DE3904889A1 - DEVICE AND METHOD FOR SEPARATING MOVABLE CELLS FROM LESS MOVABLE OR IMMOVABLE CELLS IN A SAMPLE CONTAINING A CELL MIXTURE - Google Patents

Abstract

The device comprising a tubular member which includes a sample loading area (P), a motile cell collecting area (4) and an elongate separation area (1) connecting the sample loading area and the cell collecting area, in which the separation area is substantially upright in use, whereby motile cells are able to move from the sample loading area in the separation area through medium preferably alkaline, placed in the device, into the collecting area, the non motile, or less motile, cells being left in the sample loading area and separation area. The sample loading area may include an area 11 of convex glass protrusions H which can protect the fluid from flowing back out of the tube and reflects light to the upper level (4). <IMAGE>

Description

The invention relates to an apparatus and a method for Separate moving cells from less moving or immobile cells in a mixed cell sample.

The usual modern procedures for the treatment of patients sterility and in reproductive medicine suggest that artificial insemination in utero, the extracorporeal Be fertility (in vitro), embryo transplantation and Sperm / ovum transplantation into fallopian tubes. With these The sperm samples used can be a variety of methods Bacteria or other microbes because the collected, unprocessed sperm is contaminated; it's with them conventional methods not possible, bacteria and others To separate microbes from sperm. Common methods of collecting of sperm samples include the following:

1. the simple floating method by Makler et al. in year 1983,

2. the usual floating method,

3. the descent method,

4. the use of a discontinuous Percoll gradients;

5. Centrifugation methods for use in artificial Insemination in utero.

In the following list, the above Ver drive and the present procedure for separating sperm and compared microbes.  

table

The numbers given in the list refer to the procedures discussed above. Z means one directly per proportional increase, A a directly proportional decrease; CFU means colony forming units.

Each cubic centimeter of 90% normal raw seeds contains about 1 × 10 ⁵ bacteria, of which Staphylococcus epidermis occurs most frequently at about 77%; Corynebacterium sp. occurs in about 66% of cases. Viridans streptococci occurs at about 50%; there are also some aerobic bacteria such as alpha-hemolytic Streptococcus, Neisseria sp. (not Neisseria gonorroeae), Pseudomonas aeruginosa, diphytheriods and some anaerobic bacteria such as Peptococcus prevotic, Pepto-streptococcus and the like.

The above lists show that it is difficult is to get sperm samples without microbes. The doctors are seated hence the sperm samples for insemination in utero or for the intra-fallopia transmission of gametes antibiotics. The microbes or bacteria can be from the antibiotic destroyed, the destroyed or weakened microbes however, they can lyse and release endotoxins that are harmful Lich for sperm, eggs and embryos and are a mistake perform surgery; they can also get the infection risk for women treated for sterility Increase patients so that clinical or subclinical compli cations arise. The method and the device of Invention is therefore directed to solving these problems.

The separation system on which the invention is based is based on the following principles:

• 1. In culture media with low viscosity sperm can not does not transmit microbes, so germ-free sperm samples can be obtained without the addition of antibiotics.
• 2. Taking advantage of the physiological properties of the A new floating method was developed at Spermas the sperm from right to left and from bottom to bottom above and then horizontally from left to right floats up, taking the sperm to remove the back and toxic chemical substances at two angles be directed.
• 3. According to the biological principle of sperm activity, at the one line in the horizontal direction the insulation allowed by more sperm than in the vertical direction, a gradually narrowing pipe becomes available made with a weakly alkaline culture medium low viscosity to carry out the process used so that the sperm well under a microscope  can be viewed; the sperm can be effectively separated be so that you have a sufficient amount of liquid receives enough germ-free sperm. Here applies that the longer the separation effect the better separator tube used and the longer the incubation period is.

The apparatus and method of the invention provide the following advantages:

• 1. You avoid contamination with microbes or inflammatory infections.
• 2. The number of successful artificial inseminations will increased.
• 3. The psychological burden and the financial expenditure for the patient is reduced.
• 4. There are fewer surgeries and therapies for the patients required.
• 5. There will be targeted sex control Offspring possible; the risk of gender-related diseases decreased.

