• 专利附录
  • 专利说明
  • 权利要求
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    • 专利摘要:
    A method is described for producing a portion unit from a gel-like carrier material with at least one biological cell that is embedded in the carrier material or at least one cell organelle that is embedded in the carrier material. Furthermore, a portion unit obtainable by the method is described. Various uses of the portion unit are also described.
    公开号:CH714874B1
    申请号:CH01232/19
    申请日:2018-03-23
    公开日:2021-11-30
    发明作者:Marc Josse Jean；Gajewski Martin；Arangio Mario；Woizenka Eduard
    申请人:Hamilton Bonaduz Ag；
    IPC主号:
    专利说明:

    Field of invention
    The invention relates to a method for producing a portion unit from a gel-like carrier material with at least one biological cell that is embedded in the carrier material, or at least one cell organelle that is embedded in the carrier material. The invention further relates to a portion unit obtainable by the method. The invention also relates to various uses of the portion unit.
    Background of the invention
    In the development of cancer, major structural changes in the chromosomes, so-called chromosome or DNA rearrangements, often play a decisive role. Examples of DNA rearrangements are duplications, translocations, inversions and deletions of certain chromosome sections as well as DNA strand breaks and subsequent fusions of the DNA strands in a different arrangement. The identification of DNA rearrangements is used to diagnose cancer and can serve as the basis for personalized cancer therapy.
    In order to identify DNA rearrangements, a method known as “molecular combing” is used. “Molecular combing” requires intact, long DNA fragments that are combed onto a glass surface. Combing it aligns and stretches the DNA fragments. This then makes it possible to bring the DNA fragments into contact with hybridization probes and to identify DNA rearrangements by image analysis of the hybridized probes. Accordingly, “molecular combing” is often combined with hybridization techniques such as fluorescence in situ hybridization (FISH).
    For “molecular combing”, DNA fragments with a length of more than 1000 kilobase pairs are required. Such DNA fragments are referred to as high molecular weight DNA and are also becoming increasingly important for other molecular biological techniques such as next generation sequencing.
    In order to isolate high molecular weight DNA from cells or cell nuclei, the DNA must be protected from shear forces during its isolation. Shear forces can cause the DNA to break into small fragments that would make image analysis to detect DNA rearrangements impossible. To protect against shear forces, the DNA is immobilized during isolation from cells, for example in agarose. The cells are manually embedded in agarose. To do this, the cells are first mixed with a flowable agarose solution. A certain amount of the mixture is placed in a target vessel and solidified there before the isolation of the high molecular weight DNA begins. The solidified mixture can be referred to as a portion unit.
    For the use of “molecular combing” in cancer diagnosis methods, a standardization of all process steps and a high level of precision are required in order to prevent incorrect results or misinterpretations. The manual production of the portion units cannot consistently meet these requirements, since it has poor reproducibility. On the one hand, a certain variability of the portion units from person to person cannot be avoided due to differences in the individual handling of the portion units. On the other hand, the portion units are very fragile. For this reason, there may be differences between the portion units even if they were made by one and the same person. Thus, it is difficult to manually produce standardized portion units for a large number of samples. As a result, there are also differences in the amount and quality of the high molecular weight DNA that is isolated from the cells in the serving units.
    There is therefore a need for an automatable method for producing the portion units.
    Summary of the invention
    The present invention relates to a method for producing a portion unit from a gel-like carrier material with at least one biological cell that is embedded in the carrier material, or at least one cell organelle that is embedded in the carrier material, the method comprising the steps: a) Providing a flowable, solidifiable mixture comprising the carrier material and the at least one cell or the at least one cell organelle, under a first reaction condition, b) introducing a predetermined amount of the mixture into a pipette tip with a longitudinal end with an opening for introducing and discharging fluid , wherein the opening is large enough to dispense the portion unit through the opening, c) solidifying the mixture in the pipette tip by incubating the longitudinal end of the pipette tip under a second reaction condition in which the mixture solidifies so that the portion unit forms, and d) Removal of the portion unit from the pipette tip.
    The present invention further relates to a portion unit made of a gel-like carrier material with at least one biological cell that is embedded in the carrier material, or at least one cell organelle that is embedded in the carrier material, obtainable by a method according to the invention.
    The present invention also relates to the use of a portion unit according to the invention for isolating a nucleic acid.
    The present invention also relates to the use of a portion unit according to the invention for isolating a protein.
    The present invention also relates to the use of a portion unit according to the invention for the cultivation or cocultivation of cells.
    Detailed description of the invention
    The invention relates to a method for producing a portion unit from a gel-like carrier material with at least one biological cell embedded in the carrier material or at least one cell organelle embedded in the carrier material, the method comprising the steps of: a) providing a flowable, solidifiable mixture which comprises the carrier material and the at least one cell or the at least one cell organelle, under a first reaction condition, b) introducing a predetermined amount of the mixture into a pipette tip with a longitudinal end with an opening for introducing and discharging fluid, wherein the opening is large enough to dispense the portion unit through the opening, c) solidifying the mixture in the pipette tip by incubating the longitudinal end of the pipette tip under a second reaction condition in which the mixture solidifies so that the portion unit forms, and d ) Application the portion unit from the pipette tip.
