Method of enriching a cell population containing intact and damaged cells in a liquid medium

Abstract

The invention relates to a method of enriching a cell population containing intact and damaged cells in a liquid medium, comprising the following steps: a.) providing the liquid medium containing the cell population, b.) sedimenting the cell population out of the liquid medium, c.) separating the liquid medium and the sedimented cell population, d.) resuspending the sedimented cell population in a suspension solution to produce a cell suspension, e.) admixing the cell suspension with a ferromagnetic material that binds specifically to the damaged cells, f.) incubating the cell suspension admixed with the ferromagnetic material, g.) introducing the cell suspension into a magnetic field in order to separate the bound cells and the unbound cells in the cell suspension, and h.) separating a fraction of the cell suspension enriched with unbound intact cells, to the use of intact sperm cells enriched in the method, and to an apparatus for separating cells bound to magnetic particles in a cell suspension.

Description

[0001] Method for enriching a cell population containing intact and damaged cells in a liquid medium

[0002] The application relates to a method for enriching a cell population containing intact and damaged cells in a liquid medium, the use of intact sperm cells enriched in the method, and a device for separating cells bound to magnetic particles in a cell suspension.

[0003] Artificial reproductive technologies (ART) include techniques such as in-vitro fertilization (IVF), artificial insemination (AI), intracytoplasmic sperm injection (ICSI), techniques using enucleated cells, multiple ovulation, embryo transfer (MOET) and other embryo transfer techniques, gamete transfer into the fallopian tubes (GIFT) and zygote transfer into the fallopian tubes (ZIFT).

[0004] These techniques are regularly used in mammals, including humans. ART methods are generally expensive, time-consuming, and, given the vulnerability of germ cells outside their natural environment, more or less successful. The limited availability of genetically desirable gametes (sperm or eggs) presents an additional challenge to the commercially viable use of ART in animal breeding. One way to reduce the cost of ART, and thus expand its commercial viability, particularly in human reproductive medicine, is to increase the efficiency of the procedures and processes used by improving the viability and overall quality of the gametes used in ART.

[0005] In commercial animal breeding, but also in the use of ART techniques in human reproductive medicine, there is therefore a great need to improve the quality of gametes for use in ART. This includes, for example, improving viability, motility, fertility, the genetic health of germ cells, and general longevity traits. When using AI (artificial insemination), a very high number of sperm per dose must be used to achieve successful fertilization. This high dose can be due to the fertilization process itself, as is the case with mammals, especially pigs, and the common use of intracervical insemination in this and related species. Furthermore, depending on the number of non-viable or non-viable sperm, the risk of fertilization may be significantly increased.Due to the presence of non-fertilizable sperm cells in the total ejaculate, a correspondingly high dose is necessary for meaningful and, especially in commercial applications, efficient insemination fertility. Sperm exhibit significant losses in fertility, viability, and motility when collected, handled, and transported, and also experience significant stress when mixed with a cryoprotectant, frozen, and thawed. A high number of infertile sperm alone (dead, apoptotic, acrosome-reacting, etc.) negatively impacts the fertility of the remaining intact cell population in the ejaculate.Commonly used and necessary gamete processing procedures (conventional or sex-sorted), such as egg or sperm flushing, prior to their use in ART, represent an enormous stress factor for gamete cells and negatively impact their cellular integrity and membrane structure. This, in turn, is reflected in reduced viability, motility, and fertility. Another example of gamete processing prior to ART is sex-sorting of sperm. Sex sorting includes processes in which X- and Y-carrying sperm are physically separated into distinct subpopulations, as well as processes in which sperm carrying the unwanted sex chromosome in a sperm sample are selectively killed, compromised, deactivated, immobilized, or otherwise rendered infertile, e.g., by smearing or smearing.Through laser ablation / photodamage techniques to create a sex-enriched sperm population. The sex sorting process severely stresses and damages the cells, producing a low percentage of viable sperm. While these sperm are capable of fertilizing mature eggs, they may exhibit reduced viability, motility, and fertility compared to unprocessed cells. Sex-selective sperm sorting is a technique used in animal breeding to influence the sex of offspring. This technique allows for the separation of sperm of the desired sex to control the likelihood of conception of a male or female offspring.

[0006] Several methods of sex-specific sperm sorting are known (D. Rath et al. 2013; Prakash et al. 2014; Dominguez E. et al. 2018), including, but not limited to:

[0007] Flow cytometry: This technique utilizes the difference in DNA content between male (Y chromosome) and female (X chromosome) sperm. Using fluorescent dyes, a flow cytometer can sort the sperm by sex.

[0008] Colloidal Silica Method: This method is based on the different binding properties of X and Y sperm to colloidal silicon dioxide. The sperm are passed through the medium, which causes the sorting.

[0009] Ericsson method: In this method, the sperm are filtered through a special substance placed in a tube. Since Y sperm (male) are faster than X sperm (female), more Y sperm should pass through the filter.

[0010] Preimplantation genetic diagnosis (PGD): In human reproductive medicine, sex selection can be performed directly at the embryo level. This is made possible by removing a cell from an embryo before transferring it to the uterus.

[0011] Free-flow electrophoresis (FFE): This is a separation technique that uses electric fields to separate charged particles in a liquid phase. In the case of sex-specific sorting of sperm cells, it utilizes the fact that sperm containing X and Y chromosomes have different sperm head sizes due to the different sizes of the respective chromosomes. Since sperm carry a net negative surface charge due to charged membrane components, the respective sperm cells exhibit sex-specific negative charges of varying strengths. FFE is based on the fact that charged particles, when moved in an electric field, migrate towards the electrode. In a free-flow electrophoresis device, the sample solution moves in a continuous flow through a narrow capillary tube or chamber. The different charges of the sperm cells separate them according to sex.

[0012] Sex-specific sperm separation mediated by magnetic nanoparticles (MNPs): Sperm containing X and Y chromosomes exhibit different zeta potentials and can be separated due to their different charges via electrostatic binding to MNPs.

[0013] In animal breeding, sex-specific sperm sorting is used to control the genetic improvement of breeding animals and to influence the sex ratio in a population.

[0014] Each processing step causes stress, which reduces the overall motility, viability, and / or fertility of sperm. This can result in less efficient samples for use in ART, such as IVF and AI, and other types of post- or post-processing.

[0015] Removing infertile sperm from the total sperm population generally optimizes the preparation processes prior to ART, significantly reduces the dose required for successful fertilization, and expands its commercial applicability across all animal species. There is a general need to improve currently used ART, particularly through optimized sperm processing techniques, to make the procedures more reliable, efficient, faster, more effective, and commercially viable, especially for smaller breeders for whom, for example, sex selection breeding can be a risky and expensive option.

[0016] One object of the invention is the preparation of cell populations in order to provide an improved starting material for subsequent ART procedures.

[0017] This problem is solved by a method according to independent claim 1, a use according to independent claim 16 and a device according to independent claim 17.

[0018] Advantageous further developments of the invention are the subject of the dependent claims.

[0019] The described processes and methods are primarily described below in connection with mammalian sperm, without, however, limiting the invention to this. The described processes and methods can be applied in the same way in human reproductive medicine with human sperm. The described processes and methods can encompass a multitude of aspects that can be combined in a wide variety of ways and applied to a wide variety of cell populations with different cells or cell materials. The following descriptions serve to present the invention comprehensively, without limiting it to this. The processes and embodiments of the invention are presented with regard to sperm, in particular sperm of animal and human origin.The invention should be understood to mean that any permutation and combination of all disclosed aspects can also be applied in connection with other cell populations, cells, or cell structures. The described embodiments and exemplary embodiments should not be interpreted as limiting the described methods and processes to only the explicitly described systems, techniques, and applications. Furthermore, this description should be understood to encompass all exemplary embodiments with any number of the disclosed elements, with each element alone, and also with any and all different permutations and combinations of all elements.

[0020] According to the invention, a method for enriching a cell population containing intact and damaged cells in a liquid medium is proposed. The method comprises the steps: a) providing the liquid medium containing the cell population, b) sedimenting the cell population from the liquid medium, c) separating the liquid medium and the sedimented cell population, d) resuspending the sedimented cell population in a suspension solution to produce a cell suspension, e) adding a ferromagnetic material to the cell suspension that binds specifically to the damaged cells, f) incubating the cell suspension containing the ferromagnetic material, g) placing the cell suspension in a magnetic field to separate the bound and unbound cells in the cell suspension, and h) separating a portion of the cell suspension enriched with unbound, intact cells.

[0021] The procedure is used to remove or identify cells or cell structures with damaged membranes or any cell alterations that lead to a reduction in cell health, from those with intact membranes or without any cell alterations that lead to a reduction in cell health. The procedure enriches the sample or cell viability of a cell population. The procedure can be applied to cells or cell populations contained in freshly drawn samples, or to those obtained after dilution, during and after cooling, or during and after other cell or system procedures that may be applied prior to cryopreservation, or to frozen / thawed cell samples or cell populations.

[0022] The method can also be applied immediately after obtaining cell samples or cell populations. It can also be used with cell populations or cell samples that can be stored for a specific period or extended in buffers or other substances. For example, the method can be applied with cell populations or cell samples that have been kept at 4 °C to 40 °C for at least approximately 12, 24, 30, 36, 48, 60, or 72 hours or longer. The method can also be used for cell samples present in a solution with an osmolarity of 250–375 mOsm.

[0023] The enriched cell populations can be used for routine procedures, before or after other processing techniques, before or after shipping samples, and before or after long-term cryopreservation or other processes.

[0024] In one embodiment, the method according to the invention provides that the cell population is formed by intact or damaged sperm cells, in particular mammalian sperm cells of human and animal origin. Preferably, the liquid medium is formed by ejaculate fluid, in particular mammalian ejaculate fluid. When applying the method with populations of sperm cells, the method comprises removing sperm cells with damaged membranes or with any cell alteration that leads to a reduction in cell health from those with intact membranes or without cell alteration that leads to a reduction in cell health. This results in an improvement in the viability, cell health, genetic health, fertility, motility, and general longevity characteristics of the sperm in a sperm sample or sperm population.

[0025] The method can be applied to sperm contained in freshly collected ejaculates, after dilution, during and after cooling, or during and after other semen processing procedures that may be applied prior to cryopreservation, or to frozen or thawed sperm. The method can also be used for native or diluted semen samples intended for immediate use. Furthermore, the method can be used for native or diluted semen samples stored for up to 30 hours at 4°C to 40°C, or diluted in buffer systems with an osmolarity of 250–375 mOsm.It proves advantageous to minimize the oxygen content of all solutions used in the buffer systems, either after production, before storage and / or shortly before use, or during production by means of, for example, degassing by applying a vacuum, or degassing by applying a vacuum and subsequent gassing with nitrogen, or only gassing with nitrogen.

[0026] The sperm populations enriched in the process can be used for routine artificial insemination, before or after sex sorting of sperm, before or after shipment of sperm for routine or sperm sex sorting processes or cryopreservation purposes, or for in-vitro fertilization or for ICSI or other ART for all mammalian species, including humans.

[0027] In a preferred embodiment, it is provided that a determination of the sperm cell count and / or sperm cell motility is carried out before step b). In this context, it is considered advantageous that, with knowledge of the values ​​and information thus obtained, the subsequent process steps as well as the buffer composition and quantity can be adjusted in order to further improve the process and the process result.

[0028] It is known that damaged cells, particularly those with damaged membranes, can cause damage to intact cells. The method according to the invention enables the separation of cells with damaged membranes or with any cell alterations that lead to a reduction in cell health from those with intact membranes or without any cell alterations that lead to a reduction in cell health. It can thus help to reduce membrane damage or any cell alteration that leads to a reduction in the cell health of intact cells by reducing or preventing harmful effects caused by damaged cells.For example, DNA fragmentation, oxidative damage through peroxidation, and the premature release of proteolytic and hydrolytic enzymes are examples of such effects, which shorten lifespan and impair cell health both in vitro and in vivo, and reduce desired cellular functionality. In sperm, in particular, the damage to intact sperm by non-intact sperm described above can shorten the lifespan of individual sperm or the entire sperm population both in vitro and in vivo, reduce fertilizing capacity, and result in poor embryo quality.

[0029] Mammalian sperm with good fertility should contain a high number of morphologically normal, viable sperm. Current semen preparation methods, such as sex sorting and storage (e.g., refrigeration or cryopreservation), can adversely affect sperm metabolism and motility, as well as the status of sperm membrane domains. The result of these effects can be reduced sperm functionality. Magnetic removal of damaged or compromised cells or cell structures can mitigate the adverse effects on the quality of live and normal sperm caused by dead and abnormal sperm.

[0030] Another example of cellular changes is the progressive capacitation of sperm cells, a necessary preparatory cell developmental step prior to fertilization of the egg. Capacitation (Brewis A. et al. 2005; Kqtska-Ksiqzkiewicz L. 2007) can be characterized by the removal of coating materials from the sperm surface. Capacitation can be the penultimate step of fertilization, leading to increased permeability of the plasma membrane to Ca2+ ions and enabling the sperm to undergo the acrosome reaction (Breitbart H. 2002) or to die if fertilization does not occur. Premature capacitation of sperm can lead to ovarian failure. Viable mammalian sperm can exhibit a net negative surface charge at the plasma membrane.When sperm undergo capacitation, followed by the acrosome reaction, their net charge can become less negative or more positive, for example, due to the loss of negative groups such as sialic acid groups. Capacitation can be prematurely triggered by—but not exclusively by—parameters such as temperature, pH, oxidative treatments, before and after ejaculation, or the individual composition of the seminal plasma. Naturally, no sperm population exhibits a uniform cell maturation pattern; different cell counts have different stages of cell maturation.

[0031] Furthermore, damage to sperm chromatin can lead to poor embryo quality. Since fertilization, like many other cellular functions in this class, can be a time-critical event, and good embryo quality is essential for timely embryonic development, both can be affected by sperm quality. Factors released by damaged sperm or other cells can be partly responsible for further cellular damage to the remaining subpopulation of cells, such as normal sperm. For example, freshly killed sperm can reduce sperm viability in diluted bovine semen. Additionally, freshly ejaculated sperm exposed to elevated temperatures prior to ejaculation may exhibit high levels of reactive oxidative species (ROS).Therefore, the toxic effect of dead cells, including but not limited to sperm, can be attributed to amino acid oxidase activity. Dead and abnormal cells, such as sperm, can have toxic effects on accompanying cells. Dead and abnormal cells can negatively affect the functionality of cellular samples, the overall cell population, or the ejaculate, such as sperm fertility.

[0032] There are several methods that can be used for cell purification (Henkel R. et al. 2003):

[0033] Cell cleansing generally refers to the process of cleaning cells, whether in biological, medical, or laboratory contexts. Here are some common methods:

[0034] Centrifugation: This is a widely used method in which cells are separated by centrifugal forces. Harmful substances can be removed in this way, and the purified cells can be used further.

[0035] Filtration: Filtration allows cells to be separated from unwanted particles and impurities. Filters with different pore sizes are used for this purpose.

[0036] Density gradient centrifugation: In this process, cells are placed on a density gradient and separated by centrifugation. This allows for separation based on the density of the cells.

[0037] Fluorescence-activated cell sorting (FACS): FACS enables the isolation of cells based on fluorescent labels. The cells are identified and sorted using laser beams.

[0038] Ultrafiltration: This is a method in which cells are passed through a semipermeable membrane to remove unwanted substances.

[0039] Enzymatic digestion: Through the use of enzymes, cells can be detached from tissues or other substances in order to isolate them.

[0040] Electrophoresis: Electrophoretic techniques can be used to separate cells based on their charge.

[0041] Swim-up method: This method utilizes the sperm's ability to swim against gravity. The semen sample is placed in a culture medium, and the sperm move to the top, where they are separated and collected.

[0042] Microfluidic chips: Microfluidic devices can be used to separate sperm based on their physical properties. This enables precise sperm isolation.

[0043] Immunomagnetic cell separation: This method uses antibodies bound to magnetic particles to identify and isolate specific cells. It is important to note that the optimal cell purification method depends heavily on the cell type, the starting material, and the specific requirements of the experiment. Each method has its own specific advantages and disadvantages. For example, FACS is not suitable for high-throughput applications and, due to its methodological characteristics (UV radiation, high pressure, use of potentially mutagenic substances, etc.), causes significant damage to living cells.