The attached drawings are briefly discussed below tert.

Fig. 1 is a top view of a duck-shaped tube according to the invention.

Fig. 2 is a top view of a U-shaped separation tube according to the invention.

Fig. 3 shows a U-shaped separation tube according to the invention in practical use.

Fig. 4 shows a top view of a separator tube according to the invention of a four-hole type.

Fig. 5 shows a separation pipe according to the invention of a four-hole type.

Fig. 6 shows a fourth embodiment according to the invention.

The separation system of the present invention essentially comprises lichen a transparent tube and a ball made of natural or synthetic rubber or silicone rubber. For the better Understanding four different types are preferred Embodiments described below.

As can be seen from Fig. 1, the enthals-shaped separating tube 1 comprises a glass tube and a rubber ball, in particular made of silicone rubber. The part H represents a hemispherical jump 11 before, which extends over the inner tube wall inwards. The number of such hemispherical projections 11 can be 1 or more. The rubber ball, in particular made of silicone rubber, is located at the upper end of the separating tube 1 . The rest of the separation tube 1 comprises a tube 3 , which contains a fluid culture medium for sperm. The fluid culture medium can be a BWW medium or Ham's F-10 medium with 10% human serum, collected before ovulation, or with 10% serum, collected from umbilical cords. The medium is given up at point A ; Raw seeds are introduced at point P. The point H of the convex glass protrusion 11 protects the fluid from a backward flow out of the tube and reflects light, so that the upper level 4 of the duck-neck-shaped tube is examined more clearly or better under the microscope when examining sperm.

In part F the inner diameter of the tube is 6 mm, ie the distance from M to N is 6 mm. An air collection area from point A to point Q also has a dimension of 6 mm. The tube is slightly inclined from point Q and becomes narrower and is then curved upward so that an air collection area is formed. The rear part of the tube is U-shaped and then horizontal to point D. The inside diameter at point D is 1 mm. The tube is cut into a ring shape by means of a glass cutter at points C , D and J , so that it can be easily broken during clinical use and selected sperm fractions can be collected.

The volume from point P to point C is approximately 0.15 ml, from point C to point D approximately 0.12 mm, from point D to point E approximately 0.06 ml, and from point A to point P approximately 0 .5 ml; the volume of the rubber ball is 0.5 ml. The length of each segment from point N to point B is 3 cm, from point M to point L 3 cm, from point A to point B 1 cm, from point Q to point L 1 cm, from point B to the outer surface of point C 2 cm; the distance from point K to point L is 1 cm (range 0.5 to 1.0 cm), from point C to point D 6 cm, from point D to point E 4 cm, and from point A to point D 9 cm . The point G serves to support four pivot points of the pipe so that the pipe can be set up stably and upright.

The U-shaped tube from point D to point E serves to protect against operating errors or contamination with air. The sterile sperm can be collected for further processing after an incubation period of 30 minutes to 1 hour. The prepared tube is incubated in a 37 ° C incubator with high air humidity (96% and more) and 5% carbon dioxide. The concentration of the sperm at the CD section is 20 to 30 × 10 ⁶ or more. The rubber ball protects the culture medium from backflow; it can also be used to express sperm samples contained in the medium. When raw semen is deposited at the sample position in the tube, air bubbles can sometimes be present. The air collection area at point Q therefore prevents the air bubbles from floating upwards and transporting bacteria into the CD area. Point B is the place where the semen sample is placed. Since the seed sample has a higher density than the culture medium, the introduced seed is firmly on the ground between points A and B.

