light and stable form. Cryoprotectants are usually used during lyophilization. These substances prevent the structure of cells from deteriorating. In said method, cells can be damaged during the freezing and drying process. Therefore, careful monitoring of process protocols is of utmost importance.

[0008] Patent application no. US2020032199A1 included in the state of the art, discloses a movable cell separation device comprising two reservoirs shared by a perforated sheet. In this sheet, at least one channel has a narrowed diameter and a conical shape. A sample containing cells is placed in the first reservoir and the second reservoir is filled with culture medium. This creates a flow towards the first reservoir. The device is used to select the most suitable spermatozoa for assisted reproductive techniques from a semen sample.

[0009] Reasons such as limitations and shortcomings of the present art solutions, inadequate containers and / or methods suitable for transport and storage of low concentrations of ejaculates, centrifugation and / or filtration of sperm prior to sperm storage methods resulting in complexity and prolongation of the process, the lack of a storage container that allows real-time monitoring to be used in the sperm separation process, as well as techniques such as centrifuge filtration used in the transportation and separation processes causing some physical damage to the sperm and also in terms of DNA fragmentation have made it necessary to make a development in the relevant technical field.

[0010] Summary and Objects of the Invention

[0011] The invention describes a container for transporting, separating, and storing for use in the preservation of motile cells.

[0012] The object of the invention is the storage and transport of sperms with less cell loss. In the container subject to the invention, the motile cells in the samples are forced to move to a certain location by means of the medium and channels used. In this way, healthy and motile cells can move away from other dead cells, and thus can be transported and stored with less exposure to oxidative stress and with less loss.

[0013] An object of the invention is to provide a structure that provides space for the motile cells to move around during transportation and protects them from external factors.

[0014] Another object of the invention is the real-time imaging of sperm cells during separation. The container subject to the invention is compatible with all microscopes, enabling imaging of the cells during the separation process.

[0015] An object of the invention is to separate sperm cells even at low concentrations. As approximately 20 pl of sperm is placed in each inlet port, it is also suitable for very small amounts of ejaculate.

[0016] An object of the invention is to provide a sperm transport and storage container which is easy to use and which enables separation in a short time. Sperm from all channels are channeled into a single area, increasing the amount of concentration and realizing the collection process from a single point; thus providing ease of use. Furthermore, the container subject to the invention offers a structure that can be used in routine laboratory processes and does not require a pump. Additionally, since the 6 different inlet ports are connected to 1 large outlet port via channels approximately 10-15 mm long and 3-6 mm wide, the dimensions in which the highest possible recovery is achieved to ensure that the time does not exceed 30 minutes when considering spermsized cells. If the channels are made shorter, more sperm will pass through, but their quality will decrease. If they are made longer, more high-quality sperm will pass through, but the number of sperm that can cover that distance in a short time will decrease.

[0017] Description of the Drawings

[0018] Fig. 1. The top representative view of the container subject to the invention.

[0019] Fig. 2. The bottom representative view of the container subject to the invention.

[0020] Fig. 3. The representative illustration of the cross-sectional views of the container subject to the invention. Description of the References in the Drawings

[0021] 1. Construction

[0022] 2. Inlet port

[0023] 3. Collection chamber

[0024] 4. Channel

[0025] 5. Petri dish top lid

[0026] 6. Petri bottom

[0027] 7. Channel

[0028] 8. Microscopy area

[0029] 9. Ultrasonic bonding zone

[0030] Detailed Description of the Invention

[0031] The invention relates to a transport, separation, and storage container for use in the preservation of motile cells. The container subject to the invention allows cells to accumulate separately from other dead cells, thereby improving sperm quality and ensuring better results in subsequent processes and analyses, as well as higher viability. The container subject to the invention is suitable for use with sperm, plant spores, or motile fungi.

[0032] The container subject to the invention comprises a petri dish of a structure (1) made from copolyester material, comprising a petri dish top lid (5), and a petri dish bottom (6). By attaching the structure (1) made from copolyester material to the surface of the petri dish using an adhesive, an environment is created in which motile cells are preserved during transport. The invention comprises at least 6 inlet ports (2) and at least one collection chamber (3) as the final center where the motile cells are collected after the separation process. Said structure (1) provides protection from external factors that may have a negative impact on the motile cells. In this way, motile cells are preserved and transported in this product in a healthy way.