[0044] Immunomagnetic cell separation (Phiphattanaphiphop C. et al. 2021; Peter A. et al. 1993) is a technique based on the selective isolation of cells using antibodies and magnetic particles. This method allows for the precise separation of specific cell types from a heterogeneous cell population. The procedure comprises the following basic steps:

[0045] Antibody binding: First, specific antibodies are selected that target the surface of the target cells. These antibodies can bind to specific proteins, markers, or receptors present on the target cell.

[0046] Magnetic particle labeling: The selected antibodies are conjugated with magnetic particles, which are usually made of a material such as iron oxide. These magnetic particles serve as a label for the target cells.

[0047] Incubation with cell sample: The antibodies labeled with magnetic particles are then incubated with the cell sample. The antibodies bind specifically to the target cells in the sample.

[0048] Magnetic separation: After incubation, the cell sample is placed in a magnetic field. The target cells labeled with magnetic particles are attracted by the magnetic field and remain attached, while unlabeled cells are washed away.

[0049] Isolation of the target cells: The magnetically labeled target cells are then isolated from the other cells by removing the magnetic field or by washing steps. The isolated cells can be used for further analyses or experiments.

[0050] This method has the advantage of enabling highly selective isolation of target cells without significantly impairing cell function. It is frequently used in research and diagnostics, particularly when isolating rare cell types or obtaining highly purified cell populations. Immunomagnetic cell separation is applied in various fields such as cancer research, immunology, hematological studies, and stem cell isolation. However, this method has the disadvantage that, due to the typically very complex antibody development and synthesis of the magnetic particles, its commercial application is limited to select areas.

[0051] Furthermore, patent EP2890498 describes the application of specially surface-derivatized (silanized) magnetic particles with a negative zeta potential for the enrichment of intact sperm cells.

[0052] Patent application US2023228750A1 describes the necessary combination of magnetic particles with polymers such as polycaprolactone, polyolefin, and polysaccharides with a negative zeta potential as an enrichment substance. In one embodiment of the method according to the invention, the ferromagnetic material is selected from iron (Fe), cobalt (Co), nickel (Ni), neodymium (Nd), samarium (Sm), cobalt ferrite (CoFe2O4), gadolinium (Gd), alnico alloys, ferrites (magnetite, Fe3O4), dysprosium, holmium, erbium, terbium, maghemite (Fe2O3), alloys such as SmCo, Nd2Fel4B, Ni80Fe20 (permalloy), NiFeCo (mumetal), CrO2, magnesium arsenide, EuO or mixtures thereof, wherein the use of magnetite (Fe3O4) or of material containing magnetite (Fe3O4) as a component is considered particularly advantageous.Preferably, the magnetic material is provided in the form of magnetic particles comprising or formed from the ferromagnetic material, wherein the magnetic particles have a size distribution of between 30 nm and 15,000 nm, particularly between 80 nm and 1000 nm, preferably between 100 nm and 7,000 nm. Magnetic particles with a size or diameter in at least one direction of extension of <100 nm are referred to as magnetic nanoparticles (MNPs). In the process according to the invention, magnetic particles (MPs) with a diameter in at least one direction of extension of greater than or equal to 100 nm are preferably used. However, the use of magnetic nanoparticles (MNPs) with a diameter in at least one direction of extension of less than 100 nm or mixtures with a corresponding size distribution is also possible and equally encompassed by the invention.The use of magnetic particles without coating or derivatization proves particularly advantageous. The size of the particles used is preferably in the range of 100 nm to 500 nm, particularly between 150 nm and 400 nm, and preferably between 200 nm and 350 nm. Prior to using the particles, a deposition process can be performed in which particles with diameters of less than 100 nm are removed from the particle suspension in at least one direction.

[0053] Step c) of the process involves separating the liquid medium from the sedimented cell population. In embodiments where the cell population consists of intact or damaged sperm cells, particularly mammalian sperm cells, the liquid medium consists of ejaculate fluid, particularly mammalian ejaculate fluid, containing large amounts of seminal plasma. Surprisingly, it has been found that an enrichment of intact sperm cells by applying surface-untreated magnetic particles is possible, regardless of the zeta potential of the magnetic particles used, if the liquid medium, i.e., primarily the seminal plasma, is substantially removed before their application in the process according to the invention.In the process according to the invention, particles are advantageously and with surprising effect used that do not have any surface derivatizations, in particular no coatings with dextran, silicates, silanization, and / or binding to bioactive proteins such as, in particular, antibodies, annexin, etc. The particles also do not have any modifications via functional groups, such as NH2, COOH, Si-OH, or PEG-COOH. The process according to the invention thus enables the simple, rapid, and cost-effective enrichment of a cell population containing intact and damaged cells in a liquid medium and the separation of the damaged cells.

[0054] Furthermore, the effectiveness and ease of use of this method are significantly improved by the application of certain additives. Surprisingly, it has been shown that, when applying the method according to the invention, a significant improvement in the enrichment of intact cells in a cell population can generally be achieved for all types of magnetic particles, particularly those made from the aforementioned materials, regardless of the zeta potential of the magnetic particles used. Surprisingly, it has also been shown that cell binding occurs to particles without any surface alteration or modification. For example, in particles made of iron (Fe) or particles containing iron (Fe), direct cell binding to iron (Fe) or Fe-O cell binding can occur. Furthermore, it has been surprisingly found that, particularly, but not exclusively, in Fe3O4 particles without any surface alteration or modification, cell binding to iron (Fe) can occur.Modification: Binding to cells occurs as soon as the cells exhibit a membrane defect. The type and location of the membrane defect do not influence the binding effect, or only to a negligible extent. A possible cause for this surprising effect could be, for example, a change in the cell surface charge occurring in combination with a defect, which causes the binding between the cell and the particle surface via charge differences. Other effects are also conceivable.

[0055] Therefore, there is no opposing electrostatic bond between the cell membrane and the magnetic particle leading to the enrichment of intact cells and the depletion of non-intact cells. It has been found that magnetic particles with sizes in the p-range can also be used effectively. Advantageously, the inventive method can thus be carried out much more cost-effectively than methods that rely on surface-modified particles and / or employ expensive and complex-to-produce nanoparticles (MNPs). The use of larger magnetic particles (MPs), particularly those produced using standardized processes without modifications, significantly expands and simplifies the application range of the inventive method and enables the easy and successful separation of intact and damaged cells from a cell population in a liquid medium.

[0056] In step c) of the inventive process, a reduction in seminal plasma is achieved when applied with cell populations derived from sperm cells. Seminal plasma, as a secretion, serves as a protective, transport, and nutrient medium for sperm and comprises approximately 90% of the ejaculate (Zoea G. et al. 2021). It thus determines the volume of the total ejaculate and provides the sperm with an individually optimal metabolic level. Seminal plasma consists of organic (primarily peptides and proteins) and inorganic (salts) components, with the components with a high molecular mass, the proteins, being particularly noteworthy (Tedeschi et al., 2000). Seminal plasma proteins play a significant role in capacitation, acrosome reaction, motility and sperm-oocyte fusion (Gwathmey et al., 2003; Suarez, 2006; Stival C. et al. 2016).For example, a high proportion of the seminal plasma proteins 26 kDa and 55 kDa in the ejaculate leads to better fertility in bull sperm, while a high proportion of the 16 kDa protein leads to lower fertility in bulls (Killian et al., 1993). The protein PDC-109 triggers capacitation of sperm (Desnoyers and Manjunath, 1992). The individual composition of the seminal plasma therefore directly influences the qualitative fertility properties of the sperm. Certain components of the highly complex composition of the seminal plasma cause the surfaces of magnetic particles to become blocked, preventing certain binding sites on the sperm from being used.The removal of the seminal plasma allows the use of these specific binding sites, eliminating the need for electrostatic bonding between the magnetic particles and the cells, and making purification more efficient through these specific bonds.

[0057] The substantial reduction of the seminal plasma in the sperm cell population is achieved, for example, but not exclusively, by centrifugation to sediment the cell population, as described in step b of the procedure. During centrifugation, the semen sample (cell population in liquid medium) is placed in a centrifuge tube. The centrifugal forces acting during centrifugation cause the sperm, i.e., the cell population, to sediment at the bottom of the tube. The supernatant, i.e., the liquid medium containing the other components, particularly the seminal plasma, is then removed, i.e., separated from the sedimented cell population, and the sedimented cell population, i.e., the concentrated sperm, is then collected.

[0058] The sperm sample is subsequently suspended, as provided in particular in step d) of the method according to the invention. For this purpose, a physiological suspension solution, also referred to as a buffer solution or washing solution, is used, and the sedimented cell population is diluted in it. Suspension solutions, buffer solutions, or washing solutions can be used that have the modification and mixture indicated for the respective application or cell populations.

[0059] Suspension solutions containing the following are proposed as exemplary and advantageous embodiments:

[0060] Phosphate-buffered physiological saline (PBS), Hanks' Balanced Salt Solution (HBSS), Good's Buffers, Tris buffer, Phosphate-buffered saline (PSS), Earle's Balanced Salt Solution (EBSS), Ringer's solutions, TES-Tris buffer, HEPES buffer, sperm buffer (SP), SP-TALP, FC-TALP, TCA, citrates, acetates, lactates, carbonates, phosphates and combinations thereof, or a solution containing at least one salt, at least one carbohydrate or a combination thereof; milk and derivatives thereof; MES; diluent systems compatible with the respective animal species, such as Andromed, BioXCell, BullXcell, INRA 96, PRIMXcell, TRIXcell, NUTRIXcell, OviXcell, Galap, CU-N Igel, Triladyl, Biladyl, Steridyl, OviPlus, Bovidyl, M III, BTS, Androstar, Novistar, OptiXCell, Beyond, EquiPlus, Gent or CaniPlus, or mixtures thereof; additives selected from the group consisting of coconut water, ascorbic acid, beta-carotene, quercetin, rutin, taurine, 5-HMF, glutathione, ubiquinone,Superoxide dismutase, glutathione peroxidase, catalase, L-carnitine, zinc, polyphenols, folic acid, alpha-lipoic acid, melatonin, astaxanthin, zeaxanthin, lucopins, anthocyanins, bioflavonoids, pyruvate, serum albumin, regucalcin, curcumin (diferuloylmethane), growth factors such as epidermal growth factor, oviductal proteins (such as non-luteal isthmic oviductal proteins (NLIP)), Diospyros kaki (persimmon) extract, coenzyme Q10, cobalamin, silymarin (Silybum marinanum), glycine, cholesterol-loaded cyclodextrins, docosahexaenoic acid (DHA), vitamin E, deuterated polyunsaturated fatty acids, butylhydroxytoluene, butylhydroxyanisole, ferrostatin, lipoxstatin, CoQlO, idebenone XJB-5-131 (mitochondria-directed nitroxide), JP4-039, baicalein, PD-146176 (15-LOX-1 inhibitor), AA-861 (LOX-1 inhibitor), zileuton, deferoxamine, ciclopirox, deferiprone, cycloheximide, 2-mercaptoethanol, dopamine, vildagliptin, alogliptin, linagliptin or mixtures thereof; antibiotics,Antimicrobial or antibiotic agents selected from the group consisting of tetracycline, penicillin, sulfonamide, aminoglycoside, fluoroquinolone, macrolide, cetiofur, apramycin, streptomycin, ticarcillin, polymyxin B or their derivatives, tylosin, gentamicin, lincomycin, spectinomycin, Linco-Spectin® (lincomycin hydrochloride-spectinomycin), or mixtures thereof; pyruvate; Liposome-forming or non-liposome-forming lipids (e.g., phospholipids such as PA, PC, PE, LPC, LPA, PG, LPG, PI, PIP, LPI, PS, LPS, cardiolipin, bis(monoacylglycero)phosphates (BMP), ether lipids, sterol-modified phospholipids, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(l-glycerol) (DOPG), l,2-dioleoyl-sn-glycero-3-ethyl phosphocholine (EPC), dioleoylphosphosphatidylethanolamine (DOPE), phosphatidylcholine (PC), cholesteryl hemisuccinate (cholesteryl hydrogen succinate) (CHEMS), cholesterol and its derivatives; aliphatic carboxylic acids (low as long-chain fatty acids) such as caproic acid, Sorbic acid, capric acid,n-Valerianic acid, myristic acid, pelargonic acid, octanoic acid, or polymeric carboxylic acids or polymeric amines of different chain lengths such as polyacrylic acid, polyethyleneimines, poly-L-lysine, or transfection-active substances such as dioleoyl-3-trimethylammonium propane (DOTAP), DOTMA, DODMA, DOGS, DOSPA, DC-cholesterol, DLinDMA, liposomal or nanomolecular lipids such as Lipofectamine, Lipofectamin 2000, Metafectene Pro, Fugene, GeneJuice, etc., or mixtures thereof; serum albumins: HSA, BSA, ESA, CSA, FSA, OSA, MSA, RSA, or PSA; EDTA (ethylenediaminetetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), DMSA (dimercaptosuccinic acid), deferoxamine, citric acid, EGTA (ethylene glycol bis(aminoethyl ether)tetraacetic acid), Chelex® resins, transferrin, deferasirox, penicillamine, alpha-lipoic acid, DMPS, dimercaprol and aminopolycarboxylic acids (complexones), including but not limited to fura-2, IDA, NTA, DTPA, BAPTA, NOTA,DOTA and nicotianamine and derivatives thereof; polymyxin B; lecithins, milk casein, beta-lactoglobulin, alpha-lactalbumin, glycodelin S, glucose, fructose, glycosyllable sugars; pyruvate; ethane-1,2-diol, formamide, dimethyl sulfoxide, Greg Fahy's VS41A, M22, propylene glycol, ethylene glycol, polyethylene glycol, polyvinylpyrrolidone, propane-1,2,3-triol, X-1000 (Copolymer of polyvinyl alcohol and vinyl acetate), mannitol, K12, Z-1000, VM3, CI-VM-1, 3-methoxy-1,2-propanediol, N-methylformamide, cysteine ​​and its derivatives; Contains polydatin (resveratrol), selenium-associated glutathione peroxidase, glutathione disulfide reductase, heme oxygenase, or mixtures thereof.

[0061] The aforementioned substances can be used individually or in mixtures with varying ratios adapted to the intended use in buffer systems, also known as physiological suspension solutions, buffer solutions, or washing solutions. Physiological buffer systems, referred to as physiological suspension solutions, buffer solutions, or washing solutions, are specifically buffer systems with a pH value in the range of 6.5 to 8.5 and an osmolarity in the range of 250 to 375 mOsm.

[0062] The sedimentation of the cell population from the liquid medium, and the separation of the liquid medium and the sedimented cell population from the liquid medium (which, in the case of sperm populations, consists primarily of seminal plasma), is carried out, in particular, within a temperature range of 4 to 40°C, preferably within a temperature range of 16 to 24°C. If the sedimentation of the cell population from the liquid medium is performed by centrifugation, the centrifugal force is specified by its magnitude, referred to as the "g-number" or "relative centrifugal force (RCF)." The g-number is important to ensure that the cell population, especially the sperm, sediments effectively and separates from other components. In practice, the g-number can vary depending on the specific requirements of the experiment or clinical application.Sperm centrifugation for reproductive technologies such as in-vitro fertilization (IVF) often uses a specific number of grams to achieve optimal sperm separation and concentration. Controlling centrifugation conditions is crucial to preserve sperm integrity and ensure suitability for the intended applications. Typical gram values ​​used range from 300 to 4000 g, 500 to 3500 g, 600 to 3400 g, 700 to 3300 g, 800 to 3200 g, 900 to 3100 g, or 1000 to 3000 g. Centrifugation time ranges from 1 to 60 minutes.

[0063] In a preferred embodiment of the method according to the invention, in step b.) sedimentation by centrifugation is provided, in particular with a centrifugation duration of between 1 min and 60 min, preferably with a centrifugal acceleration of between 300g and 4000g.

[0064] Centrifuge tubes that can be preferably used in the process according to the invention are specially designed tubes intended for use in centrifuges. They are used in various laboratory applications, particularly in cell culture, molecular biology, diagnostics, and other areas of biological and chemical research. Here are some important aspects of centrifuge tubes:

[0065] Material: Centrifuge tubes are made from various materials, including polypropylene, polyethylene, or polystyrene. The choice of material depends on the specific application.

[0066] Size and volume: Centrifuge tubes are available in various sizes and volumes to meet the needs of different experiments. Sizes range from small-volume microcentrifuge tubes to centrifuge bottles for larger volumes.

[0067] Maintaining strict sterility is of paramount importance to prevent contamination and cross-contamination. Containers must be autoclavable and undergo a washing and sterilization process after each use. Sterile, single-use containers are typically used.