The U-shaped tube according to Figures 2 and 3 is made of glass; the inside diameter is 3.5 mm and the thickness of the tube wall is less than 0.5 mm. The length from point A to point C is 2 mm, from point B to point C 15 mm, from point C to point D 25 mm; the volume from point A to point B about 0.02 ml, from point A to point C about 0.15 ml, from point C to point D about 0.25 ml, from point D to point E about 0.25 ml; A solid rod is provided from point E to the end of the separating tube. The total capacity of the U-shaped tube is approximately 0.65 ml. The tube is circularly incised by means of a glass cutter at points C and D , so that the tube is easy to collect the segments CD and DE , which contain culture medium and selected sperm, can be collected. According to the method, a culture medium is added to the U-shaped tube at the opening (point A ). A suitable seed sample is placed in a borosilicate tube 10 with a length of 75 mm and an inner diameter of 22 mm. The U-shaped tube is then suspended from the upper opening of the borosilicate tube, the front section being immersed from point A to point B in the central region of the seed sample. The incubator is set to 37 ° C in high humidity and with 5% carbon dioxide. The tube prepared in this way is incubated for about 1 h. After the incubation, the tube can be broken at point C for the collection of the segment CD , which is designated with number 1. Then also for the collection of segment DE , which is referred to as No. 2. The concentration in segment No. 1 is approximately 6 to 12 × 10 ⁶ / ml with a total volume of 0.25 ml; therefore segment No. 2 contains approximately 20 × 50 ⁵ sperm. Segment No. 2 contains about 75 × 10 ⁵ sperm. Both segments No. 1 and No. 2 contain no bacteria. The sample from segment # 1 can be used to fertilize 10 to 40 eggs; the average is about 25 eggs. The sample from segment No. 2 can be used to fertilize 3 to 12 eggs, on average around 7 eggs.

The four-hole type separator tube shown in Figs. 4 and 5 comprises a glass tube and a hollow rubber ball; the inside diameter of the tube is approximately 3.5 mm, the thickness of the tube wall is less than 0.5 mm, the length of the segment from point A to point B is 2 mm, from point A to point C 15 mm from point C to to point D 25 mm, from point D to point E 25 mm and from point E to point F 7 mm. The thickness of the part G is 3 mm with a diameter of 25 mm. The diameter of each hole on part G is 2 mm. The length from point H to point J is 15 mm, from point A to point F 72 mm. The total length of the tube is 90 mm. The volume of each segment is approximately 0.02 ml from point A to point B , 0.15 ml from point A to point C , 0.25 ml from point C to point D , 0.25 ml from point D to point E. , 0.07 ml from point E to point F , 0.18 ml from point F to point J and 0.72 ml from point A to point F. The volume of the rubber ball is about 0.65 ml; the volume from point A to point E is 0.65 ml.

The four holes in part C of the tube are used to allow carbon dioxide to enter the sperm section of the tube during incubation. The sperm should be supplied with dextrose, amino acids and carbon dioxide as energy suppliers. The tube wall at point C should be pre-scored using a glass cutter so that the tube can be easily broken when collecting the segment AG . The rubber ball is used to express the liquid medium for the semen sample from the CD segment and from the DE segment.

Since the capacity of the rubber ball is 0.65 ml, ie equal to that of the segment from point A to point E , the medium will be sucked up to point E when the culture medium is sucked in from the opening of the separating tube at point A.

In practical use, a raw seed sample is placed on the bottom of a borosilicate tube with a length of 75 mm and an inner diameter of 22 mm. Then, the separating tube of the four-hole type is inserted into the above-mentioned borosilicate tube, the part G covering the opening of the borosilicate tube and the front end of the separating tube being immersed from the point A to the point B in the central area of the seed sample introduced . The borosilicate tube and the attached separating tube are then placed in an incubator which contains high air humidity and 5% carbon dioxide; then incubated for one hour so that the sperm float in the separation tube. After a suitable incubation, the tube is broken off at point C. The liquid medium in segment CD and segment DE is expressed; the fractions are named No. 3 and No. 4. Segment No. 3 is the same as segment No. 1 in the U-shaped separator described above and segment No. 4 is the same as segment No. 2, with all segments containing no microbes or seed residues and the addition an antibiotic is not required. The sperm are preserved in a form and are ideal for fertilization.

The above statements relate to a method for separating sperm from microbes. However, X / Y sperm can also be separated by means of the glass tube shown in FIG. 1.

Since the sperm Y chromosome is about 1/6 the size of X- Has sperm, the Y chromosome is relatively smaller and lighter than that of the X chromosome. The head of Y- Sperm is small and more resistant to alkaline solutions than the X sperm. Y sperm have a higher rate Survival rate in weakly alkaline media with relatively high pH within a given period of time so that Y- Sperm usually swim up for a longer distance can as X sperm. For these reasons, the separation  of X sperm and Y sperm from each other the better the longer the separation tube is. This explains why on the back More Y sperm collected at the end of the separation tube of the invention can be.

