WO2018177914A1

WO2018177914A1 - Method for producing a portion unit

Info

Publication number: WO2018177914A1
Application number: PCT/EP2018/057411
Authority: WO
Inventors: Jean Marc JOSSE; Martin Gajewski; Mario Arangio; Eduard WOIZENKO
Original assignee: Hamilton Bonaduz Ag
Priority date: 2017-03-30
Filing date: 2018-03-23
Publication date: 2018-10-04
Also published as: DE102017106867B4; CH714874B1; CH714874B8; DE102017106867A1

Abstract

A description is given of 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. Also described is a portion unit that can be obtained by the method. Furthermore, various uses of the portion unit are described.

Description

Process for producing a portion unit

Field of the invention

The invention relates to a method for producing a portion unit from a gelatinous carrier material having 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 invention further relates to a portion unit obtainable by the method. The invention further relates to various uses of the portion unit. Background of the invention

In the development of cancer often major structural changes of the chromosomes, so-called chromosome or DNA rearrangements play a crucial role. Examples of DNA rearrangements include duplications, translocations, inversions and deletions of certain chromosome segments as well as DNA strand breaks and subsequent fusion of the DNA strands in a different arrangement. The identification of DNA rearrangements is used to diagnose cancer and may serve as the basis for personalized cancer therapy. To identify DNA rearrangements, a method known as "molecular combing" is used, which requires intact, long DNA fragments that are combed onto a glass surface. By combing the DNA fragments are aligned and stretched. As a result, it is then 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 which "molecular combing" requires DNA fragments with a length of more than 1000 kilobase pairs. Such DNA fragments are called high mo- Lekulare DNA referred to and gain for other molecular biology techniques such as Next Generation Sequencing increasingly important. In order to isolate high molecular weight DNA from cells or nuclei, the DNA must be protected from shear forces during its isolation. Shearing forces can cause the DNA to break up into small fragments that would render image analysis impossible for the detection of DNA rearrangements. For protection against shear forces, the DNA is immobilized from cells during isolation, for example in agarose. The cells are manually embedded in Aga rose. For this purpose, 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 starting to isolate the high molecular weight DNA. The solidified mixture may be referred to as a serving unit.

The use of "molecular combing" in cancer diagnostic procedures requires standardization of all process steps and high precision to prevent false results or misinterpretations.Manually producing the portion units can not consistently meet these requirements because it has poor reproducibility On the other hand, the serving units are very fragile, so even between the serving units, even if they differ from one-on-one and one-to-one Thus, it is difficult to manually prepare standardized unit portions for a variety of samples, and thus there are differences in the amount and quality of high molecular weight DNA that are released from the cells in the portio units is isolated. 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 gelatinous carrier material having 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:

a) Providing a flowable, solidifiable mixture that the Trägerermateria! and the at least one cell or the at least one cell organelle comprises, in a first reaction condition,

b) introducing a predetermined amount of the mixture into a pipette tip having a longitudinal end with an opening for introducing and dispensing fluid, the opening being large enough to expose the portion unit through the opening;

c) solidifying the mixture in the pipette tip by incubating the longitudinal end of the pipette tip in a second reaction condition where the mixture solidifies to form the portion unit; and d) dispensing the portion unit from the pipette tip.

The present invention further relates to a portion unit of a gelatinous carrier material having 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.

The present invention further relates to the use of a portion unit according to the invention for isolating a nucleic acid.

The present invention further relates to the use of a portion unit according to the invention for the isolation of a protein.

The present invention further relates to the use of a portioning unit according to the invention for culturing or cocultivation of cells. Detailed description of the invention

In a first aspect, the invention relates to a method for producing a portion unit from a gelatinous carrier material having 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:

a) providing a flowable, solidifiable mixture comprising the support material and the at least one cell or the at least one cell organelle, in a first reaction condition,

By means of the method according to the invention, a portion unit is produced from a gel-like carrier material having at least one biological cell embedded in the carrier material or at least one cell organelle embedded in the carrier material. The term "serving unit" as used herein refers to 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 cell organelle being enclosed by the carrier material, since the carrier material of the portion unit is solidified, Due to the gelatinous carrier material, the portion unit is elastic, which means that the portion unit can change its shape under the action of force and return to its original shape when the acting force is eliminated. The term "biological cell" as used herein refers to 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 containing the cytoplasm and The term "cell organelle" as used herein refers to a component of a cell surrounded by a single or a double membrane. The cell organelles include, for example, 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 in a first reaction condition. Suitable carrier material is any gel-like material which, depending on a reaction condition, is present as a flowable, solidifiable mass or in solidified form. In a preferred embodiment, the carrier material is Aga rose. 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 comprising the support material and the at least one cell or the at least one cell organelle, the properties of the support material are retained, so that the mixture is also flowable and solidifiable.