    By means of the method according to the invention, a portion unit is produced from a gel-like carrier material with at least one biological cell that is embedded in the carrier material, or at least one cell organelle that is embedded in the carrier material. The term “portion unit” as used here denotes a solidified predetermined amount of the carrier material in which the at least one cell or the at least one cell organelle is embedded. The cell or the cell organelle is enclosed by the carrier material. Since the carrier material of the portion unit is solidified, the cell or the cell organelle is immobilized in the carrier material of the portion unit. Due to the gel-like carrier material, the portion unit is elastic. This means that the portion unit can change its shape under the influence of force and can return to its original shape when the force is removed.
    The term “biological cell” as used here denotes a cell of an animal, a plant, a fungus, a bacterium or an archaeon. The animal is, for example, a mammal, preferably a human. A biological cell has a cell membrane that surrounds the cytoplasm and the other components of the cell contained therein, such as cell organelles. The term “cell organelle” as used here refers to a component of a cell that is surrounded by a single or a double membrane. The cell organelles include, for example, the nucleus and mitochondrion.
    For the method according to the invention, a flowable, solidifiable mixture which comprises the carrier material and the at least one cell or the at least one cell organelle is first provided under a first reaction condition. Any gel-like material that is present as a flowable, solidifiable mass or in solidified form, depending on a reaction condition, is suitable as a carrier material. In a preferred embodiment, the carrier material is agarose. If the carrier material is in the form of a flowable mass, the at least one cell or the at least one cell organelle can be mixed into the carrier material. In the resulting mixture, which comprises the carrier material and the at least one cell or the at least one cell organelle, the properties of the carrier material are retained, so that the mixture is likewise flowable and solidifiable.
    The term “reaction condition” as used here denotes at least one parameter or a combination of parameters in which at least one of the method steps is carried out. The parameters preferably include a temperature, a salt concentration, an ionic strength, a pH value and / or an addition of reagents. The choice of the reaction condition depends in particular on which support material is used in the process. The reaction condition is, in particular, a temperature.
    The first reaction condition is the reaction condition in which the flowable, solidifiable mixture comprising the carrier material and the at least one cell or the at least one cell organelle is provided. The first reaction condition is therefore chosen so that the mixture is in a flowable, solidifiable state. For this purpose, the first reaction condition is chosen such that the carrier material is in a flowable, solidifiable state. The first reaction condition is therefore selected as a function of the gelling temperature of the carrier material used. The setting temperature is the temperature at which the carrier material solidifies to form a gel or a gel-like body. In a preferred embodiment, the first reaction condition comprises a temperature of about 45 ° C.
    In the method according to the invention, a predetermined amount of the mixture is then introduced into a pipette tip with a longitudinal end with an opening for introducing and discharging fluid. A pipette tip has two opposite longitudinal ends, each with an opening, of which one longitudinal end is designed for introducing and discharging fluid and the other longitudinal end for attaching the pipette tip to a pipette shaft of a pipette or to a pipetting head of a pipetting robot. In known pipette tips, the longitudinal end for introducing and discharging fluid typically has the shape of a truncated cone, the diameter of which decreases towards the opening of the longitudinal end. These pipette tips are suitable for the method according to the invention. Furthermore, pipette tips with a cylindrical longitudinal end for introducing and discharging fluid are suitable for the method according to the invention.
    The opening of the pipette tip for introducing and dispensing fluid is large enough to dispense the portion unit through the opening from the pipette tip. Due to the gel-like carrier material, the portion unit is elastic. Due to the elasticity, the portion unit can be temporarily deformed. Thus, the portion unit can be deformed while being dispensed from the pipette tip and can return to its original shape after dispensing. The portion unit is removed from the pipette tip while maintaining the original shape of the portion unit.
    A temporary deformation of the portion unit during the discharge from the pipette tip occurs, for example, when the longitudinal end of the pipette tip for introducing and discharging fluid has the shape of a truncated cone. In this case, the portion unit is also frustoconical and that part of the portion unit facing the interior of the pipette tip must deform when it is removed from the pipette tip so that the portion unit can be brought through the opening of the pipette tip.
    The deformation of the portion unit can be brought about, for example, by exerting pressure on the portion unit in the direction of the pipette tip opening. The more elastic the portion unit, the less pressure is required to dispense the portion unit from the pipette tip with the same size of the opening for introducing and dispensing fluid. The pressure is preferably evenly distributed over the surface of the portion unit facing the interior of the pipette tip.
    The opening of the pipette tip for introducing and discharging fluid must therefore have a certain minimum size so that the portion unit can be discharged from the pipette tip through the opening. This minimum size depends on the elasticity and the size of the portion unit. For example, the opening of the pipette tip for introducing and discharging fluid can be smaller, the more elastic the portion unit is. The elasticity of the portion unit depends primarily on the type and composition of the carrier material.
    The opening of the pipette tip must be large enough to dispense the portion unit undamaged through the opening. The portion unit must not be damaged while it is being dispensed from the pipette tip so that the portion unit returns to its original shape after dispensing. If the portion unit returns to its original shape after being removed from the pipette tip, it can be assumed that the cell or cell organelle and its / its components, in particular DNA, were protected from shear forces while the portion unit was being removed from the pipette tip.