The practical application for the separation of X and Y sperm is explained below with reference to FIG. 1. First, a weakly alkaline medium with low viscosity is introduced into the tube up to point A. The medium used can be a BWW medium or Ham's F-10 medium with 10% human serum, collected before ovulation, or with 10% serum, collected from umbilical cords. The medium should be adjusted to pH 8.0 before use. When the prepared medium has been introduced into the separation tube, the raw seed, which originally contained microbes, is deposited at the location P of the seed application area. Part H serves to protect the liquid medium from backflow or overflow. The inner diameter of the tube is 6 mm and the shape and size and the volume have the same values as explained above in connection with FIG. 1. The sperm in the semen sample takes about one to 1.5 hours to float in the culture medium. After completing this step, the ratio of Y sperm and X sperm collected at the rear part of the CD segment is approximately 4 to 5: 1 to one another.

For better understanding, the following is comparative Listed.

As seen from Fig. 6, 3 of the invention comprises the bottom of the separation tube 1 in accordance with a plurality of concave indentations 5, which appear as convex lenses, as they are seeks be from above. The separating tube 1 is a hollow tube with a wider bottom, which is tapered until it is surrounded by a correspondingly smaller diameter. The separating tube 1 also includes a spherically formed region 6 at its upper end, which is preferably arranged perpendicularly above the lower convex lenses, so that light on the liquid medium during observation under the influence of the convex lenses can be obtained during observation by a microscope can. The upper region 4 of the separating tube 1 has a horizontal bottom; a rubber ball 2 is provided at its bent end. Eggs are placed in the spherical region 6 . When the sperm swim up through the curved tube, they can fertilize the eggs in the ball 6 , whereby an artificial insemination is carried out in vitro. The inseminated eggs begin to divide within the sphere 6 . The doctor can use a glass cutter to remove embryos from the inseminated eggs for transplantation into the uterus of a female patient or to remove mammals for further fertilization treatment. The in-vitro insemination method which can be carried out with the present preferred embodiment can be carried out in the manner described above, the incubation method at 37 ° C. first being carried out in an incubator with high atmospheric humidity and 5% carbon dioxide.

Claims (24)