[0068] Sperm cells are subjected to impaired functionality by mechanical influences such as centrifugation, as well as by chemical and physical influences such as atmospheric oxygen, UV radiation, etc. (Agarwal A. et al. 2003), potentially leading to apoptosis and cell death. This problem can be reduced or even prevented by adding certain additives. In a preferred embodiment of the inventive process, a washing solution is added to the liquid medium after step a.). In one embodiment, the washing solution comprises: Phosphate-buffered physiological saline (PBS), Hanks' Balanced Salt Solution (HBSS), Good's Buffers, Tris buffer, Phosphate-buffered saline (PSS), Earle's Balanced Salt Solution (EBSS), Ringer's solutions, TES-Tris buffer, HEPES buffer, sperm buffer (SP), SP-TALP, FC-TALP, TCA, citrate, acetate, lactate, carbonate.Phosphates and combinations thereof, or a solution containing at least one salt, at least one carbohydrate or a combination thereof; milk and derivatives thereof; MES; diluent systems compatible with the respective animal species, such as Andromed, BioXCell, BullXcell, INRA 96, PRIMXcell, TRIXcell, NUTRIXcell, OviXcell, Galap, CUNIgel, Triladyl, Biladyl, Steridyl, OviPlus, Bovidyl, M III, BTS, Androstar, Novistar, OptiXCell, Beyond, EquiPlus, Gent or CaniPlus, or mixtures thereof; Additives selected from the group consisting of coconut water, ascorbic acid, beta-carotene, quercetin, rutin, taurine, 5-HMF, glutathione, ubiquinone, superoxide dismutase, glutathione peroxidase, catalase, selenium, L-carnitine, zinc, polyphenols, folic acid, alpha-lipoic acid, melatonin, astaxanthin, zeanxanthin, lucopins, anthocyanins, bioflavonoids, pyruvate, serum albumin, regucalcin, curcumin (diferuloylmethane), growth factors such as epidermal growth factor, oviductal proteins,such as non-luteal isthmic oviductal proteins (NLIP), Diospyros kaki (persimmon) extract, coenzyme Q10, cobalamin, silymarin (Silybum marianum), glycine, cholesterol-loaded cyclodextrins, docosahexaenoic acid (DHA), vitamin E, deuterated polyunsaturated fatty acids, butylhydroxytoluene, butylhydroxyanisole, ferrostatin, lipoxstatin, CoQlO, idebenone, XJB-5-131 (mitochondria-directed nitroxide), JP4-039, baicalein, PD-146176 (15-LOX-1 inhibitor), AA-861 (LOX inhibitor), zileuton, deferoxamine, ciclopirox, deferiprone, cycloheximide, 2-mercaptoethanol, dopamine, vildagliptin Alogliptin, linagliptin or mixtures thereof; antibiotics, antimicrobial or antibiotic agents selected from the group consisting of tetracycline, penicillin, sulfonamide, aminoglycoside, fluoroquinolone, macrolide, cetiofur, apramycin, streptomycin, ticarcillin, polymyxin B or their derivatives, tylosin, gentamicin, lincomycin, spectinomycin, Linco-Spectin® (lincomycin hydrochloride-spectinomycin),or mixtures thereof; pyruvate; Liposome-forming or non-liposome-forming lipids (e.g., phospholipids such as PA, PC, PE, LPC, LPA, PG, LPG, PI, PIP, LPI, PS, LPS, cardiolipin, bis(monoacylglycero)phosphates (BMP), ether lipids, sterol-modified phospholipids, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(l-glycerol) (DOPG), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EPC), dioleoylphosphatidylethanolamine (DOPE), phosphatidylcholine (PC), cholesteryl hemisuccinate (cholesteryl hydrogen succinate) (CHEMS), cholesterol and its derivatives; aliphatic carboxylic acids (low as long-chain fatty acids) such as caproic acid, sorbic acid, Capric acid, n-valeric acid, myristic acid, pelargonic acid, octanoic acid, or polymeric carboxylic acids or polymeric amines of different chain lengths such as polyacrylic acid, polyethyleneimines, poly-L-lysine, or transfection-active substances such as dioleoyl-3-trimethylammonium propane (DOTAP), DOTMA, DODMA, DOGS, DOSPA, DC-cholesterol, DLinDMA,Liposomal or nanomolecular lipids such as Lipofectamine, Lipofectamin 2000, Metafectene Pro, Fugene, GeneJuice, etc., or mixtures thereof; serum albumins: HSA, BSA, ESA, CSA, FSA, OSA, MSA, RSA, or PSA; EDTA (ethylenediaminetetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), DMSA (dimercaptosuccinic acid), deferoxamine, citric acid, EGTA (ethylene glycol bis(aminoethyl ether)tetraacetic acid), Chelex® resins, transferrin, deferasirox, penicillamine, alpha-lipoic acid, DMPS, dimercaprol, and aminopolycarboxylic acids (complexones), including but not limited to fura-2, IDA, NTA, DTPA, BAPTA, NOTA, DOTA, and nicotianamine, as well as derivatives thereof; polymyxin B; Lecithins, milk casein, beta-lactoglobulin, alpha-lactalbumin, glycodelin-S, glucose, fructose, glycosatable sugars; pyruvate; Ethane-1, 2-diol, formamide, dimethyl sulfoxide, Greg Fahy's VS41A, M22, propylene glycol, ethylene glycol, polyethylene glycol, polyvinylpyrrolidone, propane-1, 2, 3-triol,X-1000 (Copolymer of polyvinyl alcohol and vinyl acetate), mannitol, K12, Z-1000, VM3, Cl-VM-1, 3-methoxy-1,2-propanediol, N-methylformamide, cysteine ​​and its derivatives; polydatin (resveratrol), selenium-associated glutathione peroxidase, glutathione disulfide reductase, heme oxygenase, or mixtures thereof. The aforementioned substances may each be used individually or in mixtures with varying ratios adapted to the intended use in the buffer systems, also known as physiological suspension solutions, buffer solutions, or washing solutions.

[0069] It may be advantageous, when applying the procedure with a cell population formed from sperm cells, to add sialidase inhibitors such as 2,3-dehydro-2-deoxy-N-acetyl-neuraminic acid (DANA) after obtaining the ejaculate, i.e., after step a.), particularly in cases where enrichment is not carried out immediately and more than 10 minutes elapse between ejaculate collection and the start of the preparation process.

[0070] The additives in all solutions used in the process according to the invention include antioxidants (Chi H. et al. 2008). Antioxidants can play an important role in sperm health, particularly because sperm are susceptible to oxidative stress. Oxidative stress can arise from an imbalance between the production of reactive oxygen species (ROS) and the protective effects of antioxidants. Here are some antioxidants that may be important for sperm health, such as, but not limited to, ascorbic acid, tocopherol, beta-carotene, quercetin, rutin, taurine, 5-HMF, glutathione, ubiquinone, superoxide dismutase, glutathione peroxidase, catalase, selenium, L-carnitine, zinc, polyphenols, folic acid, alpha-lipoic acid, melatonin, astaxanthin, zeanxanthin, lucopins, anthocyanins, or bioflavonoids or mixtures thereof.

[0071] Another additive to all solutions used in the process according to the invention can be pyruvate. Pyruvate is a molecule that plays an important role in the metabolism of cells, including sperm. It is a compound that acts as a substrate and intermediate in various biochemical processes. Pyruvate and pyruvate metabolism are of interest in the context of sperm and their energy production. Pyruvate is a key substrate in the process of cellular respiration, specifically in the citric acid cycle (tricarboxylic acid cycle) and the electron transport chain. These processes are crucial for the production of adenosine triphosphate (ATP), the cell's main energy currency. Sperm are rich in mitochondria, the cell's powerhouses. Pyruvate is metabolized in the mitochondria and can therefore influence mitochondrial activity and function.

[0072] Several studies have indicated that the addition of pyruvate to culture media can improve sperm motility, particularly rapid progressive motility. The additives can be used individually or in optimized mixing ratios. Further additives to all solutions used in the inventive process can be antibiotics. Antibiotics are used to inhibit or prevent bacterial growth. The choice of antibiotic, the dosage, and the duration of use can vary depending on individual circumstances and the medical context. Here are some examples, not limited to the invention, of antibiotics commonly used in animal husbandry:

[0073] Tetracyclines: Tetracyclines are used to treat a variety of bacterial infections in livestock. They can also be used as growth promoters.

[0074] Penicillins: Penicillins, such as penicillin G, are used to treat bacterial infections. They are among the oldest and best-known antibiotics.

[0075] Sulfonamides: Sulfonamides are used in animal husbandry to treat bacterial infections.

[0076] Aminoglycosides: Aminoglycosides such as gentamicin and amikacin are used to treat infections.

[0077] Fluoroquinolones: Fluoroquinolones such as enrofloxacin are used in animals to treat certain bacterial infections.

[0078] Macrolides: Macrolides such as erythromycin and tylosin are used to fight bacterial infections.

[0079] The antibiotics described above are examples only and not exclusive; they can also be used in any combination. The concentration range of the antibiotics used in all solutions employed in the process according to the invention is, in particular, 50 pg to 800 pg per ml of solution.

[0080] Furthermore, so-called decapacitation factors can also be used in all solutions employed in the process according to the invention. These include, but are not limited to, lecithins, milk casein, beta-lactoglobulin, alpha-lactalbumin, glycodelin S, glucose or fructose, or generally glycosylizable sugars.

[0081] The following description relates to a preferred embodiment of the invention with cell populations formed from sperm, but does not limit the invention thereto and can be applied in the same or adapted manner to other cell types or cell populations.

[0082] After removing the main components of the liquid medium, in the case of sperm samples the seminal plasma, the sperm cells are resuspended in a physiological buffer system, the separation solution. The buffer system may be the same as or different from the washing solution described in step a), depending on the individual characteristics of the cell population used. The following buffer systems and their modifications and mixtures suitable for the respective application can be used as suspension solutions, by way of example, but not exclusively: Phosphate-buffered physiological saline (PBS), Hanks' Balanced Salt Solution (HBSS), Good's Buffers, Tris buffer, Phosphate-buffered saline (PSS), Earle's Balanced Salt Solution (EBSS), Ringer's solutions, TES-Tris buffer, HEPES buffer, sperm buffer solution (SP), SP-TALP, FC-TALP, TCA, citrates, acetates, lactates, carbonates, phosphates and combinations thereof, or a solution thereof.containing at least one salt, at least one carbohydrate or a combination thereof; milk and its derivatives; MES; diluent systems compatible with the respective animal species, such as Andromed, BioXCell, BullXcell, INRA 96, PRIMXcell, TRIXcell, NUTRIXcell, OviXcell, Galap, CUNIgel, Triladyl, Biladyl, Steridyl, O-viPlus, Bovidyl, M III, BTS, Androstar, Novistar, OptiXCell, Beyond, EquiPlus, Gent or CaniPlus, or mixtures thereof; Additives selected from the group consisting of glycine, cholesterol-loaded cyclodextrins, docosahexaenoic acid (DHA), vitamin E (α-tocopherol, trolox, tocotrienol), deuterated polyunsaturated fatty acids (D-PUFAs), butylhydroxytoluene, butylhydroxyanisole, ferrostatins, liproxstatins, CoQlO, idebenone, XJB-5-131 (mitochondrially directed nitroxide), JP4-039 (broadly directed intracellular antioxidant), baicalein, PD-146176 (15-LOX-1 inhibitor), AA-861 (LOX inhibitor), zileuton, deferoxamine, ciclopirox, deferiprone, cycloheximide,2-Mercaptoethanol, Dopamine, Selenium, Vildagliptin, Alogliptin, Linagliptin, Coconut water, Ascorbic acid, Beta-carotene, Quercetin, Rutin, Taurine, 5-HMF, Glutathione, Ubiquinone, Superoxide dismutase, Glutathione peroxidase, Catalase, L-Carnitine, Zinc, Polyphenols, Folic acid, Alpha-lipoic acid, Melatonin, Astaxanthin, Zeanxanthin, Lucopene, Anthocyanins, Bioflavonoids, Pyruvate, Serum albumin, Regucalcin, Curcumin (Diferuloylmethane), Growth factors such as epidermal growth factor, Oviductal proteins (such as non-luteal isthmic oviductal proteins (NLIP)), Diospyros kaki (persimmon) extract, Coenzyme Q10, Cobalamin, Silymarin (Silybum) marianum) or mixtures thereof; antibiotics, antimicrobial or antibiotic agents selected from the group consisting of tetracycline, penicillin, sulfonamide, aminoglycoside, fluoroquinolone, macrolide, cetiofur, apramycin, streptomycin, ticarcillin, polymyxin B or their derivatives, tylosin, gentamicin, lincomycin, spectinomycin,Linco-Spectin® (lincomycin hydrochloride-spectinomycin), or mixtures thereof; pyruvate; Liposome-forming or non-liposome-forming lipids (e.g., phospholipids such as PA, PC, PE, LPC, LPA, PG, LPG, PI, PIP, LPI, PS, LPS, cardiolipin, bis(monoacylglycero)phosphates (BMP), ether lipids, sterol-modified phospholipids, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(l-glycerol) (DOPG), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EPC), dioleoylphosphatidylethanolamine (DOPE), phosphatidylcholine (PC), cholesteryl hemisuccinate (cholesteryl hydrogen succinate) (CHEMS), cholesterol and its derivatives; aliphatic carboxylic acids (low as long-chain fatty acids) such as caproic acid, sorbic acid, capric acid, n-Valerianic acid, myristic acid, pelargonic acid, octanoic acid, or polymeric carboxylic acids or polymeric amines of different chain lengths such as polyacrylic acid, polyethyleneimines, poly-L-lysine, or transfection-active substances such as dioleoyl-3-trimethylammonia propane (DOTAP), DOTMA, DODMA, DOGS,DOSPA, DC cholesterol, DLinDMA, liposomal or nanomolecular lipids such as Lipofectamine, Lipofectamin 2000, Metafectene Pro, Fugene, GeneJuice, etc., or mixtures thereof; serum albumins: HSA, BSA, ESA, CSA, FSA, OSA, MSA, RSA, or PSA; EDTA (ethylenediaminetetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), DMSA (dimercaptosuccinic acid), deferoxamine, citric acid, EGTA (ethylene glycoside tetraacetic acid), Chelex® resins, transferrin, deferasirox, penicillamine, alpha-lipoic acid, DMPS, dimercaprol and aminopolycarboxylic acids (complexones), including but not limited to fura-2, IDA, NTA, DTPA, BAPTA, NOTA, DOTA and nicotianamine and derivatives thereof; polymyxin B; lecithins, milk casein, beta-lactoglobulin, alpha-lactalbumin, glycodelin S, glucose, fructose, glycolyzable sugars; pyruvate; Ethane-1, 2-diol, formamide, dimethyl sulfoxide, Greg Fahy's VS41A, M22, propylene glycol, ethylene glycol, polyethylene glycol, polyvinylpyrrolidone, propane-1, 2, 3-triol,X-1000 (Copolymer of polyvinyl alcohol and vinyl acetate), mannitol, K12, Z-1000, VM3, CI-VM-1, 3-methoxy-1,2-propanediol, N-methylformamide, cysteine ​​and its derivatives; polydatin (resveratrol), selenium-associated glutathione peroxidase, glutathione disulfide reductase, heme oxygenase, or mixtures thereof. The aforementioned substances can each be used individually or in mixtures with varying ratios adapted to the intended use in buffer systems, also known as physiological suspension solutions, buffer solutions, or washing solutions. Physiological buffer systems are understood to be, in particular, those buffer systems that have a pH value in the range of 6.0 to 8.5 and an osmolarity in the range of 250 to 375 mOsm.

[0083] The separation or purification process, i.e., the resuspension of the sedimented cell population in a suspension solution to produce a cell suspension, particularly according to step d) of the process according to the invention, is carried out in a temperature range of 4 to 40°C, particularly in the range between 16 and 24°C.

[0084] The aforementioned additives can be added to the suspension solution, individually or in optimized mixing ratios, to reduce or prevent the stress-related negative effects on sperm cells caused by the purification process. These additives, among other things, maintain the membrane integrity and fluidity of the cells. The substances can be positively charged, negatively charged, or uncharged at neutral pH.

[0085] Other additives that promote the purification process during suspension are, in particular, serum albumins. Albumins are proteins found in the blood plasma of vertebrates. They belong to the group of serum proteins and fulfill various vital functions in the organism. Albumins are globular proteins with a complex three-dimensional structure. They consist of a single polypeptide chain.