As used herein, the term "reaction condition" refers to at least one parameter or combination of parameters that performs at least one of the process steps. The parameters preferably include a temperature, a salt concentration, an ionic strength, a pH, and / or a Addition of Reagents The choice of reaction condition depends in particular on which support material is used in the process, the reaction condition being in particular a temperature.

The first reaction condition is the reaction condition in which the flowable, solidifiable mixture containing the support material and the at least one cell or which comprises at least one cell organelle. 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 so that the carrier material is present in a flowable, solidifiable state. The first reaction condition is therefore chosen as a function of the gelling temperature of the carrier material used. The gelling temperature is the temperature at which the carrier material solidifies into a gel or 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 having a longitudinal end with an opening for introducing and discharging fluid. A pipette tip has two opposite longitudinal ends, each with an opening, one of which has a longitudinal end for introducing and dispensing 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 dispensing fluid typically has the shape of a truncated cone whose diameter 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 expose the portion unit through the opening from the pipette tip. Due to the gelatinous carrier material, the portion unit is elastic. Due to the elasticity, the portion unit can be temporarily deformed. Thus, the portioning unit may be deformed from the pipette tip during dispensing and returned to its original shape after dispensing. The dispensing of the portion unit from the pipette tip thus takes place while retaining the original shape of the portion unit. Transient deformation of the serving unit during dispensing from the pipette tip occurs, for example, when the longitudinal end of the pipette tip for introducing and dispensing fluid has the shape of a truncated cone. In this case, the portion unit is also frusto-conical and the portion of the serving unit facing the interior of the pipette tip must deform when dispensed 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 caused for example by exerting a pressure on the portion unit in the direction of the pipette tip opening. The more elastic the portion unit is, the less pressure is required for dispensing the portion unit from the pipette tip for the same size of opening for introducing and dispensing fluid. The pressure is preferably distributed uniformly over the surface of the portion unit facing the interior of the pipette tip.

The opening of the pipette tip for introducing and dispensing fluid must therefore have a certain minimum size so that the portion unit can be dispensed through the opening from the pipette tip. This minimum size depends on the elasticity and the size of the portion unit. For example, the more elastic the portion unit, the smaller the opening of the pipette tip for introducing and dispensing fluid. 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 dislodge the portion unit undamaged through the opening. The serving unit should not be damaged during dispensing from the pipette tip so that the serving unit returns to its original shape after dispensing. If the portion unit returns to its original shape after being dispensed from the pipette tip, then it can be assumed that the cell or the cell organelle and / or its constituents, in particular DNA, during the dispense gens the portion unit from the pipette tip have been protected against shear forces.

In a preferred embodiment, the opening of the pipette tip for introducing and dispensing fluid has an inner diameter of 2 mm to 5 mm, preferably 3 mm to 4 mm.

To avoid contamination of the serving unit, the pipette tip is preferably a disposable pipette tip.

The introduction of the predetermined amount of the mixture into the pipette tip can take place 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 into a vessel containing the mixture , Upon immersion of the pipette tip into the mixture in the vessel, the mixture is introduced by capillary forces into the pipette tip. In this case, the amount of the introduced mixture 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 inventive method further comprises solidifying the mixture in the pipette tip so that the portion unit is formed. This process step is accomplished by incubating the longitudinal end of the pipette tip at a second reaction condition where the mixture solidifies. For this purpose, the second reaction condition is chosen such that the carrier material solidifies. The second reaction condition is therefore selected depending on 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, which has been 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 forms in the pipette tip. The portion unit is formed by solidifying the predetermined amount of the mixture.

The method according to the invention further comprises the dispensing of the portioning unit from the pipette tip. The dispensing of the portion unit can be done in various ways, for example by blowing or knocking out the portion unit from the pipette tip or by destroying the pipette tip. The dispensing of the portion unit from the pipette tip is carried out while maintaining the original shape of the portion unit. It is possible that the portion unit deforms during dispensing from the pipette tip and returns to its original shape after deployment. Alternatively, the portioning unit is not deformed during dispensing from the pipette tip.

The solidified carrier material protects the cell embedded therein or the cell organelle embedded therein and its constituents, in particular DNA, from shear forces.