1. Device for separating moving cells from less movable or immobile cells in a one cell mixed sample, characterized by a tubular element that has a section to the task of Sample, a section for collecting the moving cells and an elongated separating section which comprises the Feeding section and the collecting section connects, wherein when arranging the device with the collecting section higher level than the loading section the moving Cells from the feed section through one in the device existing medium via the separation section in the Collection section can hike and the immovable or less mobile cells in the feed section and / or the separation section remain. 2. Device according to claim 1, characterized in that the feed section is arranged transversely to the separating section is.   3. Device according to claim 1 or 2, characterized net that the collecting section transverse to the separating section is arranged. 4. The device according to at least one of claims 1 to 3, characterized in that the feed section and the Collection section substantially parallel to each other are arranged. 5. The device according to at least one of claims 1 to 4, characterized in that the tubular element in Area of the collecting section is designed to be weaker by collecting the cleaned, mobile cells Break open the tubular element on the weakened To facilitate the job. 6. The device according to claim 5, characterized in that the tubular element by notches or scratches its surface is structurally weakened. 7. The device according to at least one of claims 1 to 6, characterized by facilities for ejecting the gerei ned, movable cells from the collection section. 8. The device according to at least one of claims 1 to 7, characterized in that the facilities with a one end of the tubular element connected fle xiblen ball for ejecting air from the ball and for Ejection of the mobile cells from the collection section include. 9. The device according to at least one of claims 1 to 8, characterized in that the inner diameter of the Collection section less than that of the sample is cut.   10. The device according to at least one of claims 1 to 9, characterized in that the wall of the tubular Element with projections for reflection of light after is equipped above for practical use. 11. The device according to claim 10, characterized by Lin sensor arrangements in the pipe wall. 12. The device according to at least one of claims 1 to 11, characterized in that the feed section, the Separating section and the collecting section of one in essential straight section of the tube are formed. 13. The apparatus according to claim 12, characterized in that it comprises at least one solid section. 14. Method for separating moving cells from unbe mobile or less mobile cells in a ge mixed cell sample, characterized in that the Sample in the receiving section according to a device at least one of Claims 1 to 13, which is suitable contains, introduces, ensuring that that the device is in substantially vertical Arrangement is located, and that the loaded device incubated for a certain period of time and the pure, movable cells from the collection section after the expiry of mentioned period of time collects. 15. The method according to claim 14, characterized in that the mobile cells are sperm cells. 16. The method according to claim 15, characterized in that the mobile cells are Y-sperm cells and the less movable and / or immobile cells X sperm cells and / or microbial contamination.   17. The method according to any one of claims 14 to 16, characterized characterized in that the medium is alkaline. 18. A method for separating sperm / germ systems and germ-free Y, X sperm systems, characterized in that in the separation system for sperm / germs, a fluid culture medium is first placed in the separation tube and then a sperm sample in the separation tube, in particular a duck neck -shaped separation tube, is given at a point P for the absorption of the nutrients of the fluid medium by the sperm, so that the sperm automatically float and rid of adhering microbes or bacteria, and after a certain period of time germ-free sperm on a section CD of the duckbill-shaped separation tube or on a section CD and DE of a separation tube provided with four holes is obtained. 19. The method according to claim 18, characterized in that a sample tube in the separation system for X / Y sperm complete with a weakly alkaline, fluid cool is filled with the medium, the filled sample tube with provided with a stopper and incubated in a suitable manner an untreated semen sample in a volume ver ratio of 1: 3 is added to the fluid culture medium, the mixed semen sample and the fluid medium thoroughly stirred and to separate liquid floating from above centrifugation and a sperm pellet, so obtained sperm pellet to the bottom of the sample tube is brought to the sperm with the alkaline, fluid Medium to contact for 1 hour or more that the upper segment of the sample tube using a microscope is examined to determine the number of swims Sperm determine that one is at the time the sperm in the upper section in a suitable There is volume to cut off the rear part of the upper volume is the rear part of the upper one  Segment of the sample tube using a glass cutter cuts off, and that with the help of the rubber ball contained fluid medium to maintain a germ-free Y sperm pushes out. 20. The method according to claim 18 or 19, characterized in net that the duckbill-shaped tube one comprises an upper and a lower section, the lower section has a transparent support on it Opening and one or more crescent-shaped, convex Has projections that extend from the inner wall of the Tube inward at the front end thereof ken, the lower segment is bent into a U-shape and a gradually decreasing inner diameter with the upper segment parallel to the lower Segment is arranged and its rear end U-shaped Attachment of a hollow and elastic rubber ball is formed, and wherein the duck neck-shaped training Detected pipe using a glass cutter with circular notches on the front and rear end is equipped so that the pipe to separate a preferred section can be canceled. 21. The method according to claim 18 or 19, characterized in that the U-shaped tube is rod-shaped from its lower end to a height of 65 mm as a tube and at its upper part and at points C and D for the removal of sperm is provided with circular cutters provided with a glass cutter. 22. The method according to claim 18, characterized in that one uses a separating tube of a four-hole type, which comprises a glass tube with a rubber ball at its upper end and a cover 72 mm above the glass tube sheet, the cover having four holes as air outlets and the outer wall of the glass tube is provided at points c and d with circular incisions made with a glass cutter to facilitate the breaking of the tube and to collect the desired sperm. 23. The method according to at least one of claims 18 to 20, characterized in that the duck-neck-shaped separating tube has a section for collecting air between points A and Q to prevent air bubbles from floating upwards, the air collecting section so it is formed that the upper tube wall is inclined downward from point Q while reducing the inner diameter and is then curved upward in a curve. 24. The method according to claim 18, characterized in that the separation tube a variety of symmetrically arranged Spheres that are perpendicular to that of concave Indentations formed convex lenses are arranged and a microscope for observation purposes menes sperm or an embryo or a dividing, fertilized egg can be straightened.