[0086] Their function is, for example:

[0087] Transport of molecules: Albumins serve as transport proteins for various substances in the blood, including hormones, fatty acids, bilirubin, drugs and many other molecules.

[0088] Maintaining osmotic pressure: Albumins help maintain the osmotic pressure of blood plasma, which is important for maintaining fluid balance between blood and tissues.

[0089] Buffer function: They can stabilize the pH value of the blood and act as a buffer.

[0090] Immunological functions: Albumins may be involved in the regulation of the immune system. The addition of albumin to a sperm solution may be considered in certain situations in reproductive medicine or laboratory research. Serum albumin is often used as a protein source and stabilizer in medical and reproductive applications. In scientific research, particularly in the cultivation of cells for studies of spermatogenesis or sperm function, the addition of albumins to the culture medium can be used to promote cell growth and survival. The following serum albumins may be used, though not exclusively: HSA, BSA, ESA, CSA, FSA, OSA, MSA, RSA, or PSA. Other protein sources may include, for example, commercially available, mostly synthetic protein supplements.

[0091] Another additive to the suspension solution can be antibiotics. Antibiotics are used to inhibit or prevent bacterial growth. It's important to note that there may be guidelines that prescribe or prohibit the use and dosage of certain antibiotics. Therefore, it's crucial to remember that the use of antibiotics with sperm should be approached carefully and with consideration of the specific circumstances. The choice of antibiotic, dosage, and duration of treatment can vary depending on individual conditions and the medical context. Here are some of the antibiotics commonly used in animal husbandry:

[0092] Tetracyclines: Tetracyclines are used to treat a variety of bacterial infections in livestock. They can also be used as growth promoters.

[0093] Penicillins: Penicillins, such as penicillin G, are used to treat bacterial infections. They are among the oldest and best-known antibiotics.

[0094] Sulfonamides: Sulfonamides are used in animal husbandry to treat bacterial infections.

[0095] Aminoglycosides: Aminoglycosides such as gentamicin and amikacin are used to treat infections.

[0096] Fluoroquinolones: Fluoroquinolones such as enrofloxacin are used to treat certain bacterial infections.

[0097] Macrolides: Macrolides such as erythromycin and tylosin are used to fight bacterial infections.

[0098] The antibiotics described above are examples, do not limit the invention to them, and can be used in any combination. The concentration range of the antibiotics used is 50 pg to 800 pg per ml of suspension solution. Furthermore, the use of chelating agents as an additive to the suspension solution can be advantageous. Chelating agents can play a role in various contexts, particularly with regard to sperm. Here are some aspects in which chelating agents could be relevant in relation to sperm:

[0099] Sperm cryopreservation: In the cryopreservation of sperm, particularly in reproductive medicine, chelating agents such as EDTA (ethylenediaminetetraacetic acid) can be used as a component of dilution solutions. They can help to chelate metal ions and thereby reduce oxidative damage during the freezing process.

[0100] Sperm preparation for in-vitro fertilization (IVF): When preparing sperm for IVF procedures, chelating agents can be used in various sperm preparation media and solutions. These solutions can serve to purify sperm of unwanted substances and ensure better quality for fertilization. Unwanted substances include, for example, so-called endotoxins (lipopolysaccharides of bacterial origin).

[0101] Intracellular calcium regulation: Calcium plays an important role in regulating sperm functions such as the acrosome response and hyperactivation. Chelating agents can influence the intracellular concentration of calcium, which in turn can affect sperm physiological processes.

[0102] Metal ions and sperm function: Metal ions such as zinc, iron, and copper can affect sperm function. Chelating agents can be used in this context to bind metal ions and modulate their effects on sperm.

[0103] Some of the most common chelating agents that may be used, but are not limited to, are EDTA (ethylenediaminetetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), DMSA (dimercaptosuccinic acid), deferoxamine, citric acid, EGTA (ethylene glycol bis(aminoethyl ether)tetraacetic acid), Chelex® resins, transferrin, deferasirox, penicillamine, alpha-lipoic acid, DMPS, dimercaprol, and aminopolycarboxylic acids (complexones), including but not limited to fura-2, IDA, NTA, DTPA, BAPTA, NOTA, DOTA, and nicotianamine, as well as derivatives thereof.

[0104] Common agents for removing endotoxins include, but are not limited to, chelating agents such as polymyxin B.

[0105] Furthermore, so-called decapacitation factors can be used as additives. These include, but are not limited to, lecithins, milk casein, beta-lactoglobulin, alpha-lactalbumin, glycodelin-S, glucose or fructose, or generally glycosylizable sugars.

[0106] In step e) of the method according to the invention, the cell suspension is treated with a ferromagnetic material that binds specifically to the damaged cells. Materials with ferromagnetic properties are added to the cell suspension; these materials have a special binding capacity to non-intact cells, particularly sperm cells. Ferromagnetic materials are substances capable of forming permanent magnetic dipoles and exhibiting strong magnetic properties. Unlike paramagnetic materials, ferromagnetic materials retain their magnetic properties even without an external magnetic field. The following are some examples of ferromagnetic materials, which are not limited to the invention:

[0107] Iron (Fe): Iron is one of the best-known ferromagnetic materials. It is frequently used in industry for the manufacture of magnets.

[0108] Cobalt (Co): Cobalt is a ferromagnetic metal and is often used together with other materials for the manufacture of permanent magnets.

[0109] Nickel (Ni): Nickel is a ferromagnetic element and is also used in the manufacture of magnets. Neodymium (Nd): Neodymium is a rare-earth metal and is used in combination with iron and boron to produce strong permanent magnets.

[0110] Samarium (Sm): Samarium is another rare-earth metal used in permanent magnets. Cobalt ferrite (CoFe₂O₄): A ferromagnetic oxide used in technology for various applications, including magnetic materials.

[0111] Gadolinium (Gd): Gadolinium is ferromagnetic at low temperatures and is used in some special applications, such as magnetic resonance imaging (MRI).

[0112] Alnico alloys: Alnico (an alloy of aluminum, nickel, cobalt and iron) is a group of ferromagnetic alloys used in magnets.

[0113] Ferrites (e.g., magnetite, Fe3O4; maghemite, Fe2O3): Ferrites are ferromagnetic ceramics that contain iron oxide as their main component.

[0114] Other elements with ferromagnetic properties at lower temperatures: Dysprosium, Holmium, Erbium, Terbium.

[0115] Other alloys: SmCo, Nd2Fel4B, Ni80Fe20 (Permalloy), NiFeCo (Mumetal), CrO2, Magnesium arsenide, EuO, etc.

[0116] It is important to note that the magnetic properties of materials can depend strongly on factors such as temperature, pressure, and structure. Ferromagnetic materials, in particular, exhibit their best magnetic properties at room temperature.

[0117] Nanoparticles are frequently used to bind to and deposit cells and cell structures.

[0118] Nanoparticles are particles with dimensions in the nanometer range, meaning they have a size of 1 to 100 nanometers in at least one dimension. These tiny particles can be made of various materials, including metals, semiconductors, polymers, or organic molecules, and they often exhibit unique physical and chemical properties compared to their macroscopic counterparts.

[0119] Here are some types of nanoparticles and their applications:

[0120] Silver nanoparticles: Applications: Antibacterial coatings, medical implants, wound dressings, water disinfection.

[0121] Gold nanoparticles: Applications: Medical imaging (e.g. cancer therapy), catalysis, sensors, diagnostics.

[0122] Iron oxide nanoparticles (magnetite or hematite): Applications: Magnetic resonance imaging (MRI) in medicine, magnetic nanoparticles for drug delivery, environmental monitoring.

[0123] Titanium dioxide nanoparticles: Applications: Sunscreens, catalysts, self-cleaning surfaces.

[0124] Quantum dots: Applications: Optoelectronics, imaging, LEDs (light-emitting diodes).

[0125] Carbon nanotubes (CNTs): Applications: Lightweight materials, electronics, reinforcement of composite materials.

[0126] Graphene: Applications: Electronics, sensors, medical imaging, supercapacitors.

[0127] Liposomes: Applications: Drug delivery systems.

[0128] Polymer nanoparticles: Applications: Medical implants, drug delivery, diagnostics.

[0129] Ferrofluids: Applications: Sealing technology, vibration damping, cooling in electronics.

[0130] Quantum dots: Applications: Imaging, solar cells, LED technology.

[0131] The unique properties of nanoparticles make them attractive for a wide range of applications in various industries, including medicine, electronics, materials science, environmental technology, and energy. However, it is important to consider potential health and environmental impacts when developing and applying nanomaterials. Therefore, intensive work is being done in nanotechnology to develop safety guidelines and procedures.

[0132] Magnetic particles are particles that exhibit magnetic properties and can react to an external magnetic field. These particles can consist of various materials and are used in a variety of applications. Magnetic particles with a size or diameter of less than 100 nm in at least one direction are referred to as magnetic nanoparticles (MNPs). In the process according to the invention, magnetic particles (MPs) with a diameter of greater than or equal to 100 nm in at least one direction are preferably used. However, the use of magnetic nanoparticles (MNPs) with a diameter of less than 100 nm in at least one direction, or mixtures with a corresponding size distribution, is also possible and equally covered by the invention. The use of magnetic particles without a coating or derivatization proves to be particularly advantageous.The size of the particles used is preferably in the range of 100 nm to 500 nm, particularly between 150 nm and 400 nm, and preferably between 200 nm and 350 nm. To provide a particle suspension with a defined diameter distribution or particle size, a separation process can be performed prior to the use of the particles in the process, removing particles with diameters of, for example, less than 100 nm from the particle suspension in at least one direction of extension.

[0133] Here are some examples of magnetic particles and their applications:

[0134] Iron oxide nanoparticles: These nanoparticles consist of iron oxide (Fe2O3 or Fe3O4) and are used in medicine for magnetic resonance imaging (MRI). They are also used for targeted drug delivery and hyperthermia treatments in cancer therapy.

[0135] Magnetite (Fe3O4): Magnetite is a naturally occurring magnetic material. In nanotechnology, magnetite nanoparticles are used in various applications, including the production of magnetic fluids and magnetic sensors.

[0136] Nickel-iron alloys: Alloys such as Permalloy (Ni80Fe20) are used in electronics, especially in magnetic sensors and circuits, due to their magnetic properties.

[0137] Manganese-zinc ferrite: Ferrites based on manganese, zinc and iron are used in electrical engineering and electronics, especially in transformers and inductors.

[0138] Neodymium-iron-boron (NdFeB): NdFeB is a very strong, artificial magnetic material and is used in the manufacture of permanent magnets, which are used in electric motors, loudspeakers and various electronic devices.

[0139] Soft magnetic composites (SMCs): SMCs consist of powdered soft magnetic materials held together in a binder. They are used for applications such as inductive components in electric vehicles and power electronics.

[0140] Ferromagnetic nanoparticles in liquids: These particles are referred to as magnetic fluids or ferrofluids and consist of ferromagnetic nanoparticles suspended in a liquid. Ferrofluids are used in applications such as sealing technology, vibration damping, and magnetic cooling.

[0141] The interaction forces between the individual magnetic spin moments in ferromagnetic materials are so strong that they align uniformly despite thermal motion. This process is called spontaneous magnetization. Nevertheless, the body does not appear magnetized externally, as this magnetization only occurs within regions formed by aligned electron spins, which initially cancel each other out. Only when an external magnetic field is applied do the differently oriented regions align parallel to this magnetic field, and with increasing magnetic field strength, they all tilt in the same direction. Ferromagnetism is therefore a collective phenomenon that occurs only in solids. The following physical relationship exists between the magnetization M and the magnetic field strength H: M = x * H, where x = magnetic susceptibility.

[0142] Elementary magnets, once oriented in a specific direction by an external field, do not completely return to their random state after the external field disappears; that is, these materials retain an inherent magnetization. Demagnetization and remagnetization can be achieved by applying an opposing field. Magnetic susceptibility indicates how easily a material can be magnetized when exposed to an external magnetic field. Ferromagnetic materials typically have high magnetic susceptibility, meaning they are easily magnetized and exhibit strong magnetic properties.

[0143] The applications of magnetic particles are diverse, ranging from medical applications and electronics to industrial processes. It is important to consider the specific properties and compositions of the magnetic particles according to the intended application. Magnetic nanoparticles can be functionalized through various surface coatings to improve their stability, increase their biocompatibility, or bind specific molecules.

[0144] Magnetic particles can be functionalized with specific antibodies or ligands that bind to cell surface binding sites. By applying an external magnetic field, the labeled cells are selectively attracted and can thus be separated from unlabeled cells. This method is frequently used for cell sorting in laboratories and biomedical applications. This approach is highly effective, provided that the appropriate specific antibodies and ligands for a particular application are researched and available. Antibody production and the specific derivatization of magnetic particles are generally complex and relatively expensive, and therefore often unsuitable for widespread market use or only appropriate for specialized cases.

[0145] One of the present inventions is to provide a simple yet efficient method using materials that are easy to manufacture or available on the commercial market.

[0146] The invention provides a method that enables highly efficient enrichment of intact cells, in particular sperm cells, without specific derivatization of magnetic particles.

[0147] The ferromagnetic or superparamagnetic particles used can consist of any ferromagnetic or superparamagnetic material.

[0148] Possible monodisperse examples, which do not limit the invention to these, are in particular: ferrites such as magnetite, zinc ferrite or manganese ferrite; ferrofluids; metals such as iron, nickel, cobalt or their alloys or oxides, or chromium dioxide or Mn3O4. By way of example, magnetite (Fe3O4), maghemite (Fe2O3) or substances such as other iron oxide-based particle materials, including composites with the general structure MFe2O4 (where M can be Au, Ba, Co, Cu, Cr, Mg, Mn, Ni, Pt, Ti or Zn) or heteroparticles such as, for example, gold@iron oxide or FePt@MnO.

[0149] The following are examples of possible synthesis methods for iron-containing ferromagnetic nanoparticles:

[0150] Nanocrystalline magnetic particles made of Fe3O4, gamma-Fe2O3, or corresponding hydroxides are obtained by converting an acidic iron(II) and / or iron(III) salt solution into iron(II) and / or iron(III) carbonate by adding an equivalent amount of alkali carbonates such as sodium bicarbonate, sodium carbonate, or ammonium carbonate, followed by thermal oxidation to magnetic iron hydroxide and then to magnetic iron oxide. The particle size can be controlled by the thermal reaction rate and the concentration of the iron salt solution. Small diameters of 20–100 nm are obtained, for example, by the separate formation of iron(II,III) carbonate at temperatures of 1–50 °C and subsequent heating, while larger particles of 100–1000 nm are obtained at reaction temperatures of 60–100 °C and the associated faster conversion of iron(II,III) carbonate to iron(II,III) hydroxide.The conversion of an iron(II) and / or iron(III) salt solution into an iron(II) and / or iron(III) complex by adding one or more complexing agents such as ethylenediaminetetraacetic acid, citric acid, tartaric acid, or their salts, the subsequent neutralization with moderately basic reagents such as ammonia or alkali carbonates, and the precipitation of the iron hydroxides by adding strong alkalis such as sodium hydroxide to a pH of 11 also yields the desired magnetic particles. The ionization rate during alkalization determines the size of the magnetic particles. At a molar ratio of iron(II) / iron(III) salt solution of 1:2, the ferromagnetic Fe3O4 (magnetite) is formed upon alkalization. Its mild oxidation yields the also ferromagnetic gamma-Fe2O3. Examples of iron salts that can be used include...Iron (III) chloride, iron (III) sulfate, iron (III) nitrate as well as iron (II) chloride, iron (II) sulfate or the respective double salts such as iron (II) / iron (III) ammonium sulfates are used.

[0151] Another method for producing particles is the controlled mechanical comminution of large particles down to a specific size range. This grinding method, for example using planetary ball mills, is also called micronization. The magnetic or superparamagnetic particles used can be stabilized or stored sterilely by autokiaving, treatment with ethylene oxide, or suspension in a storage buffer system sterilized via a sterile filter.