    In a preferred embodiment, the opening of the pipette tip for introducing and discharging fluid has an internal diameter of 2 mm to 5 mm, preferably 3 mm to 4 mm.
    In order to avoid contamination of the portion unit, the pipette tip is preferably a disposable pipette tip.
    The introduction of the predetermined amount of the mixture into the pipette tip can be done in various ways, for example by sucking the predetermined amount of the mixture into the pipette tip with negative pressure or by immersing the longitudinal end for introducing and discharging fluid in a vessel containing the mixture contains. When the pipette tip is immersed in the mixture in the vessel, the mixture is introduced into the pipette tip by capillary forces. The amount of the mixture introduced depends on the duration of the immersion of the pipette tip, so that the predetermined amount of the mixture can be achieved by a predetermined immersion time.
    The method according to the invention further comprises solidifying the mixture in the pipette tip, so that the portion unit is formed. This process step is carried out by incubating the longitudinal end of the pipette tip under a second reaction condition in which the mixture solidifies. For this purpose, the second reaction condition is chosen so that the carrier material solidifies. The second reaction condition is therefore selected as a function of the gelling temperature of the carrier material used. In a preferred embodiment, the second reaction condition comprises a temperature of about 4 ° C.
    The mixture that was introduced into the longitudinal end of the pipette tip for introducing and discharging fluid is solidified in the pipette tip, so that the portion unit is formed in the pipette tip. The portion unit is created by solidifying the predetermined amount of the mixture.
    The method according to the invention further comprises removing the portion unit from the pipette tip. The portion unit can be dispensed in various ways, for example by blowing or knocking the portion unit out of the pipette tip or by destroying the pipette tip. The portion unit is discharged from the pipette tip while maintaining the original shape of the portion unit. It is possible that the portion unit deforms while being dispensed from the pipette tip and returns to its original shape after dispensing. Alternatively, the portion unit is not deformed while it is being dispensed from the pipette tip.
    The solidified carrier material protects the cell embedded therein or the cell organelle embedded therein and its / its components, in particular DNA, from shear forces.
    One advantage of the method according to the invention is that the portion unit is formed in the pipette tip. It is known to introduce the solidifiable mixture into a target vessel and to solidify the mixture in the target vessel so that the portion unit is formed in the target vessel. However, the portion unit then adheres to the bottom and to the walls of the target vessel, so that the surface of the portion unit accessible for reagents is reduced and is insufficient for certain uses of the portion unit. In contrast to this, in the method according to the invention, the portion unit is formed in the pipette tip and then discharged from the pipette tip. This means that the portion unit is accessible for reagents from all sides. As a result, reaction and / or washing steps can be carried out more efficiently as part of the further use of the portion unit.
    Another advantage of the method according to the invention is that the method can be automated, so that the portion unit can be produced in an automated manner. In a preferred embodiment, the portion unit is therefore produced in an automated manner. Because it can be automated, the method can be carried out partially or completely by a pipetting robot. The pipetting robot is preferably set up to carry out the method steps to be carried out by it at predetermined times. In a preferred embodiment, the method steps are therefore carried out at predetermined times.
    An automated execution of the method enables the parallel production of a large number of portion units. As a result, the method according to the invention can also be used in high throughput applications. For example, the mixture can be provided in a microtiter plate with 96 or 384 wells (wells).
    Automated execution of the method enables not only the parallelization of the method, which saves time, but also extensive control and monitoring of the individual method steps. This enables reproducible production of the portion unit. The control and monitoring of the method steps is particularly advantageous for step b), since a pipetting robot can control whether the predetermined amount of the mixture has been introduced into the pipette tip, for example by monitoring the air displaced from the pipette tip. By means of the controlled and reproducible introduction of the predetermined amount of the mixture into the pipette tip, in contrast to the manual implementation of the method step, a reproducible size of the portion unit can be ensured. The reproducible size of the portion unit is particularly important in molecular biological examinations, since the examination results can often only be correctly interpreted under this condition. Furthermore, the speed at which the predetermined amount of the mixture is introduced into the pipette tip can be specified. This allows the predetermined amount of the mixture to be introduced into the pipette tip in a controlled manner. Furthermore, any penetration of air bubbles into the mixture during step b) can thereby be avoided. The correct and complete discharge of the portion unit from the pipette tip can also be verified by a pipetting robot. These technical possibilities allow the correct and reproducible production of the portion unit to be ensured, so that standardized portion units can be produced for a large number of samples of cells or cell organelles.
    In a preferred embodiment, prior to step a), the flowable, solidifiable carrier material is mixed with the at least one cell or the at least one cell organelle under the first reaction condition in order to produce the mixture. The cell or the cell organelle is preferably already present in the first reaction condition before being mixed into the carrier material in order to avoid solidification of the carrier material when the cell or the cell organelle is added. For example, the cell or the cell organelle can be preheated or precooled to the same temperature as the flowable carrier material.
    A suspension of cells or cell organelles is mixed with the carrier material in a ratio of 1: 1 (v / v), for example. For example, 50 μl of a cell suspension are mixed with 50 μl of a solution of 2% agarose as the carrier material. The proportion of carrier material in the mixture can be increased in order to obtain stronger and thus mechanically more stable portion units. Alternatively, the concentration of the carrier material can be increased in order to produce more solid serving units.