[0033] Structurally, 6 inlet ports (2) are connected to 1 collection chamber (3) by multiple channels (7) with a width of 3-6 mm and a length of about 10-15 mm. In this way, the motile cells in the samples at the inlet port (2) are forced to move to a certain location by means of the medium and channels used. In this way, healthy and motile cells can move away from other dead cells, and thus can be transported and stored with less exposure to oxidative stress and with less loss. The channels prepared for this situation and the internal structure of the channels allow the cells to move freely, but they will need to pass through the obstacles created in the channels. In addition, in some basic analyses, these living cells will spontaneously separate from the liquid or medium in which they are present and transition to the medium of choice for analyses and treatments with known content. Here, measurements in fluids containing significant amounts of protein, such as seminal plasma, do not yield accurate results in protein and antibody tests performed on cells. In addition, in treatments such as IVF treatments, sperm must be in a liquid suitable for culture media. Seminal plasma cannot be processed. In this way, several steps will be skipped in the subsequent operations. In other words, in the present art, removing cells from their liquid environment and doing so in a way that keeps them alive is achieved through several centrifugation and washing processes. In this way, these stages are skipped.

[0034] In order to form the container subject to the invention, 6 channels are first formed with copolyester and mold injection technique and these 6 channels are connected to a collection chamber (3). The inlet port (2) is then filled with the liquid, e.g. sperm, from which it is desired to collect the motile cells, in order to create a environment where there is no ambient flow for them to swim and the pH and temperature are constant. Here, utilizing the dynamics of composite containers and microfluids, a 5-minute wait is required to ensure the stability of the liquid's movement within the containers. Since even evaporation in liquids in low volume environments will cause movement in the environment, after the sample is placed, all inlet ports and the collection chamber (3) are covered with mineral oil to ensure that the environment is a system in itself, completely independent of the external environment. Sperm from the inlet port (2) will then swim through the channel (7) to the collection chamber (3). After passing through the obstacles they will encounter in the environment, the sperms arriving at the outlet will be collected in the prepared collection chamber (3). The microscopy area (8) in the container subject to the invention is the part that allows real-time examination under a microscope, and the ultrasonic joining area (9) is the part where the parts of a structure (1) made of copolyester material made of two parts, the upper and lower parts, are joined by welding. The operation method of a storage container for use in the preservation of motile cells subject to the invention comprises the process steps of: i. filling the inlet port (2) with the liquid containing the motile cells to be collected, e.g. sperm, and preparing an environment of constant pH and temperature and with no ambient flow for the cells to swim, ii. holding for 5 minutes for the movement of the fluid to stabilize, iii. after the sample is placed, sealing all holes with mineral oil to ensure that the environment is a system in itself, completely independent from the external environment, iv. swimming of sperm from the inlet port (2) through the channel (7) to the collection chamber (3), v. collecting the sperm arriving at the outlet in the prepared collection chamber (3) after passing the obstacles encountered in the environment.

[0035] Industrial Applicability of the Invention

[0036] The invention relates to a transport, separation, and storage container for use in the preservation of motile cells, and is industrially applicable.

[0037] The invention is not limited to the above descriptions and the person skilled in the art can readily present other different embodiments of the invention. These should be considered within the protection scope of the invention claimed by the claims.

[0038] REFERENCES

[0039] [1] Narh^ay, S., & Ataman, M. B. (2022, December 23). Spermamn Uzun sureli saklanmasinda Kullanilan Guncel Yontemler. Turkish Veterinary Journal.

Claims

CLAIMS1 . A storage container for use in the preservation of motile cells, characterized in that it comprises a structure (1) to prepare an environment for the preservation of motile cells during transportation by adhering to the surface of the petri dish using adhesive, a petri dish top lid (5) and a petri dish bottom (6), at least 6 inlet ports (2) into which the cells are placed, with an environment of constant pH and temperature, with no ambient flow suitable for the swimming of motile cells and at least one collection chamber (3) as the final center where the motile cells are collected after the separation process, multiple channels (7) to connect the inlet port (2) to the collection chamber (3), at least one microscopy area (8) to allow examination under a microscope, at least one ultrasonic joining zone (9) where the parts of the structure (1 ), which is formed from two parts, the upper and lower parts, are joined by welding.

2. A container according to claim 1 characterized in that said structure (1) is made of copolyester material.

3. A container according to claim 1 , characterized in that said channel (6) is 10-15 mm long and 3-6 mm wide.

4. A container according to any one of the preceding claims, characterized in that said motile cells are sperm, vegetative spores, or motile fungi.

5. The operation method of a container according to claim 1 , characterized in that it comprises the process steps of: i. filling the inlet port (2) with the liquid containing the motile cells to be collected, e.g. sperm, and preparing an environment of constant pH and temperature and with no ambient flow for the cells to swim, ii. holding for 5 minutes for the movement of the fluid to stabilize, iii. after the sample is placed, sealing all holes with mineral oil to ensure that the environment is a system in itself, completely independent from the external environment, iv. swimming of sperm from the inlet port (2) through the channel (7) to the collection chamber (3),v. collecting the sperm arriving at the outlet in the prepared collection chamber (3) after passing the obstacles encountered in the environment.

Images