[0152] In a preferred embodiment of the method according to the invention, a solution of the ferromagnetic material in a storage and stabilization buffer, also referred to as a suspension storage buffer system, is provided prior to step a.) or step e.), wherein the storage and stabilization buffer particularly comprises: deionized water, phosphate-buffered physiological saline (PBS), Hanks' Balanced Salt Solution (HBSS), Good's Buffers, Tris buffer, phosphate-buffered saline (PSS), Earle's Balanced Salt Solution (EBSS), Ringer's solutions, TES-Tris buffer, HEPES buffer, sperm buffer solution (SP), SP-TALP, FC-TALP, TCA, citrates, acetates, lactates, carbonates, phosphates and combinations thereof, or a solution containing at least one salt, at least one carbohydrate or a combination thereof; Milk and its derivatives; MES; diluent systems compatible with the respective animal species, such as Andromed, BioXCell, BullXcell, INRA 96,PRIMXcell, TRIXcell, NUTRIXcell, OviXcell, Galap, CUNIgel, Triladyl, Biladyl, Steridyl, OviPlus, Bovidyl, M III, BTS, Androstar, Novistar, OptiXCell, Beyond, EquiPlus, Gent or CaniPlus, or mixtures thereof; Additives selected from the group consisting of glycine, cholesterol-loaded cyclodextrins, docosahexaenoic acid (DHA), vitamin E (α-tocopherol, trolox, tocotrienol), deuterated polyunsaturated fatty acids (D-PUFAs), butylhydroxytoluene, butylhydroxyanisole, ferrostatins, liproxstatins, CoQlO, idebenone, XJB-5-131 (mitochondrially directed nitroxide), JP4-039 (broadly directed intracellular antioxidant), baicalein, PD-146176 (15-LOX-1 inhibitor), AA-861 (LOX inhibitor), zileuton, deferoxamine, ciclopirox, deferiprone, cycloheximide, 2-mercaptoethanol, dopamine, selenium, vildagliptin, alogliptin, linagliptin, coconut water. Ascorbic acid, beta-carotene, quercetin, rutin, taurine, 5-HMF, glutathione, ubiquinone, superoxide dismutase, glutathione peroxidase, catalase, L-carnitine, zincPolyphenols, folic acid, alpha-lipoic acid, melatonin, astaxanthin, zeanxanthin, lucopins, anthocyanins, bioflavonoids, pyruvate, serum albumin, regucalcin, curcumin (diferuloylmethane), growth factors such as epidermal growth factor, oviductal proteins (such as non-luteal isthmic oviductal proteins (NLIP)), Diospyros kaki (persimmon) extract, coenzyme Q10, cobalamin, silymarin (Silybum marianum) or mixtures thereof; antibiotics, antimicrobial or antibiotic agents selected from the group consisting of tetracycline, penicillin, sulfonamide, aminoglycoside, fluoroquinolone, macrolide, cetiofur, apramycin, streptomycin, ticarcil I, polymyxin B or their derivatives, tylosin, gentamicin, linco- mycin, spectinomycin, Linco-Spectin® (lincomycin hydrochloride-spectinomycin), or mixtures thereof; pyruvate; liposome-forming or non-liposome-forming lipids (e.g., phospholipids such as PA, PC, PE, LPC, LPA, PG, LPG, PI, PIP, LPI, PS, LPS, cardiolipin,Bis(monoacylglycero)phosphates (BMP) ether lipids, sterol-modified phospholipids, l,2-dioleoyl-sn-glycero-3-phospho-rac-(l-glycerol) (DOPG), l,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EPC), dioleoylphosphatidylethanolamine (DOPE), phosphatidylcholine (PC), cholesteryl hemisuccinate (Cholesteryl hydrogen succinate) (CHEMS), cholesterol and its derivatives; Aliphatic carboxylic acids (low chain length, like long-chain fatty acids) such as caproic acid, sorbic acid, capric acid, n-valeric acid, myristic acid, pelargonic acid, octanoic acid, or polymeric carboxylic acids or polymeric amines of different chain lengths such as polyacrylic acid, polyethyleneimines, poly-L-lysine, or transfection-active substances such as dioleoyl-3-trimethylammonium propane (DOTAP), DOTMA, DODMA, DOGS, DOSPA, DC-cholesterol, DLinDMA, liposomal or nanomolecular lipids such as lipofectamines, lipofectamin 2000, Metafectene Pro, Fugene, GeneJuice, etc., or mixtures thereof; serum albumins: HSA, BSA, ESA, CSA, FSA, OSA, MSA.RSA or PSA; EDTA (ethylenediaminetetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), DMSA (dimercaptosuccinic acid), deferoxamine, citric acid, EGTA (ethylene glycol bis(aminoethyl ether)tetraacetic acid), Chelex® resins, transferrin, deferasirox, penicillamine, alpha-lipoic acid, DMPS, dimercaprol and aminopolycarboxylic acids (complexones), including but not limited to fura-2, IDA, NTA, DTPA, BAPTA, NOTA, DOTA and nicotianamine and derivatives thereof; polymyxin B; lecithins, milk casein, beta-lactoglobulin, alpha-lactalbumin, glycodelin S, glucose, fructose, glycolyzable sugars; pyruvate; Ethane-1,2-diol, formamide, dimethyl sulfoxide, Greg Fahy's VS41A, M22, propylene glycol, ethylene glycol, polyethylene glycol, polyvinylpyrrolidone, propane-1,2,3-triol, X-1000 (Copolymer of polyvinyl alcohol and vinyl acetate), mannitol, K12, Z-1000, VM3, CI-VM-1, 3-methoxy-1,2-propanediol, N-methylformamide, cysteine ​​and its derivatives; polydatin (resveratrol), selenium-associated glutathione peroxidase,Glutathione disulfide reductase, heme oxygenase, or mixtures thereof. The aforementioned substances can each be used individually or in mixtures with different mixing ratios adapted to the intended use in the buffer systems, also known as physiological suspension solutions, buffer solutions, or washing solutions. In particular, a particle concentration of between 1 mg and 100 mg of particles per 100 ml of storage and stabilization buffer is required.

[0153] Surprisingly, the addition of certain substances to the ferro- or superparamagnetic particles has proven particularly advantageous. In the purification process according to the invention, these substances, when a magnetic field is applied, significantly improve pellet formation on the vessel wall and thus the separability of the particle-cell conglomerates from the suspension containing the intact cells. Typically, the magnets are positioned laterally against the vessels for the deposition of the magnetic particles, causing a pellet of magnetic particles with the bound non-intact sperm to form on the vessel wall facing the magnet. The separation of the suspension containing intact sperm from the magnetic particles is achieved, for example, by pipetting. However, the particle-cell pellet is usually not compact enough, so a small amount of magnetic particles is carried back into the pipette.Furthermore, the particle-cell pellet slides downwards towards the bottom of the container due to gravity. Consequently, this procedure typically needs to be repeated several times to achieve sufficient separation of the magnetic particles from the suspension containing the intact cells. This repeated separation process places additional stress on the delicate sperm cells, resulting in significantly reduced purification efficiency, as intact cells are damaged by the additional stress. By adding certain substances, a much more compact particle-cell pellet is obtained, which no longer slides down the container wall, or does so to a significantly lesser extent, and parts of it are no longer, or to a significantly lesser extent, suspended in the pipette during pipetting and reintroduced into the cell suspension containing the intact cells.It is often even possible to decant the suspension containing the intact cells. The separation procedure thus made possible is significantly simplified and gentler on cells according to the invention, thereby substantially improving the purification quality of intact cells. Substances that can be used in the storage and stabilization buffer in this context include, in particular but not exclusively, ethane-1,2-diol, formamide, dimethyl sulfoxide, Greg Fahy's VS41A, M22, propylene glycol, ethylene glycol, polyethylene glycol, polyvinylpyrrolidone, propane-1,2,3-triol, X-1000 (copolymer of polyvinyl alcohol and vinyl acetate), mannitol, K12, Z-1000, VM3, CI-VM-1, 3-methoxy-1,2-propanediol, N-methylformamide, cysteine, and its derivatives. Furthermore, this includes substances classified as non-cell-penetrating proteins and sugars, including those of natural origin such as sucrose or trehalose. More generally, these substances can be classified as cryonic substances.These substances can be added to the storage and stabilization buffer, as well as to all other solutions used in the process according to the invention, as additives in a suitable concentration range. The pH range of the storage and stabilization buffer is, in particular, 6.0 to 8.5.

[0154] The zeta potential of ferro- or superparamagnetic particles can be negative, positive, or neutral.

[0155] The storage temperature of the ferro- or superparamagnetic particles is in the range of 4°C to 40°C. Before using the ferro- or superparamagnetic particles as a separating agent with the cells, especially sperm cells, the particle suspension must be vortexed, shaken, or ultrasonically treated.

[0156] The concentration of ferro- or superparamagnetic particles in the storage and stabilization buffer is preferably between 1 mg and 100 mg per ml.

[0157] The average size of the binding-active ferro- or superparamagnetic particles is typically between 100 nm and 7 pm. Binding-active particle size refers to the average size of those ferro- or superparamagnetic particles that are added to the cell population, particularly sperm cells, or to the shape in which the magnetic particles in the cell suspension bind and separate non-viable or infertile sperm from healthy cells. These magnetic particles are either present in the storage and stabilization buffer or form with the cells after the magnetic particles are added to the suspension solution.

[0158] In some embodiments of the invention, the magnetic particles have a size distribution of approximately 800 nm to approximately 5 pm; approximately 150 nm to approximately 8 pm; approximately 80 nm to approximately 7 pm; approximately 200 nm to approximately 7 pm; approximately 30 nm to approximately 7 pm; approximately 70 nm to approximately 7 pm; approximately 90 nm to approximately 7 pm; approximately 300 nm to approximately 7 pm; approximately 700 nm to approximately 7 pm; approximately 900 nm to approximately 7 pm; approximately 1.10 pm to approximately 7 pm; approximately 1.50 pm to approximately 7 pm; approximately 100 nm to approximately 7 pm; approximately 1 pm to approximately 4 pm; approximately 500 nm to approximately 7 pm; approximately 800 nm to approximately 7 pm; approximately 400 nm to approximately 7 pm; approximately 40 nm to approximately 7 pm; from about 20 nm to about 7 pm; from about 700 nm to about 3 pm; from about 800 nm to about 3 pm; from about 60 nm to about 600 nm; from about 1 pm to about 5 pm; from about 100 nm to about 900 nm; from about 1 pm to about 3 pm; from about 600 nm to about 7 pm; from about 40 nm to about 300 nm; from about 500 nm to about 6.5 pm; from about 60 nm to about 2 pm;from about 1.50 pm to about 5 pm; from about 60 nm to about 7 pm; from about 150 nm to about 7 pm; from about 50 nm to about 500 nm; from about 1.1 pm to about 4 pm; from about 300 nm to about 3 pm; from about 700 nm to about 3 pm;from approximately 600 nm to approximately 3 pm. Magnetic particles with a size or diameter of <100 nm in at least one direction are referred to as magnetic nanoparticles (MNPs). In the process according to the invention, magnetic particles (MPs) with a diameter greater than 100 nm in at least one direction are used in particular. However, the use of magnetic nanoparticles (MNPs) with a diameter of less than 100 nm in at least one direction, including mixtures or mixtures with corresponding size distributions, is not excluded. To provide particle suspensions with a defined diameter distribution or particle size, a deposition process can be performed prior to the use of the particle suspension in the process, in which particles with undesired diameters, for example, with diameters of less than 100 nm in at least one direction, are removed from the particle suspension.

[0159] The following ratios of ferro- or superparamagnetic particles to cells, especially sperm cells, are used:

[0160] 25 to 200 pg of ferro- or superparamagnetic particles per 1 x 10*8 cells, preferably with a sperm motility of 60 to 80%; 50 to 400 pg of ferro- or superparamagnetic particles per 1 x 10*8 cells, preferably with a sperm motility of less than 60%.

[0161] In one embodiment, step f.) provides for an incubation time of between 1 and 120 min, i.e., the contact time of the ferro- or superparamagnetic particles with the cells, in particular sperm cells, is between 1 and 120 min. This process is preferably repeated as required until no more ferro- or superparamagnetic particles are present, visible, or optically detectable in the cell suspension.

[0162] The temperature of the suspension solution during the entire purification process of the ferro- or superparamagnetic particles with the cells, in particular sperm cells, is between 4 and 40 °C, preferably between 16 and 24 °C.

[0163] A further development of the invention, considered advantageous, provides for the use of intact sperm cells, in particular mammalian sperm cells, enriched in a process as described above, for assisted reproduction (ART), in particular for artificial insemination, intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF) of mammals.

[0164] This involves transferring the purified cells, particularly sperm cells, to further ART processing steps such as AI, IVF, or ICSI. This may require buffering to a cryoprotective buffer system or other buffer system necessary for the specific application. This buffering can be achieved, for example, by cell-friendly centrifugation. The typical g-weight range is between 300 and 4000 g, 500 and 3500 g, 600 and 3400 g, 700 and 3300 g, 800 and 3200 g, 900 and 3100 g, or 1000 and 3000 g. Centrifugation time ranges from 1 to 60 minutes. The addition of autologous or allogeneic seminal plasma can be beneficial for stabilizing cell health.Seminal plasma contains various nutrients such as fructose, which provides energy to the sperm, enzymes and proteins that support sperm motility and aid in fluid dilution, and which may also possess immunomodulatory properties. Reintroduction of autologous seminal plasma can be beneficial because its components are individually tailored to the cells. In particular, the administration of a seminal plasma conglomerate of allogeneic, autologous, or mixed origin can be advantageous.

[0165] Possible, but not exclusive, uses of the cells enriched in the inventive process, in particular sperm cells, are listed below, without limiting the use to these:

[0166] AI (Artificial insemination):

[0167] In animal breeding, artificial insemination (artificial fertilization) is used as a reproductive technique to promote the inheritance of specific genetic traits, improve the health of breeding animals, and increase the efficiency of breeding programs. Some key aspects of artificial insemination in animal breeding are the following: Semen collection: In male mammals, including humans, semen is collected through masturbation or electrical stimulation. The collected semen is then processed in a laboratory.

[0168] Egg collection: Female animals can be prepared for egg collection in various ways. This may include the use of hormones to stimulate egg maturation, transvaginal egg retrieval, or laparoscopy.

[0169] Semen preparation: The collected semen is processed in the laboratory to concentrate the motile and fertilizable sperm. This processing may include the removal of ejaculate fluid and the selection of the best sperm.

[0170] Insemination: The prepared sperm is then introduced into the female reproductive organs. This can be done through artificial insemination, in which the sperm is inserted directly into the cervix or uterus.

[0171] Embryo transfer: In some cases, fertilized eggs or embryos are produced through in-vitro fertilization (IVF) and then transferred into the reproductive tract or uterus.

[0172] Use of sperm banks: In animal breeding, sperm banks (insemination stations) can be set up to store and distribute the sperm of high-quality breeding animals.

[0173] Artificial insemination makes it possible to obtain genetically superior offspring from outstanding parent animals that may be located in different places. This technique also has the advantage of controlling the spread of hereditary diseases and maintaining genetic diversity in animal populations.

[0174] Intracytoplasmic Sperm Injection (ICSI):

[0175] Intracytoplasmic sperm injection (ICSI) is an advanced method of artificial insemination that can be used in animal breeding to achieve targeted genetic improvements or to enhance fertility in animals with limited natural reproductive capacity. Here are some key aspects of ICSI in animal breeding: Sperm collection: Sperm is collected from a male animal. This can be done through masturbation or electrical stimulation.

[0176] Egg retrieval and maturation: Eggs are retrieved from female animals. The eggs are then matured in a laboratory.

[0177] ICSI procedure: In ICSI, a single sperm is injected directly into a mature egg. This is done under a microscope using a fine glass needle that guides the sperm into the egg. Incubation and development: The fertilized egg, now called a zygote, is cultured in a special incubator to support its development. After a few days, the developing group of embryos (blastocyst) can be prepared for transfer. Embryo transfer: The developed embryos can then be transferred into the reproductive tract of the female animal or surrogate mother. ICSI offers several advantages, particularly when male animals have impaired sperm quality or when targeted genetic enhancements are desired. It also allows for the overcoming of certain types of infertility in animals.

[0178] In-vitro fertilization (IVF):

[0179] In-vitro fertilization (IVF) is an advanced method of artificial insemination used in animal breeding to improve the reproductive capacity of animals, achieve genetic improvements, or overcome reproductive challenges. Here are some key aspects of IVF in animal breeding: Oocyte collection: Oocytes are retrieved from the ovaries of female animals. This can be done through transvaginal egg retrieval or laparoscopy.

[0180] Sperm collection: Sperm is collected from a male animal. This can be done through masturbation or electrical stimulation.