An 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 forms in the target vessel. However, the portioning unit then adheres to the bottom and walls of the target vessel such that the reagent-accessible surface area of the portioning unit is reduced and insufficient for certain portion unit uses. In contrast to the portion unit is formed in the inventive method in the pipette tip and then discharged from the pipette tip. This makes the portion unit accessible from all sides for reagents. As a result, reaction and / or washing steps can be carried out more efficiently during 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 automatically can. In a preferred embodiment, the portion unit is therefore produced automatically. Due to the automation capability, 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.

Automated execution of the method allows parallel production of a plurality of portion units. As a result, the method according to the invention can also be used in high-throughput applications. For example, the mixture may be provided in a microtiter plate with 96 or 384 wells (wells). Automated execution of the method not only enables the parallelization of the method, which saves time, but also extensive control and monitoring of the individual method steps. This enables a 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 check 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 the controlled and reproducible introduction of the predetermined amount of the mixture into the pipette tip, in contrast to the manual performance of the method steps, a reproducible size of the portion unit can be ensured. The reproducible size of the portion unit is of particular importance in molecular biological investigations, since the test results can often be correctly interpreted only under this condition. Furthermore, the speed at which the introduction of the predetermined amount of the mixture into the pipette tip takes place can be predetermined. As a result, the predetermined amount of the mixture can be introduced in a controlled manner in the pipette tip. Furthermore, this can prevent any penetration of air bubbles into the mixed during step b) are avoided. The correct and complete dispensing of the portion unit from the pipette tip can also be verified by a pipetting robot. These technical possibilities ensure the correct and reproducible production of the portion unit, 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 in the first reaction condition to produce the mixture. Preferably, the cell or the cell organelle is already in the first reaction condition prior to mixing into the carrier material in order to avoid solidification of the carrier material upon addition of the cell or the cell organelle. For example, the cell or cell organelle may be preheated or pre-cooled to the same temperature as the flowable carrier material.

For example, a suspension of cells or cell organelles is mixed with the carrier material in a ratio of 1: 1 (v / v). For example, 50 .mu.l of a cell suspension are mixed with 50 .mu.l of a solution of 2% agarose as carrier material. The proportion of carrier material in the mixture can be increased in order to obtain firmer and thus mechanically more stable portion units. Alternatively, the concentration of the carrier material may be increased to produce more solid portion units.

In a preferred embodiment, step b) is carried out by sucking the predetermined amount of the mixture into the pipette tip with negative pressure. For this purpose, for example, a conventional microliter or Kolbenhubpipette, on which the pipette tip is attached, are used. In this case, a moving piston in the upward movement pulls the column of air under it upwards and thereby also the mixture into the attached pipette tip. In a preferred embodiment, step b) takes place at a flow rate of 1 μΙ / s to 300 μΙ / s, preferably from 1 μΙ / s to 100 μΙ / 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 dispensing 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 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 μΙ to about 300 μΙ, more preferably about 10 μΙ to about 250 μΙ, more preferably about 50 μΙ to about 200 μΙ, most preferably about 75 μΙ to about 150 μΙ. 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 serving unit. In a particularly preferred embodiment, the predetermined amount of the mixture is about 100 μΙ.

In a preferred embodiment, incubating the longitudinal end of the pipette tip in the second reaction condition is accomplished by inserting the longitudinal end of the pipette tip into a solution having the temperature of the second reaction condition. For example, the longitudinal end of the pipette tip may be immersed in a solution having a temperature of about 4 ° C. In a preferred embodiment, step d) is carried out by blowing out the portion unit from the pipette tip. For this purpose, for example, a conventional microliter or Kolbenhubpipette, on which the pipette tip is attached, are used. In this case displaces a movable piston when pressed down the underlying column of air, so that the portion unit is blown out of the pipette tip. In a preferred embodiment, the portion unit is dispensed into a target vessel with a reaction solution or onto a solid surface. This depends above all on the intended further use of the portion unit. If the portion unit is discharged into a destination vessel with a reaction solution, the reaction solution is selected as a function of the further use of the portion unit. The reaction solution can be, for example, 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 is cylindrical for introducing and dispensing fluid, the cylindrical longitudinal end having a predetermined height such 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 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 the 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. Prior to further use, the dissolved agarose is usually cooled to about 50 ° C to about 60 ° C or allowed to cool. The gelling temperature of agarose, at which the agarose solidifies into a gel, ranges from about 34 ° C to about 42 ° C with 1.5% agarose, depending on the source from which the agarose is derived. In the solidified state, agarose is present as a gel having pores. The pores allow access of reagents to the cell embedded in the serving unit or to the cell organelle embedded in the serving unit. This is useful for further use of the serving unit. Agarose is present at a temperature of about 50 ° C to about 60 ° C as a flowable, solidifiable mass. Therefore, when using agarose as the 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 present in solidified form. Thus, when Aga rose is used as the support 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 about 4 ° C. A temperature of about 0 ° C to about 10 ° C is preferred since solidification of agarose at a temperature of about 20 ° C to about 30 ° C takes longer and is more uncontrolled compared to lower temperatures. At 4 ° C, just a few seconds are sufficient for the solidification of Aga rose and thus the mixture 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 higher the agarose is concentrated, the smaller are the pores that the solidified Aga rose has in the serving unit and the firmer the serving unit. The concentration of the agarose is therefore chosen depending on the intended further use of the portion unit. If 0.5% agarose is used as carrier material, the portioning unit is very soft. At higher agarose concentrations, such as from 0.75% to 1%, the portion unit is already firmer and thus more mechanically stable, thereby facilitating handling of the portion unit.