    In a preferred embodiment, step b) takes place by sucking the predetermined amount of the mixture into the pipette tip with negative pressure. For this purpose, for example, an ordinary microliter or piston-operated pipette, onto which the pipette tip is attached, can be used. In this case, a movable piston pulls the column of air below it upwards with it in its upward movement and thereby also pulls the mixture into the attached pipette tip.
    In a preferred embodiment, step b) takes place at a flow rate of 1 pl / s to 300 μl / s, preferably from 1 pl / s to 100 μl / s. The flow rate is selected as a function of the viscosity of the mixture, the predetermined amount of the mixture to be introduced into the pipette tip and the diameter of the opening of the longitudinal end of the pipette tip for introducing and discharging fluid. By choosing the flow rate, the predetermined amount of the mixture can be introduced into the pipette tip in a controlled manner. The flow rate is preferably chosen to be sufficiently low in order to avoid any occurrence of air bubbles in the mixture during step b).
    In a preferred embodiment, the predetermined amount of the mixture is about 5 µl to about 300 µl, more preferably about 10 µl to about 250 µl, more preferably about 50 µl to about 200 µl, most preferably about 75 µl to about 150 µl . The predetermined amount of the mixture is selected depending on the available amount of cells or cell organelles and / or the intended use of the portion unit. In a particularly preferred embodiment, the predetermined amount of the mixture is about 100 μl.
    In a preferred embodiment, the longitudinal end of the pipette tip is incubated in the second reaction condition by placing the longitudinal end of the pipette tip in a solution which has the temperature of the second reaction condition. The longitudinal end of the pipette tip can, for example, be immersed in a solution which has a temperature of about 4 ° C.
    In a preferred embodiment, step d) is carried out by blowing the portion unit out of the pipette tip. For this purpose, for example, an ordinary microliter or piston-operated pipette, onto which the pipette tip is attached, can be used. In this case, a movable piston displaces the column of air below when it is pressed down, so that the portion unit is blown out of the pipette tip.
    In a preferred embodiment, the portion unit is applied to a target vessel with a reaction solution or onto a solid surface. This mainly depends on the intended further use of the portion unit. If the portion unit is applied to a target vessel with a reaction solution, the reaction solution is selected depending on the further use of the portion unit. The reaction solution can, for example, be a lysis buffer for lysing the cell or the cell organelle in the portion unit.
    In a preferred embodiment, the longitudinal end of the pipette tip for introducing and discharging fluid is cylindrical, the cylindrical longitudinal end having a predetermined height so that the free surface of the mixture facing the interior of the pipette tip does not protrude beyond the cylindrical longitudinal end of the pipette tip . Thus, the minimum predetermined height of the cylindrical longitudinal end depends on the predetermined amount of the mixture that is introduced into the pipette tip and on the diameter of the cylindrical longitudinal end. The cylindrical longitudinal end facilitates the dispensing of the portion unit from the pipette tip, since the portion unit does not have to be deformed during dispensing.
    In a preferred embodiment, the carrier material comprises agarose. Agarose is a polysaccharide that is inexpensive and easy to use. Agarose is typically purchased as a powder. The powder is placed in a suitable aqueous buffer solution and boiled for about 1 to about 5 minutes to dissolve the agarose. The dissolved agarose is typically cooled to about 50 ° C to about 60 ° C or allowed to cool before further use. The setting temperature of agarose, at which the agarose solidifies to form a gel, is in the range of about 34 ° C to about 42 ° C for 1.5% agarose, depending on the source from which the agarose originates. In the solidified state, agarose is in the form of a gel that has pores. The pores allow reagents to access the cell embedded in the portion unit or the cell organelle embedded in the portion unit. This is useful for further use of the portion unit.
    Agarose is at a temperature of about 50 ° C to about 60 ° C as a flowable, solidifiable mass. Therefore, when using agarose as carrier material, the first reaction condition preferably comprises a temperature of about 50 ° C to about 60 ° C, preferably about 52 ° C.
    At a temperature of about 0 ° C to about 30 ° C, agarose is in solidified form. Therefore, when using agarose as the carrier material, the second reaction condition preferably comprises a temperature of from about 0 ° C to about 30 ° C, more preferably from about 0 ° C to about 10 ° C, most preferably from about 4 ° C. A temperature of about 0 ° C to about 10 ° C is preferred because the solidification of agarose takes longer and is more uncontrolled at a temperature of about 20 ° C to about 30 ° C compared to lower temperatures. At 4 ° C, a few seconds are enough for the agarose and thus the mixture to solidify in the pipette tip.
    In a preferred embodiment, the carrier material comprises from about 0.5% to about 10% agarose. The concentration data for agarose are given as weight per volume (w / v). For example, for 1% agarose, 1.0 g of agarose powder is dissolved in 100 ml of buffer solution. The more concentrated the agarose, the smaller the pores that the solidified agarose has in the portion unit and the stronger the portion unit. The concentration of the agarose is therefore selected depending on the intended further use of the portion unit. If 0.5% agarose is used as the carrier material, the portion unit is very soft. At higher agarose concentrations, such as from 0.75% to 1%, the portion unit is already stronger and therefore more mechanically stable, which makes handling of the portion unit easier.