[0181] In-vitro fertilization: The egg cells and sperm are brought together in a laboratory to enable fertilization. This can take place in a special nutrient solution.

[0182] Embryo culture: The fertilized eggs, now called zygotes, are cultured in an incubator to support their development. After a few days, the developing group of embryos (blastocyst) can be prepared for transfer.

[0183] Embryo transfer: The developed embryos are transferred into the reproductive tract of the female animal or surrogate mother. This can take place in the uterus or the fallopian tube.

[0184] Storage of embryos: In some cases, excess embryos can be frozen (cryopreserved) and stored for future transfers.

[0185] IVF in animal breeding offers several advantages, including:

[0186] - Overcoming infertility in animals.

[0187] - Efficient use of genetically valuable parent animals.

[0188] - Possibility of carrying out targeted genetic enhancements.

[0189] - Maximizing reproductive efficiency.

[0190] The application of IVF in animal breeding requires expertise and specialized facilities, and it is often used in breeding programs for valuable breeds or in animals with specific reproductive challenges. Another possibility is the use of the sperm cells purified according to the invention for sex-specific cell sorting. The sperm can be used before or after, or before and after, the application for sex-specific cell sorting. In certain embodiments of the invention, the sex sorting of sperm can be carried out using any method or device known in the art for cell analysis, sorting, and / or population enrichment, including, but not limited to, the use of a flow cytometer, a microfluidic chip, or a free-flow electrophoresis method.As previously stated, sex sorting encompasses not only techniques for physically separating or isolating X- and Y-carrying sperm from each other, such as droplet sorting and fluid-switching sorting, but also sex enrichment techniques, which involve killing, immobilizing, or otherwise rendering infertile sperm carrying the unwanted sex chromosome, for example, through the use of laser ablation / photodamage techniques.

[0191] In one embodiment, a device for separating cells bound to magnetic particles in a cell suspension, particularly for use in a method as described above, is proposed. The device comprises a receptacle for at least one sample vessel and at least one magnet receptacle arranged relative to the sample vessel, wherein at least one magnet is provided in the magnet receptacle and the magnet receptacle essentially encloses the sample vessel in a semicircular fashion. An advantage of this device is that it allows for easy handling of the samples, since the magnetic particle pellet is formed in a clearly defined area of ​​the sample vessel and the supernatant containing the intact cells can be removed without contamination by the magnetic particle.

[0192] In one embodiment, the magnet is designed to generate a point- or line-shaped magnetic field within the sample vessel. The advantage of this is that the formed pellet is clearly defined and available within the sample vessel.

[0193] In one embodiment, the magnetic field is formed in the region of a circumferential wall or at the bottom of the sample vessel. The advantage of this is that the pellet formation is limited to a clearly defined area of ​​the sample vessel.

[0194] In one embodiment, the fixture is designed as a rack, in particular a centrifuge rack. The advantage of this is that the samples can remain in the fixture during the execution of the process, particularly when centrifugation steps are to be carried out, thus making handling more efficient and gentler.

[0195] In one embodiment, the device has an enclosing casing. This proves advantageous for protecting the samples from the effects of light and temperature fluctuations and creates uniform experimental and handling conditions inside the device. In some embodiments, a kit for magnetic cell manipulation is provided. The kit may include instructions. The instructions may include contacting a composition with a sperm sample to form a mixture. The instructions may also include applying a magnetic field to the mixture to manipulate the composition. The kit may also contain the buffer solutions and compositions used in the process. The compositions may comprise a variety of particles. Each particle in the variety of particles may comprise a magnetic substrate.The magnetic substrate can be characterized by a magnetic susceptibility greater than zero.

[0196] In several embodiments of the kit, the composition can be configured to form a complex with damaged or impaired sperm cells or sperm cell structures. The instructions may also include the selection of the biological sample, which comprises viable sperm cells and damaged or impaired sperm cells or sperm cell structures. The instructions may also include the separation of viable sperm from the complex, including the damaged or impaired sperm or sperm cell structures, by applying the magnetic field to the mixture.

[0197] In several embodiments, the kit can include an apparatus with which the sperm complexed to the magnetic particles can be separated from the sperm not bound to them.

[0198] In several embodiments, the kit can include aqueous solutions of certain compositions that, in particular, enable seminal plasma reduction.

[0199] In several embodiments, the kit can include aqueous solutions of certain compositions that enable complex formation with the magnetic particles.

[0200] In several versions, the kit can include aqueous solutions of specific compositions that serve to stabilize the magnetic particles.

[0201] Embodiments of the method according to the invention may include sperm or spermatozoa collected from numerous species of male mammals, and the invention should be understood as not being limited to the species of male mammals described in the specific examples of this application. Rather, the specific examples in this application are intended to illustrate the diverse and numerous species of male mammals from which sperm can be collected and used in certain embodiments of the invention. Embodiments of the invention may, for example, include human sperm. Embodiments of the invention may, for example, include the sperm of mammals that have commercial value for meat or milk production, such as pigs, sheep, cattle, horses, deer, elk, buffalo, camels, goats, etc.This may include sperm from various domesticated mammal species, including dogs and cats, as well as sperm from primates, including but not limited to chimpanzees, gorillas, and whales, dolphins, and other marine mammals. It may also include frozen and thawed sperm from all mammals described above, including humans, and including but not limited to sperm from deceased donors, rare or exotic mammals, zoological specimens, or endangered species.

[0202] The present invention thus relates to a novel and easy-to-implement method for the purification and, in particular, enrichment of sperm populations by very effectively and cost-efficiently separating non-intact cells from intact cell populations capable of successful fertilization:

[0203] The invention is explained below by means of examples, without limiting the invention to these examples.

[0204] Example 1

[0205] Standard method for small volumes (separation in a 1.5 mL reaction vessel)

[0206] Experimental approach in small volume for screening buffers, additives, etc. with a larger number of samples to be processed simultaneously.

[0207] Typically, a sample with 1 mL of separation solution yields 4 - 6 sepals for preservation in LN2.

[0208] material

[0209] Fresh bovine sperm

[0210] Wash buffer (e.g., IX PBS when a wash step is performed)

[0211] Washing solution (e.g. IX PBS)

[0212] Magnetic particles (MNP)

[0213] 1.5 mL reaction vessels with lids

[0214] Centrifuge with rotor for 1.5 mL vessels

[0215] Overhead rotary mixer

[0216] Magnetic racks for 1.5 mL reaction vessels with the strongest possible magnets

[0217] method

[0218] By default, MP separation is performed in a suspension with 2 x 108 cells / mL and 100 pg MP per 1 x 108 cells.

[0219] The method will be carried out quickly, so that there are no long waiting times between the individual writings.

[0220] The concentration of the native ejaculate is measured as precisely as possible using CASY (preferably in a 1:20 dilution of a small aliquot). Additionally, it is advisable to determine the motility and PMAI% of this native reference sample. Based on the CASY measurement, an aliquot of the native ejaculate containing the desired cell count (e.g., 2 x 10⁸ cells for a 1 mL sample) is transferred to a fresh 1.5 mL reaction tube and washed with 500–1000 pL of IX PBS.

[0221] The cells are sedimented in the centrifuge at 1500 xg for 1 min, the supernatant is carefully aspirated and the cell pellet is carefully resuspended in the suspension solution.

[0222] If the washing step is omitted, the required sperm are diluted directly in the washing solution and processed without further centrifugation.

[0223] From this initial sample (or from each individual sample if there are several different samples) before MP treatment, the cell concentration is determined again using CASY, as are motility and PMAI%.

[0224] The MPs are added to the initial sample: 100 pg MPs per 1 x 108 cells, which at an MP concentration of 25 mg / mL is 4 pL.

[0225] The containers are tightly sealed and suspended in the overhead rotary mixer, then mixed for 30 seconds at 10 rpm. If the microorganisms have collected as pellets at the bottom of the container, it is worthwhile to homogenize the suspension by briefly inverting it before placing it in the rotary mixer.

[0226] The mixed, incubated reaction vessels are placed in a magnetic rack for 5 minutes. It is advisable to open the vessels beforehand and not move them during the separation process.

[0227] Once the MP pellet has formed on the magnet, the supernatant is carefully but quickly removed and transferred to a fresh 1.5 mL container.

[0228] In some cases, the MP pellet may slip, therefore the removal of the supernatant must be visually monitored. If the pellet is observed slipping, it is advisable not to remove the entire supernatant, but to stop just before the MP would be drawn back into the pipette.

[0229] The new vessel is placed in the magnetic rack for another 5 minutes to remove the remaining MP from the suspension.

[0230] In some cases, further separation rounds are necessary, especially if MP of a sliding pellet was previously transferred.

[0231] The procedure is complete if, after removing the supernatant, only a fine reddish shadow is visible on the vessel wall.

[0232] The supernatant after the last separation round is transferred back into a fresh reaction vessel and the cell concentration is determined using CASY; motility and PMAI% are also determined.

[0233] Subsequent preservation in liquid nitrogen (LN2)

[0234] Sperm are typically frozen at a concentration of 6.5 x 10⁷ cells / mL, so a 0.25 mL vial contains a standard portion of approximately 1.5 x 10⁷ cells. OptiXcell 2 is generally used as the freeze-drying diluent, prepared as fresh as possible from concentrate (1 mL of OptiXcell is diluted with 2 mL of water).

[0235] Based on the CASY measurement of the supernatant, the amount of cells required for preservation is transferred to a fresh, suitable reaction vessel (1.5 mL or 2 mL).

[0236] 1 mL of final total volume is sufficient for 4 sequins.

[0237] At least 3 sequins should be frozen from each sample in order to perform 3 TRT.

[0238] The cells are sedimented in the centrifuge at 1500 xg for 1 min.

[0239] The supernatant is removed and the cell pellet is resuspended in OptiXcell (volume adjusted to the previously set cell quantity, e.g. 1 mL to 6.5 x 107 cells).

[0240] The cell suspensions are filled into sequins using the standardized deep-freezing method, preserved and further processed.

[0241] Example 2

[0242] Standard method for medium volumes (separation of approximately 5 mL volume in a 15 mL reaction vessel)

[0243] Approach for separating a partial ejaculate or a full ejaculate into multiple processes, similar to the application in a bull station, but offering the possibility to compare multiple conditions on the same sample.

[0244] One aliquot must be retained as an untreated reference.

[0245] For experimental purposes, it is not necessary to freeze the entire volume at the end, but only a small part.

[0246] material

[0247] Fresh bovine sperm, complete ejaculate, or a larger quantity of partial ejaculate

[0248] Wash buffer (e.g., IX PBS when a wash print is performed)

[0249] Washing solution (e.g. IX PBS)

[0250] MP

[0251] 15 mL reaction vessels with lids

[0252] Centrifuge with rotor and inserts for 15 mL vessels

[0253] Overhead rotary mixer

[0254] Magnetic racks for 15 mL reaction vessels with the strongest possible magnets

[0255] method

[0256] By default, MP separation is performed in a suspension with 2 x 10⁸ cells / mL and 100 pg MP per 1 x 10⁸ cells. The method is carried out quickly so that there are no long holding times between individual casts.

[0257] The concentration of the native ejaculate is measured using CASY (preferably in a 1:20 dilution of a small aliquot). Additionally, it is advisable to determine the motility and PMAI% of this native reference sample. Based on the CASY measurement, an aliquot of the native ejaculate containing the desired cell count (e.g., 1 x 10⁹ cells for a 5 mL reaction) is transferred to a fresh 15 mL reaction tube and washed with 2–3 mL of IX PBS. The cells are sedimented at 2000 x g for 2 min, the supernatant is carefully aspirated, and the cell pellet is gently resuspended in the suspension solution.

[0258] If the washing step is omitted, the required sperm are diluted directly in the suspension solution and processed without further centrifugation.

[0259] From this initial sample (or from each individual sample if there are several different samples) before MP treatment, the cell concentration is determined again using CASY, as are motility and PMAI%.

[0260] The MPs are added to the initial sample: 100 pg MNPs per 1 x 108 cells, which, at an MP concentration of 25 mg / mL, equates to 40 pL of MPs for 5 mL with 1 x 109 cells.

[0261] The containers are tightly sealed, suspended in the overhead rotary mixer, and mixed for 30 seconds at 10 rpm. If the microorganisms have collected as pellets at the bottom of the container, it is worthwhile to homogenize the suspension by briefly inverting it before suspending it in the rotary mixer.

[0262] The mixed, incubated reaction vessels are placed in a magnetic rack for 10 minutes. It is advisable to open the vessels beforehand and not move them during the separation process.

[0263] Once the MP pellet has formed on the magnet, the supernatant is carefully but quickly removed with a serological pipette and transferred to a fresh 15 mL container.

[0264] In some cases, the MP pellet may (partially) slide off, therefore the removal of the supernatant must be visually monitored. If the pellet is observed sliding off, it is advisable not to remove the entire supernatant, but to stop just before the MP would be drawn back into the pipette.

[0265] The new vessel is placed in the magnetic rack for another 10 minutes to remove the remaining MP from the suspension.

[0266] In some cases, further separation rounds are necessary, especially if MP of a sliding pellet was previously transferred.

[0267] The procedure is complete if, after removing the supernatant, only a fine reddish shadow is visible on the vessel wall.

[0268] The supernatant after the last round is transferred to a fresh reaction vessel and the cell concentration is determined using CASY; motility and PMAI% are also determined.

[0269] Subsequent preservation in liquid nitrogen (LN2): Sperm are frozen at a standard concentration of 6.5 x 107 cells / mL, so that a 0.25 mL vial contains a standard portion of 1.5 x 107 cells.

[0270] OptiXcell 2 is usually used as the deep-freeze thinner, which is prepared as fresh as possible from concentrate (1 mL OptiXcell is made up with 2 mL mQ water).

[0271] Since experimental purposes (e.g., TRT) never require as many sediment strands as could theoretically be obtained from such a larger separation, only a portion can actually be preserved. Nevertheless, a larger aliquot should be processed to maintain consistency with a field application where the full volume would be processed. This means that centrifugation must be performed exactly as required for a 5 mL batch, even if only 1-2 mL are used, which would theoretically fit into a smaller vessel and could be sedimented with less centrifugation.

[0272] If a deep-freeze diluent (e.g. OptiXcell) has already been used as a buffer for the separation, the aliquot required for freezing is simply taken from the supernatant and topped up to 6.5 x 107 cells / mL with fresh OptiXcell according to the CASY measurement.

[0273] The entire supernatant (treated sample) or a suitable aliquot (at least 1 mL) is sedimented in the centrifuge at 2000 xg for 2 min.

[0274] The clarified supernatant is suctioned off as completely as possible.

[0275] Based on the CASY measurement of the treated supernatant, the required volume of cryogenic diluent is calculated (1 mL / 6.5 x 107 cells) and the cell pellet is carefully resuspended in it.

[0276] The cell suspensions are filled into sequins using the standardized deep-freezing method, preserved and further processed.

[0277] Example 3

[0278] Standard method for large volumes (separation of a complete ejaculate)

[0279] Approach to separating an ejaculate using a selected separation method (method, additives, etc.) that most closely resembles the application in the bull station.

[0280] A small aliquot must be retained as an untreated reference.

[0281] For experimental purposes, it is not necessary to freeze the entire volume at the end, but only a small part.

[0282] material

[0283] Fresh bovine sperm, complete ejaculate

[0284] Washing solution (e.g. IX PBS if a wash print is performed)

[0285] Suspension solution

[0286] MP 15 and / or 50 mL reaction vessels with lids

[0287] Centrifuge with rotor and inserts for 15 / 50 mL vessels

[0288] Overhead rotary mixer

[0289] Magnetic racks for 15 / 50 mL reaction vessels with the strongest possible magnets

[0290] method

[0291] By default, MP separation is performed in a suspension with 2 x 108 cells / mL and 100 pg MP to 1 x 108 cells.

[0292] The method is carried out quickly, so that there are no long waiting times between the individual steps.

[0293] It is advisable to choose the volume of the total ejaculate to be separated in such a way that a small residue remains from which native reference samples can be frozen (e.g., with a total volume of 4 mL, only about 3.7 mL should be allocated for separation).

[0294] The concentration of the native ejaculate is measured as accurately as possible using CASY (preferably in a 1:20 dilution of a small aliquot); additionally, it is advisable to determine the motility and PMAI% from this native reference.