    In a particularly preferred embodiment, the carrier material comprises 1.5% agarose. In the case of 1.5% agarose, the portion unit is mechanically sufficiently stable so that the portion unit is easy to handle, while the pore size of the agarose allows a large number of other possible uses of the portion unit.
    In a preferred embodiment, the agarose is a low-melting agarose. Low-melting agarose has a lower melting temperature and also a lower setting temperature compared to standard agarose. The gelling temperature of low melting point agarose is in the range of about 24 ° C to about 30 ° C for 1.5% agarose. Thus, low-melting agarose is still present as a flowable, solidifiable mass at a temperature of about 40 ° C to about 50 ° C. Therefore, when using low-melting agarose as the carrier material, the first reaction condition preferably comprises a temperature of about 40 ° C to about 50 ° C, preferably about 45 ° C. This is advantageous since temperatures above 45 ° C. can damage the cell or the cell organelle in the mixture or damage the pipette tip.
    Another advantage is that low-melting agarose still remains liquid even at temperatures up to 15 ° C below 45 ° C. This is important for process reliability, as it prevents the mixture from solidifying prematurely, for example through contact with the pipette tip. The pipette tip is preferably not preheated or pre-cooled to the temperature of the mixture, but used without preheating or pre-cooling, so that the pipette tip is generally room temperature.
    At a temperature of about 0 ° C to about 20 ° C, low-melting agarose is in solidified form. Therefore, when using low-melting agarose as carrier material, the second reaction condition preferably comprises a temperature of about 0 ° C to about 20 ° C, more preferably about 0 ° C to about 10 ° C, most preferably about 4 ° C. At 4 ° C, a few seconds are enough for the low-melting agarose and thus the mixture to solidify in the pipette tip.
    In contrast to agarose, which is present as a flowable mass at higher temperatures and in a solidified state at lower temperatures, there are also carrier materials which are present as a flowable mass at lower temperatures and form a gel at higher temperatures. Gelatin is an example of such a carrier material. Gelatin is, for example, a flowable mass at 4 ° C. The gelling temperature of gelatin is in the range from about 35 ° C. to about 40 ° C., solidified gelatin becoming liquid again by further heating, for example to about 50 ° C.
    In a preferred embodiment, the at least one cell is an animal cell, preferably a mammalian cell, more preferably a human cell.
    The cell is preferably derived from a cancer patient. The portion unit produced by the method according to the invention is particularly suitable for isolating high molecular weight DNA from the cell which is embedded in the carrier material. The high molecular weight DNA is required for the identification of DNA rearrangements for the diagnosis of cancer. In addition, the identification of the specific DNA rearrangements that exist in a cancer patient serves as an essential prerequisite for personalized cancer therapy.
    In a preferred embodiment, the method is carried out under sterile conditions. This can be advantageous, for example, if the at least one cell is to be cultivated in the same after the production of the portion unit.
    In a preferred embodiment, a concentration of the at least one cell in the mixture is about 50,000 cells / ml to about 5,000,000 cells / ml, preferably about 1,000,000 cells / ml. The concentration of the at least one cell in the mixture is selected depending on the intended further use of the portion unit. The same applies to the at least one cell organelle in the mixture, the concentration of which in the mixture is preferably about 50,000 cell organelles / ml to about 5,000,000 cell organelles / ml, preferably about 1,000,000 cell organelles / ml.
    In a preferred embodiment, the at least one cell organelle is a cell nucleus, a mitochondrion, a vesicle, an endoplasmic reticulum, a Golgi apparatus, a lysosome, a peroxisome, a chloroplast, a chromoplast, a leukoplast, a cell sap vacuole, a melanosome or a phagosome.
    In a particularly preferred embodiment, the at least one cell organelle is a cell nucleus or a mitochondrion, preferably a cell nucleus. The portion unit produced by the method according to the invention is particularly suitable for isolating high molecular weight DNA. DNA can be found in cell nuclei and mitochondria, and in plants in chloroplasts. The chromosomal DNA present in the cell nuclei is of interest for most applications, for example for the identification of DNA rearrangements in cancer patients.
    In a preferred embodiment, the mixture further comprises magnetic particles. This produces a portion unit in which magnetic particles are present. The presence of magnetic particles is advantageous when the portion unit is used further. The magnetic particles make it possible to hold the portion unit in a certain position using a magnetic field. For this purpose, for example, a magnet can be attached to the side or below a reaction vessel. The portioning unit then moves to the side of the reaction vessel facing the magnet. Because the position at which the portion unit is located is known, damage or destruction of the portion unit while liquids are being moved, for example while the portion unit is being washed, is avoided. In this way, liquids in particular can be safely sucked out of the reaction vessel without damaging the portion unit.
    In a preferred embodiment, the magnetic particles do not interact with nucleic acids. An interaction of the magnetic particles with nucleic acids could interfere with the isolation of nucleic acids such as DNA from the cell or the cell organelle in the portion unit.
    In a preferred embodiment, an average particle size of the magnetic particles is about 10 nm to about 1000 nm, preferably about 50 nm to about 500 nm.
    In a second embodiment, the invention relates to a portion unit made of a gel-like carrier material with at least one biological cell embedded in the carrier material or at least one cell organelle embedded in the carrier material, obtainable by a method according to the invention.