[0295] The MP separation can now be performed in a single large batch in a 50 mL reaction vessel or divided among several 15 mL reaction vessels. The following applies: separation volumes above 10 mL are processed in the 50 mL vessel, and separation volumes below or around 10 mL are processed in the 15 mL vessel. For the washing step, it can be advantageous to use a 15 mL reaction vessel even for the large batch, as the sperm sediment better in this size. The sample can then be transferred to the larger vessel for separation using a portion of the wash solution. Due to the larger volume of the MP pellet in the 50 mL batch, there is a greater risk of the MP pellet slipping off the magnet. Therefore, it is possible that several separation steps will be necessary to clarify the supernatant.

[0296] The ejaculate to be separated is carefully resuspended for washing with at least twice the volume of IX PBS.

[0297] The cells are sedimented at 2000g for 2 min, the supernatant is carefully aspirated and the cell pellet is carefully resuspended in the required amount of suspension solution.

[0298] The required volume of the suspension solution is calculated from the previously determined total cell count: The cells are prepared for separation in such a way that approximately 2 x 108 cells / mL are present.

[0299] If the washing step is omitted, the required sperm are diluted directly in the suspension solution and processed without further centrifugation.

[0300] The cell concentration of this initial sample, prior to MP treatment, is again determined using CASY, along with motility and PMAI%. MP is added to the initial sample: 100 pg MP to 1 x 10⁸ cells, i.e., at an MP concentration of 25 mg / mL, this equates to 4 pL, resulting in a total separation volume of 8 pL / mL. The vessels are tightly sealed and placed in an overhead rotary mixer, where they are mixed for 30 seconds at 10 rpm. If the MP has settled as a pellet at the bottom of the vessel, it is advisable to homogenize the suspension by briefly inverting it before placing it in the rotary mixer. The mixed, incubated reaction vessels are placed in a magnetic rack for 10 minutes. It is recommended to open the vessels beforehand and not move them during the separation. If the suspension has not clarified after 10 minutes, the separation can be extended (20–30 minutes).Alternatively, especially with poor initial sample quality (e.g., < 50% viability), increasing the amount of microorganisms (MPs) can be helpful (e.g., doubling it) to promote pellet formation. Once the MP pellet has formed on the magnet, the supernatant is carefully but quickly removed and transferred to a fresh 15 or 50 mL tube. This new tube is placed in the magnetic rack for another 10 minutes to remove any remaining MPs from the suspension. After this final cycle, the supernatant is transferred to a fresh reaction tube, and the cell concentration is determined using CASY. Motility and PMAI% are also measured.

[0301] Subsequent preservation in liquid nitrogen (LN2)

[0302] Sperm are typically frozen at a concentration of 6.5 x 107 cells / mL, so a standard 0.25 mL packet contains a standard portion of 1.5 x 107 cells.

[0303] OptiXcell 2 is usually used as the deep-freeze thinner, which is prepared as fresh as possible from concentrate (1 mL OptiXcell is made up with 2 mL mQ water).

[0304] The cells of the complete supernatant (treated sample) or an adequate part thereof are sedimented in the centrifuge at 2000 xg for 2 minutes.

[0305] If no cell sediment has formed after 2 minutes, the centrifugation is repeated until the supernatant is sufficiently clear.

[0306] The supernatant is removed and the cell pellet is resuspended in OptiXcell (volume adjusted to the previously set cell quantity, e.g. 10 mL for 6.5 x 108 cells).

[0307] The cell suspensions are filled into sequins using the standardized deep-freezing method, preserved and further processed.

[0308] The following section explains embodiments, further developments, and examples of the invention in more detail with reference to the accompanying drawings. The figures show:

[0309] Fig. 1 PMAI data from various bull semen samples,

[0310] Fig. 2a-j Fertility data of the bull sperm samples from Fig. 1,

[0311] Fig. 3a, b Data on the total motility of sperm from a bull semen sample,

[0312] Fig. 4a shows an embodiment of a device according to the invention in a front view, and Fig. 4b shows the embodiment of the device from Fig. 4a in a first sectional view.

[0313] Fig. 4c shows the embodiment of the device from Fig. 4a in a second sectional view.

[0314] Figure 1 and Figures 2a-j show exemplary results from evaluations of a total of 10 selected bull semen samples. Figure 1 shows the PMAI data of the samples before a freezing process. Here, the data of samples not treated according to the invention (untreated) are compared with those of identical samples enriched or purified according to the invention (treated). Figures 2a-j show all fertility data of the samples depicted in the figures, representing some of the 10 bull semen samples examined. Motility values ​​were determined by computer-assisted sperm analysis (CASA). Flow cytometric data (multicolor assay) were obtained according to the dissertation (Bucher et al. 2019).

[0315] The following flow cytometric parameters, which are important indicators of sperm quality, were evaluated: Cpos: Esterase activity

[0316] Plneg: Intact plasma membrane (unstained)

[0317] PNAneg: acrosome reaction (unstained)

[0318] Fneg: intracellular Ca2+ level

[0319] Mpos: mitochondrial membrane potential

[0320] PMAI: Proportion of healthy cells (no PI, no PNA staining)

[0321] DFI: DNA fragmentation level

[0322] MOT: Total Motility

[0323] Siow: Slow motile cells

[0324] Rap: Fast motile cells

[0325] TRT: Thermal resistance test after 2 hours at 38°C following freeze / thaw procedure (OptiXcell diluent). Untreated sperm: Without separation process using magnetic particles. Treated sperm: With separation process using magnetic particles.

[0326] Significantly better values ​​were found in all samples treated with magnetic particles in the separation process according to the invention.

[0327] Figures 3a and 3b show the results of a live / dead separation of a total ejaculate in a bull semen sample. Seven milliliters of total semen with 2.4 x 10⁹ sperm cells per milliliter were analyzed. Here, too, it was demonstrated that the samples treated according to the inventive method were of higher quality than the untreated samples. This is also confirmed by the PMAI% values ​​shown in Figure 3b.

[0328] Fig. 4a shows a front view of an embodiment of a device 10 according to the invention. The device 10 serves to separate cells bound to magnetic particles in a cell suspension, in particular for use in a method as described above. The device 10 comprises a receptacle 11 for a sample vessel (not shown), which is formed by a depression 12 in the base 13 of the device 10. After being placed in the device 10, the sample vessel is enclosed in a semicircular shape by the device body 14. For a secure hold of the inserted sample vessel in the device 10, the device 10 has a collar 15 at its upper edge 17, which is adapted to the respective sample vessel. Several magnets 20 are located in the device 10, which extend radially from the receptacle 11 towards the rear wall 16 of the device 10. The magnets shown in Fig.The device 10 shown in Figure 4a serves to separate the magnetic particles from the sample solution and deposit them on a wall of the sample vessel. The widening of the base 13 on the bottom provides stability, allowing the sample vessel and the sample contained within it to be handled without having to remove the sample vessel from the device 10.

[0329] Fig. 4b shows a sectional view of the device 10 depicted in Fig. 4a along line AA. The semicircular device body 14 has a total of three magnets 20 in its interior 18, aligned relative to each other and radially towards the sample container or the receptacle 11 for the sample container. The interior 18 of the device body 14 is encased in a potting compound 19, in which the magnets 20 are embedded. The magnets 20 are thus held stably in their position within the device 10 and relative to a sample container. As soon as a sample container is inserted into the device 10, the magnetic particles contained in the sample are attracted by the magnets 10 and deposited on the wall of the sample container.Since the sample vessel is usually placed openly into the device 10, the sample vessel can remain in the device 10 while the supernatant not containing magnetic particles is removed from the sample vessel, for example by pipetting.

[0330] Fig. 4c shows a longitudinal sectional view through the device body 14. As can be seen, several magnets 20 are arranged in vertically extending rows 21 within the device body 14 and embedded in the potting compound 19. This embedding ensures the positional alignment of the magnets 20, thus enabling reproducible magnetic separation with the device 10. In the embodiment shown here, the base 13 is screwed to the device body 14. Of course, embodiments are also possible in which the base 13 is integrally formed with the device body 14. The device body 14 containing the magnets 20 is enclosed by a sleeve 22, which tightly seals the interior 18 of the device body 14. The sleeve 22 is made of a chemical-resistant and autoclavable material, thus allowing for easy cleaning and sterilization of the device 10.This also prevents material from adhering to the device 10. In addition to the device 10 shown here, it is of course also possible to design it in the form of a rack to allow the simultaneous processing of several samples. In this case, the device body 14 has several receptacles 11 arranged in a row within the device body 14.

[0331] Device 10 facilitates easy sample handling, as the MP pellet is formed in a clearly defined area of ​​the sample vessel, and the supernatant containing the intact cells can be removed without contamination by the MP. The magnets generate a point- or line-shaped magnetic field within the sample vessel, ensuring that the formed pellet is clearly defined and available within the vessel.

[0332] BIBLIOGRAPHY

[0333] Agarwal A. et al. 2003. Role of Sperm Chromatin Abnormalities and DNA Damage in Male Infertility. Human Reproduction Update 9, no. 4. 331-45.

[0334] Breitbart H. 2002. Intracellular Calcium Regulation in Sperm Capacitation and Acrosomal Reaction, Molecular and Cellular Endocrinology 187, no. 1. 139-44.

[0335] Brewis I. et al., 2005. Molecular Mechanisms during Sperm Capacitation, Human Fertility 8, no. 4, 253-61

[0336] Bucher et al. 2019. Multicolor Flow Cytometric Analysis of Cryopreserved Bovine Sperm: A Tool for the Evaluation of Bull Fertility. Dissertation Vetsuisse-Fakultät Universität Zürich.

[0337] Chi H. et al. 2008. Protective Effect of Antioxidant Supplementation in Sperm-Preparation Medium against Oxidative Stress in Human Spermatozoa. Human Reproduction (Oxford, England) 23, no. 5. 1023-28.

[0338] DESNOYERS, L. & MANJUNATH, P. 1992. Major proteins of bovine seminal plasma exhibit novel interactions with phospholipid. J Biol Chem, 267, 10149-55.

[0339] Dominguez E. et al. 2018. Sperm Sexing Mediated by Magneti c Nanoparticles in Donkeys, a Preliminary In Vitro Study. Journal of Equine Veterinary Science Volume 65, Pages 123-127.

[0340] GWATHMEY, T. M., IGNOTZ, G. G. & SUAREZ, S. S. 2003. PDC-109 (BSP-A1 / A2) promotes bull sperm binding to ovi- ductal epithelium in vitro and may be involved in forming the oviductal sperm reservoir. Biol Reprod, 69, 809-15. Henkel R et al. 2003. Sperm preparation for ART. Reproductive Biology and Endocrinology. 1:108.

[0341] Kqtska-Ksiqzkiewicz L. 2007. Recent Studies on Molecular Mechanisms Involved in Mammalian Sperm Capacitation: A Review. Journal of Animal and Feed Sciences 16, no. 3, 311-28.

[0342] KILLIAN, G. J., CHAPMAN, D. A. & ROGOWSKI, L. A. 1993. Fertility-associated proteins in Holstein bull seminal plasma. Biol Reprod, 49, 1202-7.

[0343] Peter A. et al. 1993. FRACTIONATION OF BOVINE SPERMATOZOA FOR SEX SELECTION: A RAPID IMMUNOMAGNETIC TECHNIQUE TO REMOVE SPERMATOZOA THAT CONTAIN THE H-Y ANTIGEN. Theriogenology 40: 1177-1 185.

[0344] Phiphattanaphiphop C et al. 2021. Antibody-Conjugated Magneti c Beads For Sperm Sexing By A Multi-Wall Carbon Nanotube Microfluidic Device. hftps: / / doi.org / 10.21203 / rs.3.rs-646645 / vl .

[0345] Prakash et al. 2014. Sexing of Spermatozoa in Farm Animals: a Mini Review. Advances in Animal and Veterinary Sciences 2 (4), 226 - 232.

[0346] Rath D. Sex selection of sperm in farm animals: status report and developmental prospects. Reproduction (2013) 145 R15-R30. 23.02.2024 11:18 65.

[0347] Stival C. et al. 2016. Sperm Capacitation and Acrosome Reaction in Mammalian Sperm. Advances in Anatomy, Embryology and Cell Biology. Vol. 220 Springer, Cham.

[0348] TEDESCHI, G., OUNGRE, E, MORTARING, M, NEGRI, A., MAFFEO, G. & RONCHI, S. 2000. Purification and primary structure of a new bovine spermadhesin. Eur J Biochem, 267, 6175-79.

[0349] Zoea G. et al. 2021. Influence of seminal plasma during different stages of bovine sperm cryopreservation. Reprod Dornest Anim Vol 56 (6). 872-883. REFERENCE SYMBOL LIST

[0350] 10 Device

[0351] 11th entry

[0352] 12 In-depth study

[0353] 13 Stand

[0354] 14 Device bodies

[0355] 15 collars

[0356] 16 Wall

[0357] 17 Rand

[0358] 18 Interior

[0359] 19 Potting compound

[0360] 20 magnets

[0361] 21st row

[0362] 22 Mantel

Claims

PATENT CLAIMS 1. A method for enriching a cell population containing intact and damaged cells in a liquid medium, comprising the steps of: a) providing the liquid medium containing the cell population, b) sedimenting the cell population from the liquid medium, c) separating the liquid medium and the sedimented cell population, d) resuspending the sedimented cell population in a suspension solution to produce a cell suspension, e) adding a ferromagnetic material to the cell suspension that binds specifically to the damaged cells, f) incubating the cell suspension containing the ferromagnetic material, g) placing the cell suspension in a magnetic field to separate the bound cells from the unbound cells in the cell suspension, h) separating a portion of the cell suspension enriched with unbound, intact cells.

2. Method according to claim 1, characterized in that the cell population is formed by intact or damaged sperm cells, in particular mammalian sperm cells.

3. Method according to claim 1 or 2, characterized in that the liquid medium is formed by an ejaculate fluid, in particular mammalian ejaculate fluid.

4. Method according to claim 3, characterized in that the ejaculate fluid is provided as freshly obtained ejaculate fluid or thawed cryopreserved ejaculate fluid.

5. Method according to one of claims 2 to 4, characterized in that a determination of a sperm cell count and / or a sperm cell motility is provided before step b.).

6. A method according to any one of the preceding claims, characterized in that the suspension solution comprises: phosphate-buffered physiological saline (PBS), Hanks' Balanced Salt Solution (HBSS), Good's Buffers, Tris buffer, phosphate-buffered saline (PSS), Earle's Balanced Salt Solution (EBSS), Ringer's solutions, TES-Tris buffer, HEPES buffer, sperm buffer (SP), SP-TALP, FC-TALP, TCA, citrates, acetates, lactates, carbonates, phosphates and combinations thereof, or a solution containing at least one salt, at least one carbohydrate or a combination thereof; milk and derivatives thereof; MES; diluent systems compatible with the respective animal species, such as Andromed, BioXCell, BullXcell, INRA 96, PRIMXcell, TRIXcell, NUTRIXcell, OviXcell, Galap, CUNIgel, Triladyl, Biladyl, Steridyl, OviPlus, Bovidyl, M III, BTS, Androstar, Novistar, OptiXCell, Beyond, EquiPlus, Gent or CaniPlus, or mixtures thereof; Additives selected from the group consisting of coconut water, ascorbic acid, beta-carotene, quercetin, rutin, taurine, 5-HMF, glutathione, ubiquinone, superoxide dismutase, glutathione peroxidase, catalase, selenium, L-carnitine, zinc, polyphenols, folic acid, alpha-lipoic acid, melatonin, astaxanthin, zeanxanthin, lucopins, anthocyanins, bioflavonoids, pyruvate, serum albumin, regucalcin, curcumin (diferuloylmethane), growth factors such as epidermal growth factor, oviductal proteins (such as non-luteal isthmic oviductal proteins (NLIP)), Diospyros kaki (persimmon) extract, coenzyme Q10, cobalamin, silymarin (Silybum marianum), glycine, cholesterol-loaded cyclodextrins, and docosahexaenoic acid. (DHA), Vitamin E, deuterated polyunsaturated fatty acids, butylhydroxytoluene, butylhydroxyanisole, ferrostatin, liproxstatin, CoQlO, idebenone, XJB-5-131 (mitochondria-directed nitroxide, JP4-039,Baicalein, PD-146176 (15-LOX-1 inhibitor), AA-861 (LOX inhibitor), zileuton, deferoxamine, ciclopirox, deferiprone, cycloheximide, 2-mercaptoethanol, dopamine, vildagliptin, alogliptin, linagliptin, or mixtures thereof; antibiotics, antimicrobials, or antibiotic agents selected from the group consisting of tetracycline, penicillin, sulfonamide, aminoglycoside, fluoroquinolone, macrolide, cetiofur, apramycin, streptomycin, ticarcillin, polymyxin B or their derivatives, tylosin, gentamicin, lincomycin, spectinomycin, Linco-Spectin® (lincomycin hydrochloride-spectinomycin), or mixtures thereof; pyruvate; Liposome-forming or non-liposome-forming lipids (e.g., phospholipids such as PA, PC, PE, LPC, LPA, PG, LPG, PI, PIP, LPI, PS, LPS, cardiolipin, bis(monoacylglycero)phosphates (BMP), ether lipids, sterol-modified phospholipids, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(l-glycerol) (DOPG), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EPC), dioleoylphosphatidylethanolamine (DOPE)Phosphatidylcholine (PC), Cholesteryl Hemisuccinate (Cholesteryl hydrogen succinate) (CHEMS), cholesterol and its derivatives; aliphatic carboxylic acids (low as long-chain fatty acids) such as caproic acid, sorbic acid, capric acid, n-valeric acid, myristic acid, pelargonic acid, octanoic acid, or polymeric carboxylic acids or polymeric amines of different chain lengths such as polyacrylic acid, polyethyleneimines, poly-L-lysine, or transfection-active substances such as dioleoyl-3-trimethylammonium propane (DOTAP), DOTMA, DODMA, DOGS, DOSPA, DC-cholesterol, DLinDMA, liposomal or nanomolecular lipids such as Lipofectamine, Lipofectamine 2000, Metafectene Pro, Fugene, GeneJuice etc. or mixtures thereof; Serum albumins: HSA, BSA, ESA, CSA, FSA, OSA, MSA, RSA or PSA; EDTA (ethylenediaminetetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), DMSA (dimercaptosuccinic acid), deferoxamine, citric acid, EGTA (ethylene glycol bis(aminoethyl ether)tetraacetic acid), Chelex® resins, transferrin,Deferasirox, penicillamine, alpha-lipoic acid, DMPS, dimercaprol and aminopolycarboxylic acids (complexones), including but not limited to fura-2, IDA, NTA, DTPA, BAPTA, NOTA, DOTA and nicotianamine and derivatives thereof; polymyxin B; lecithins, milk casein, beta-lactoglobulin, alpha-lactalbumin, glycodelin S, glucose, fructose, glycolyzable sugars; pyruvate; Ethane-1,2-diol, formamide, dimethyl sulfoxide, Greg Fahy's VS41A, M22, propylene glycol, ethylene glycol, polyethylene glycol, polyvinylpyrrolidone, propane-1,2,3-triol, X-1000 (Copolymer of polyvinyl alcohol and vinyl acetate), mannitol, K12, Z-1000, VM3, CI-VM-1, 3-methoxy-1,2-propanediol, N-methylformamide, cysteine ​​and its derivatives; polydatin (resveratrol), selenium-associated glutathione peroxidase, glutathione disulfide reductase, heme oxygenase; or mixtures thereof.

7. A method according to any of the preceding claims, characterized in that the ferromagnetic material is selected from iron (Fe), cobalt (Co), nickel (Ni), neodymium (Nd), samarium (Sm), cobalt ferrite (CoFe2O4), gadolinium (Gd), alnico alloys, ferrites (magnetite, (Fe3O4), maghemite (Fe2O3), dysprosium, holmium, erbium, terbium, alloys such as SmCo, Nd2Fel4B, Ni80Fe20 (permalloy), NiFeCo (mumetal), CrO2, magnesium arsenide, EuO or mixtures thereof.

8. A method according to any one of the preceding claims, characterized in that, prior to step a) or step e), a solution of the ferromagnetic material is provided in a storage and stabilization buffer, wherein the storage and stabilization buffer in particular comprises: deionized water, phosphate-buffered physiological saline (PBS), Hanks' Balanced Salt Solution (HBSS), Good's Buffers, Tris buffer, phosphate-buffered saline (PSS), Earle's Balanced Salt Solution (EBSS), Ringer's solutions, TES-Tris buffer, HEPES buffer, sperm buffer (SP), SP-TALP, FC-TALP, TCA, citrates, acetates, lactates, carbonates, phosphates and combinations thereof, or a solution containing at least one salt, at least one carbohydrate or a combination thereof; milk and derivatives thereof; MES; diluent systems compatible with the respective animal species, such as Andromed, BioXCell, BullXcell, INRA 96, PRIMXcell, TRIXcell, NUTRIXcell, OviXcell, Galap, CUNIgel,Triladyl, Biladyl, Steridyl, OviPlus, Bovidyl, M III, BTS, Androstar, Novistar, OptiXCell, Beyond, EquiPlus, Gent or CaniPlus, or mixtures thereof; Additives selected from the group consisting of coconut water, ascorbic acid, beta-carotene, quercetin, rutin, taurine, 5-HMF, glutathione, ubiquinone, superoxide dismutase, glutathione peroxidase, catalase, L-carnitine, zinc, polyphenols, folic acid, alpha-lipoic acid, melatonin, astaxanthin, zeanxanthin, lucopins, anthocyanins, bioflavonoids, pyruvate, serum albumin, regucalcin, curcumin (diferuloylmethane), growth factors such as epidermal growth factor, oviductal proteins (such as non-luteal isthmic oviductal proteins (NLIP)), Diospyros kaki (persimmon) extract, coenzyme Q10, cobalamin, silymarin (Silybum marianum), glycine, cholesterol-loaded cyclodextrins, docosahexaenoic acid (DHA). Vitamin E, deuterated polyunsaturated fatty acids, butylhydroxytoluene, butylhydroxyanisole, ferrostatin, liproxstatin, CoQlO, idebenoneXJB-5-131 (mitochondria-directed nitroxide, JP4-039, baicalein, PD-146176 (15-LOX-1 inhibitor), AA-861 (LOX inhibitor), zileuton, deferoxamine, ciclopirox, deferiprone, cycloheximide, 2-mercaptoethanol, dopamine, selenium, vildagliptin, alogliptin, linagliptin or mixtures thereof; antibiotics, antimicrobial or antibiotic agents selected from the group consisting of tetracycline, penicillin, sulfonamide, aminoglycoside, fluoroquinolone, macrolide, cetiofur, apramycin, streptomycin, ticarcillin, polymyxin B or their derivatives, tylosin, gentamicin, lincomycin, spectinomycin, Linco-Spectin® (Lincomycin hydrochloride-spectinomycin), or mixtures thereof; pyruvate;; Liposome-forming or non-liposome-forming lipids (e.g., phospholipids such as PA, PC, PE, LPC, LPA, PG, LPG, PI, PIP, LPI, PS, LPS, cardiolipin, bis(monoacylglycero)phosphates (BMP), ether lipids, sterol-modified phospholipids, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(l-glycerol) (DOPG), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EPC), dioleoylphosphatidylethanolamine (DOPE), phosphatidylcholine (PC), cholesteryl hemisuccinate (cholesteryl hydrogen succinate) (CHEMS), cholesterol and its derivatives; aliphatic carboxylic acids (low as long-chain fatty acids) such as caproic acid, sorbic acid, capric acid, n-valeric acid, myristic acid, pelargonic acid, octanoic acid, or polymeric carboxylic acids or polymeric amines of different chain lengths such as polyacrylic acid, polyethyleneimines, poly-L-lysine, or transfection-active substances, such as dioleoyl-3-trimethylammonium propane (DOTAP), DOTMA, DODMA, DOGS, DOSPA, DC-cholesterol, DLinDMA, liposomal or nanomolecular lipids such as Lipofectamine, Lipofectamin 2000, Metafectene Pro, Fugene, GeneJuice etc. or mixtures thereof; serum albumins: HSA, BSA, ESA, CSA, FSA, OSA, MSA, RSA or PSA; EDTA (ethylenediaminetetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), DMSA (dimercaptosuccinic acid), deferoxamine, citric acid, EGTA (ethylene glycol bis(aminoethyl ether)tetraacetic acid), Chelex® resins, transferrin, deferasirox, penicillamine, alpha-lipoic acid, DMPS, dimercaprol and aminopolycarboxylic acids (complexones), including but not limited to fura-2, IDA, NTA, DTPA, BAPTA, NOTA, DOTA and nicotianamine and derivatives thereof; polymyxin B; lecithins, milk casein, beta-lactoglobulin, alpha-lactalbumin, glycodelin S, glucose, fructose, glycolyzable sugars; pyruvate;Ethane-1,2-diol, formamide, dimethyl sulfoxide, Greg Fahy's VS41A, M22, propylene glycol, ethylene glycol, polyethylene glycol, polyvinylpyrrolidone, propane-1,2,3-triol, X-1000 (Copolymer of polyvinyl alcohol and vinyl acetate), mannitol, K12, Z-1000, VM3, CI-VM-1, 3-methoxy-1,2-propanediol, N-methylformamide, cysteine ​​and its derivatives; polydatin (resveratrol), selenium-associated glutathione peroxidase, glutathione disulfide reductase, heme oxygenase; or mixtures thereof, wherein in particular a concentration of the ferromagnetic material in the storage and stabilization buffer of between 1 mg and 100 mg particles per 100 ml of storage and stabilization buffer is provided.

9. Method according to one of the preceding claims, characterized in that the ferromagnetic material is provided in the form of magnetic particles (MP) comprising or consisting of the ferromagnetic material, wherein the magnetic particles have a size distribution of between 30 nm and 15000 nm, in particular between 80 nm and 1000 nm, preferably between 100 nm and 7000 nm.

10. Method according to one of the preceding claims, characterized in that after step a.) the addition of a washing solution to the liquid medium is provided.

11. The method of claim 10, characterized in that the washing solution comprises: phosphate-buffered physiological saline (PBS), Hanks 1Balanced Salt Solution (HBSS), Good's Buffers, Tris Buffer, Phosphate-Buffered Saline Solution (PSS), Earle's Balanced Salt Solution (EBSS), Ringer's Solutions, TES-Tris Buffer, HEPES Buffer, Sperm Buffer Solution (SP), SP-TALP, FC-TALP, TCA, citrates, acetates, lactates, carbonates, phosphates and combinations thereof, or a solution containing at least one salt, at least one carbohydrate or a combination thereof; milk and derivatives thereof; MES; diluent systems compatible with the respective animal species, such as Andromed, BioXCell, BullXcell, INRA 96, PRIMXcell, TRIXcell, NUTRIXcell, OviXcell, Galap, CUNIgel, Triladyl, Biladyl, Steridyl, OviPlus, Bovidyl, M III, BTS, Androstar, Novistar, OptiXCell, Beyond, EquiPlus, Gent or CaniPlus, or mixtures thereof;Additives selected from the group consisting of: coconut water, ascorbic acid, beta-carotene, quercetin, rutin, taurine, 5-HMF, glutathione, ubiquinone, superoxide dismutase, glutathione peroxidase, catalase, L-carnitine, zinc, polyphenols, folic acid, alpha-lipoic acid, melatonin, astaxanthin, zeanxanthin, lucopins, anthocyanins, bioflavonoids, pyruvate, serum albumin, regucalcin, curcumin (diferuloylmethane), growth factors such as epidermal growth factor, fallopian tube; Proteins (oviductal proteins, such as non-luteal isthmic oviductal proteins (NLIP), Diospyros kaki (persimmon) extract, coenzyme Q10, cobalamin, silymarin (Silybum marianum), glycine, cholesterol-loaded cyclodextrins, docosahexaenoic acid (DHA), vitamin E, deuterated polyunsaturated fatty acids, butylhydroxytoluene, butylhydroxyanisole, ferrostatin, liproxstatin, CoQlO, idebenone, XJB-5-131 (mitochondria-directed nitrite), JP4-039, baicalein, PD-146176 (15-LOX-1 inhibitor), AA-861 (LOX inhibitor), zileuton, deferoxamine, cyclopirox, deferiprone, cycloheximide, 2-mercaptoethanol, dopamine) Selenium, vildagliptin, alogliptin, linagliptin or mixtures thereof;Antibiotics, antimicrobial or antibiotic agents selected from the group consisting of tetracycline, penicillin, sulfonamide, aminoglycoside, fluoroquinolone, macrolide, cetiofur, apramycin, streptomycin, ticarcillin, polymyxin B or their derivatives, tylosin, gentamicin, lincomycin, spectinomycin, Linco-Spectin® (lincomycin hydrochloride-spectinomycin), or mixtures thereof; pyruvate; Liposome-forming or non-liposome-forming lipids (e.g., phospholipids such as PA, PC, PE, LPC, LPA, PG, LPG, PI, PIP, LPI, PS, LPS, cardiolipin, bis(monoacylglycero)phosphates (BMP), ether lipids, sterol-modified phospholipids, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(l-glycerol) (DOPG), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EPC), dioleoylphosphatidylethanolamine (DOPE), phosphatidylcholine (PC), cholesteryl hemisuccinate (cholesteryl hydrogen succinate) (CHEMS), cholesterol and its derivatives;Aliphatic carboxylic acids (low chain length, like long-chain fatty acids) such as caproic acid, sorbic acid, capric acid, n-valeric acid, myristic acid, pelargonic acid, octanoic acid, or polymeric carboxylic acids or polymeric amines of different chain lengths such as polyacrylic acid, polyethyleneimines, poly-L-lysine, or transfection-active substances such as dioleoyl-3-trimethylammonium propane (DOTAP), DOTMA, DODMA, DOGS, DOSPA, DC-cholesterol, DLinDMA, liposomal or nanomolecular lipids such as Lipofectamine, Lipofectamin 2000, Metafectene Pro, Fugene, GeneJuice, etc., or mixtures thereof; serum albumins: HSA, BSA, ESA, CSA, FSA, OSA, MSA, RSA, or PSA;EDTA (ethylenediaminetetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), DMSA (dimercaptosuccinic acid), deferoxamine, citric acid, EGTA (ethylene glycol bis(aminoethyl ether)tetraacetic acid), Chelex® resins, transferrin, deferasirox, penicillamine, alpha-lipoic acid, DMPS, dimercaprol and aminopolycarboxylic acids (complexones), including but not limited to fura-2, IDA, NTA, DTPA, BAPTA, NOTA, DOTA and nicotianamine and derivatives thereof; polymyxin B; lecithins, milk casein, beta-lactoglobulin, alpha-lactalbumin, glycodelin S, glucose, fructose, glycolyzable sugars; pyruvate; Ethan-l,2-diol, formamide, dimethyl sulfoxide, Greg Fahy's VS41A, M22, propylene glycol, ethylene glycol, polyethylene glycol, polyvinylpyrrolidone, propane-l,2,3-triol, CI-VM-1, 3-methoxy-l,2-propanediol, N-methylformamide, cysteine ​​and its derivatives;Polydatin (resveratrol), selenium-associated glutathione peroxidase, glutathione disulfide reductase, heme oxygenase; or mixtures thereof...; 12. Method according to one of the preceding claims, characterized in that in step f.) an incubation period of between 1 and 120 min is provided.

13. Method according to one of the preceding claims, characterized in that in step b.) sedimentation by centrifugation is provided, in particular with a centrifugation time of between 1000 and 60 minutes, preferably with a centrifugal acceleration of between 300g and 4000g.

14. Method according to one of the preceding claims, characterized in that at least one repetition of steps f.) to h.) is provided.

15. Method according to one of the preceding claims, characterized in that after step h.) cryopreservation of the portion of the cell suspension enriched with the intact cells is carried out.

16. Use of intact sperm cells, in particular mammalian sperm cells, enriched in a method according to any one of claims 2 to 15, for assisted reproduction, in particular for artificial insemination, intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF) of mammals.

17. Device (10) for separating cells bound to magnetic particles (MP) in a cell suspension, in particular for use in a method according to one of claims 1 to 15, comprising a receptacle (11) for at least one sample vessel and at least one magnet receptacle arranged relative to the sample vessel, characterized in that at least one magnet (20) is provided in the magnet receptacle and the magnet receptacle encloses the sample vessel in a substantially semicircular manner.

18. Device (10) according to claim 17, characterized in that the magnet (20) forms a point- or line-shaped magnetic field in the sample vessel.

19. Device (10) according to one of claims 17 or 18, characterized in that the magnetic field is formed in the area of ​​a circumferential wall or at the bottom of the sample vessel.

20. Device (10) according to one of claims 17 to 19, characterized in that the receiving device is designed as a rack, in particular a centrifuge rack.

21. Device (10) according to one of claims 17 to 20, characterized in that a casing (22) enclosing the device (10) is provided.

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