    In a third embodiment, the invention relates to the use of a portion unit according to the invention for isolating a nucleic acid. The nucleic acid is protected from shear forces by the carrier material, so that intact, long nucleic acid fragments can be isolated from the cell or the cell organelle. The nucleic acid can be DNA or RNA. In a preferred embodiment, the nucleic acid is DNA, more preferably high molecular weight DNA. The term “high molecular weight DNA” as used here refers to DNA fragments with a length of more than 1000 kilobase pairs. During the isolation, the DNA is immobilized in the portion unit and protected from shear forces. This makes the portion unit particularly advantageous for isolating high molecular weight DNA. High molecular DNA is required for various molecular biological processes, in particular for so-called “molecular combing”, which is used to identify DNA rearrangements in the diagnosis of cancer.
    In order to isolate high molecular weight DNA from the at least one cell that is embedded in the carrier material, the portion unit can be applied, for example, into a target vessel. The target vessel preferably contains a buffer solution. In a next step, the cell is digested or lysed. The cell is lysed within the portion unit so that the DNA is further protected by the carrier material. The lysis of the cell is often temperature-dependent, so that this step is preferably carried out at a predetermined temperature that is kept constant. The temperature is 37 ° C, for example. The lysis step is carried out, for example, overnight, that is to say for about 16 hours.
    The portion unit is then washed in order to remove the lysed cell material, cell debris, enzymes and other contaminants which could interfere with the further use of the DNA from the portion unit. When washing the portion unit, the portion unit must be carefully mixed with the washing solution and moved in this in order to ensure efficient washing of the portion unit without the portion unit being damaged or destroyed in the process. If the portion unit were damaged or destroyed during washing, the DNA would be exposed to shear forces that could break the high molecular weight DNA into small fragments. To move the portion unit in the washing solution, the target vessel can be carefully turned upside down and back up again, for example, once or several times. The washing solution is then removed. The washing of the portion unit can be repeated several times, for example 5 to 50 times. The washing of the portion unit is preferably repeated 20 to 50 times, more preferably 50 times.
    After washing, the carrier material of the portion unit is removed. For this purpose, the carrier material can, for example, be brought back into a flowable state and / or broken down. This step is often temperature-dependent and is therefore preferably carried out at a predetermined temperature that is kept constant. The degradation is preferably achieved by enzymatic digestion of the carrier material. Agarose can be broken down, for example, by incubating with the enzyme beta-agarase, preferably at 42 ° C. The isolated high molecular weight DNA is now available for further use. All steps of DNA isolation are preferably carried out by means of a pipetting robot in order to ensure a controlled, precise and reproducible process sequence.
    In a further embodiment, the invention relates to the use of a portion unit according to the invention for isolating a protein, preferably a protein with at least two subunits. Subunits of proteins are held together by hydrogen bonds, van der Waals forces and Coulomb forces. The carrier material of the portion unit offers protection against shear forces not only for nucleic acids but also for proteins. This means that the portion unit is also suitable for isolating proteins and protein complexes.
    In a further embodiment, the invention relates to the use of a portion unit according to the invention for the cultivation or cocultivation of cells. When cells are co-cultivated, at least two different types of cells, for example cancer cells and fibroblasts, are cultivated together.
    Due to the three-dimensional environment of the cells in the portion unit, the portion unit is particularly suitable for three-dimensional cultivation or co-cultivation of cells. For this use, the carrier material is selected such that it is in a solidified state under the reaction condition in which the cells are to be cultivated or co-cultivated. Human cells and other mammalian cells are typically cultured at 37 ° C (body temperature), so that the carrier material is preferably in a solidified state at 37 ° C. The carrier material is preferably present as a flowable mass at lower temperatures, for example at about 4 ° C. Suitable carrier materials for three-dimensional cultivation or cocultivation of cells are known. For example, hydrogels made from structural proteins such as collagen or gelatin methacrylate can be used as carrier material. In this context, the carrier material is also referred to as a matrix and its composition is preferably similar to the naturally occurring, complex extracellular environment of cells in tissues.
    In a preferred embodiment, the cells cultivated or co-cultivated in the portion unit release a compound which can diffuse through the carrier material and thus exit the portion unit. For example, the cells can secrete proteins that inhibit the growth and / or proliferation of cancer cells. In this case, the serving unit can be used to treat cancer.
    In a further embodiment, the use of a serving unit according to the invention for releasing a compound into the surroundings of the serving unit is disclosed. The portion unit is preferably surrounded by a fluid, for example by a medium, into which the compound is released from the portion unit. As described above, the compound is released, for example, from the cell, which is embedded in the carrier material, and diffuses to the outside through the carrier material. In another embodiment, a lysis of the cell that is embedded in the carrier material or of the cell organelle that is embedded in the carrier material is carried out. During the lysis, the membrane that surrounds the cell or the cell organelle is dissolved, so that the content of the cell or the cell organelle in the carrier material is released and diffuses through the carrier material into the vicinity of the portion unit. By controlling the rate at which the cell or cell organelle is lysed, the rate at which the compound is released into the vicinity of the serving unit can be controlled. This enables a controlled release of the compound into the surroundings of the portion unit, for example a particularly slow release.
    For a quick release of the contents of the cell or the cell organelle in the vicinity of the portion unit, the carrier material of the portion unit can be broken down during or after the lysis of the cell or the cell organelle and / or brought into a flowable state, for example by adding the portion unit to the is exposed to the first reaction condition.
    In a further embodiment, the use of a portion unit according to the invention for releasing the cell or the cell organelle into the surroundings of the portion unit is disclosed. For this purpose, the carrier material of the portion unit is degraded and / or brought into a flowable state, for example by exposing the portion unit to the first reaction condition.
    In a further embodiment, a pipette tip with a cylindrical longitudinal end for introducing fluid is disclosed, wherein the cylindrical longitudinal end is so high that a predetermined amount of fluid can be introduced into the cylindrical longitudinal end. Thus, the minimum predetermined height of the cylindrical longitudinal end depends on the predetermined amount of fluid that is introduced into the pipette tip and on the diameter of the cylindrical longitudinal end.
    In a preferred embodiment, the entire pipette tip is cylindrical.
    In a preferred embodiment, the pipette tip is a disposable pipette tip.
    Examples
    example 1
    A portion unit is produced from low-melting agarose with cancer cells which are embedded in the low-melting agarose. For this purpose, cancer cells originating from a lung tumor of a cancer patient are provided as a suspension with a concentration of 2,000,000 cells / ml in an aqueous buffer solution containing 10 mM Tris, pH 7.2, 20 mM NaCl and 50 mM EDTA. The cell suspension is preheated to 45 ° C. for 5 minutes. Furthermore, 1.5 g of low-melting agarose in powder form are added to 100 ml of an aqueous buffer solution, which also contains 10 mM Tris, pH 7.2, 20 mM NaCl and 50 mM EDTA, and boiled for about 3 minutes in order to dissolve the low-melting agarose . The solution is then cooled to 45 ° C. 100 µl of the preheated cell suspension are mixed with 100 µl of the 1.5% agarose at 45 ° C. The flowable mixture is kept at a temperature of 45 ° C. 100 μl of the mixture are sucked in by a pipetting robot at a flow rate of 50 μl / s into a pipette tip with a cylindrical longitudinal end for introducing and discharging fluid. The cylindrical longitudinal end of the pipette tip is so high that the free surface of the 100 μl of the mixture facing the interior of the pipette tip does not protrude beyond the cylindrical longitudinal end of the pipette tip. The cylindrical longitudinal end of the pipette tip is immersed by the pipetting robot for 20 seconds in a pre-cooled buffer solution at a temperature of 4 ° C. This solidifies the mixture so that the portion unit is formed. The portion unit is blown out of the pipette tip by the pipetting robot into a microreaction vessel which contains 500 μl of lysis buffer for lysing the cells. The portion unit is then used to isolate high molecular weight DNA from the cancer cells that are embedded in the low-melting agarose.
    Example 2
    A portion unit is produced from low-melting agarose with cell nuclei which are embedded in the low-melting agarose. For this purpose, cell nuclei originating from leukemia cells of a leukemia patient are provided as a suspension with a concentration of 4,000,000 cell nuclei / ml in an aqueous buffer solution containing 10 mM Tris, pH 7.2, 20 mM NaCl and 50 mM EDTA. The cell nucleus suspension is preheated to 45 ° C. for 5 minutes. Furthermore, 1.5 g of low-melting agarose in powder form are added to 100 ml of an aqueous buffer solution, which also contains 10 mM Tris, pH 7.2, 20 mM NaCl and 50 mM EDTA, and boiled for about 3 minutes in order to dissolve the low-melting agarose . The solution is then cooled to 45 ° C. 100 µl of the preheated cell nucleus suspension are mixed with 100 µl of the 1.5% agarose at 45 ° C. The flowable mixture is kept at a temperature of 45 ° C. 75 μl of the mixture are sucked in by a pipetting robot at a flow rate of 50 pl / s into a pipette tip with a longitudinal end with an opening for introducing and discharging fluid. The longitudinal end of the pipette tip is in the shape of a truncated cone and the opening has an inner diameter of 2 mm. The long end of the pipette tip is immersed by the pipetting robot for 10 seconds in a pre-cooled buffer solution at a temperature of 4 ° C. This solidifies the mixture so that the portion unit is formed. The portion unit is elastic and is blown out of the pipette tip by the pipetting robot into a microreaction vessel that contains 500 μl of lysis buffer for lysing the cell nuclei. While the portion unit is being blown out of the pipette tip, the portion unit deforms in order to pass the opening of the pipette tip. After blowing out, the portion unit returns to its original shape and is used to isolate high molecular weight DNA from the cell nuclei that are embedded in the low-melting agarose.
    Example 3
    A portion unit made of gelatin methacrylate with cancer cells which are embedded in the gelatin methacrylate is produced. For this purpose, cancer cells from a breast tumor of a transgenic mouse are provided as a suspension with a concentration of 2,000,000 cells / ml. The cell suspension is precooled to 4 ° C. for 5 minutes. 100 μl of the pre-cooled cell suspension are mixed with 100 μl of a 4 ° C. solution of 5% (w / v) gelatin methacrylate in phosphate-buffered saline (PBS) at 4 ° C. The flowable mixture is kept at a temperature of 4 ° C. 150 μl of the mixture are sucked into a pipette tip with a cylindrical longitudinal end for introducing and discharging fluid. The cylindrical longitudinal end of the pipette tip is so high that the free surface of the 150 μl of the mixture facing the inside of the pipette tip does not protrude beyond the cylindrical longitudinal end of the pipette tip. The cylindrical longitudinal end of the pipette tip is incubated for 5 minutes at 37 ° C. This solidifies the mixture so that the portion unit is formed. The portion unit is blown out into a cell culture dish which contains 5 ml of cell culture medium for culturing the cells. The portion unit is then used to cultivate the cancer cells that are embedded in the gelatin methacrylate.
    Example 4
    Portion units are produced with agarose or low-melting agarose as the carrier material. The predetermined amount of the mixture that is introduced into a pipette tip with a longitudinal end with an opening for introducing and discharging fluid is in each case 100 μl. Table 1 gives the minimum size of the inner diameter of the opening of the pipette tip for different concentrations of agarose or low melting point agarose in the mixture. The concentration is given as weight per volume (w / v).
    Table 1:
    0.25 1.8 0.5 2.0 0.75 2.2 1.0 2.5 1.5 3.0 2.0 3.5 2.5 4.0 3.0 4, 5 3.5 5.0 4.0 6.0
    权利要求:
    Claims (14)
    [1]
    1. A method for producing a portion unit from a gel-like carrier material with at least one biological cell which is embedded in the carrier material or at least one cell organelle which is embedded in the carrier material, the method comprising the steps:a) providing a flowable, solidifiable mixture which comprises the carrier material and the at least one cell or the at least one cell organelle, under a first reaction condition,b) introducing a predetermined amount of the mixture into a pipette tip with a longitudinal end with an opening for introducing and discharging fluid, the opening being large enough to dispense the portion unit through the opening,c) solidifying the mixture in the pipette tip by incubating the longitudinal end of the pipette tip under a second reaction condition in which the mixture solidifies so that the portion unit is formed, andd) Removal of the portion unit from the pipette tip.
    [2]
    2. The method according to claim 1, wherein step b) is carried out by sucking the predetermined amount of the mixture into the pipette tip with negative pressure.
    [3]
    3. The method according to claim 1 or 2, wherein step b) takes place at a flow rate of 1 µl / s to 300 µl / s, preferably from 1 µl / s to 100 pl / s.
    [4]
    4. The method according to any one of claims 1 to 3, wherein the predetermined amount of the mixture is from 5 µl to 300 µl, preferably 100 µl.
    [5]
    5. The method according to any one of claims 1 to 4, wherein step d) is carried out by blowing the portion unit out of the pipette tip.
    [6]
    6. The method according to any one of claims 1 to 5, wherein the carrier material comprises agarose, preferably low-melting agarose.
    [7]
    7. The method according to any one of claims 1 to 6, wherein the at least one cell is an animal cell, preferably a mammalian cell, more preferably a human cell.
    [8]
    8. The method according to any one of claims 1 to 6, wherein the at least one cell organelle is a cell nucleus or a mitochondrion, preferably a cell nucleus.
    [9]
    9. The method according to any one of claims 1 to 8, wherein the mixture further comprises magnetic particles, which preferably do not interact with nucleic acids.
    [10]
    10. The method according to claim 9, wherein an average particle size of the magnetic particles is 10 nm to 1000 nm, preferably 50 nm to 500 nm.
    [11]
    11. Portion unit made of a gel-like carrier material with at least one biological cell which is embedded in the carrier material, or at least one cell organelle which is embedded in the carrier material, obtained by a method according to any one of claims 1 to 10.
    [12]
    12. Use of the portion unit according to claim 11 for isolating a nucleic acid.
    [13]
    13. Use of the portion unit according to claim 11 for isolating a protein.
    [14]
    14. Use of the portion unit according to claim 11 for the cultivation or cocultivation of cells.
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    同族专利:
    公开号 | 公开日
    WO2018177914A1|2018-10-04|
    DE102017106867A1|2018-10-04|
    CH714874B8|2022-01-31|
    DE102017106867B4|2021-12-02|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    GB0315991D0|2003-07-08|2003-08-13|Dakocytomation Denmark As|Standard|
    SG185988A1|2009-05-15|2012-12-28|Univ Nanyang Tech|Composition for manufacturing a scaffold for tissue engineering, and a method of making it|
    DE102010040687A1|2010-09-14|2012-03-15|Hamilton Bonaduz Ag|Process for producing active agent beads|
    US9090866B2|2012-01-31|2015-07-28|The Rogosin Institute|Method for manufacture of macrobeads|
    KR102054034B1|2012-07-24|2020-01-08|닛산 가가쿠 가부시키가이샤|Culture medium composition, and method for culturing cell or tissue using said composition|
    法律状态:
    2021-12-15| PK| Correction|Free format text: BERICHTIGUNG |
    2022-01-31| PK| Correction|Free format text: BERICHTIGUNG B8 |
    优先权:
    申请号 | 申请日 | 专利标题
    DE102017106867.8A|DE102017106867B4|2017-03-30|2017-03-30|Method for producing a portion unit|
    PCT/EP2018/057411|WO2018177914A1|2017-03-30|2018-03-23|Method for producing a portion unit|
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