In a particularly preferred embodiment, the carrier material comprises 1, 5% agarose. With 1.5% agarose, the portion unit is mechanically sufficiently stable so that the portion unit is easy to handle, while the pore size is easy to handle. large of the Aga rose allows a variety of other 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 a lower gelling temperature compared to standard agarose. The gelling temperature of low melting Aga rose ranges from about 24 ° C to about 30 ° C at 1.5% Aga rose. Thus, low-melting agarose is still present at a temperature of about 40 ° C to about 50 ° C as a flowable, compactable mass. 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 because temperatures above 45 ° C can damage the cell or cell organelle in the mixture or damage the pipette tip.

Another advantage is that low-melting agarose remains liquid even at temperatures of up to 15 ° C below 45 ° C. This is important for process safety because it prevents premature solidification of the mixture, for example, by 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, so that the pipette tip is typically at room temperature. At a temperature of about 0 ° C to about 20 ° C is low melting agarose in solidified form. Thus, when using low melting Aga rose as the support material, the second reaction condition preferably comprises a temperature of from about 0 ° C to about 20 ° C, more preferably from about 0 ° C to about 10 ° C, most preferably about 4 ° C. At 4 ° C, just a few seconds are sufficient for the solidification of low-melting Aga rose and thus the mixture in the pipette tip. In contrast to Aga rose, which is at higher temperatures than flowable mass and at lower temperatures in solidified state, there are also support materials that are at lower temperatures than flowable mass and form a gel at higher temperatures. An example of such a carrier material is gelatin. Gelatine is for example at 4 ° C as a flowable mass. The gelatin temperature of gelatin ranges from about 35 ° C to about 40 ° C, with solidified gelatin becoming fluid again upon 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 from a cancer patient. The portion unit produced by the method according to the invention is particularly suitable for the isolation of high molecular weight DNA from the cell, which is embedded in the carrier material. The high molecular weight DNA is needed for the identification of DNA rearrangements for the diagnosis of cancer. In addition, identification of the specific DNA rearrangements present in a cancer patient is an essential prerequisite for personalized cancer therapy.

In a preferred embodiment, the process is carried out under sterile conditions. This may 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 organogel in the mixture whose concentration in the mixture is preferably wa is 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, an itochondrium, a vesicle, an endoplasmic reticulum, a Golgi apparatus, a lysosome, a peroxisome, a chloroplast, a chromoplast, a leucoplast, a cell sac vacuole, a melanosome, or a phagosome ,

In a particularly preferred embodiment, the at least one cell organ part is a cell nucleus or a mitochondrium, preferably a cell nucleus. The portion unit produced by the method according to the invention is particularly suitable for the isolation of high molecular weight DNA. DNA is found in cell nuclei and in mitochondria, in plants also in chloroplasts. For most applications, the chromosomal DNA present in the cell nuclei is of interest, for example for the identification of DNA rearrangements in cancer patients.

In a preferred embodiment, the mixture further comprises magnetic particles. As a result, a portion unit is produced in which magnetic particles are present. The presence of magnetic particles is advantageous in the further use of the portion unit. Due to the magnetic particles, it is possible to hold the portion unit by a magnetic field at a certain position. For this purpose, for example, a magnet can be attached to the side or below a reaction vessel. The portion unit then moves to the side of the reaction vessel facing the magnet. By knowing the position at which the portion unit is located, damage to or destruction of the portion unit during the movement of liquids, for example during washing of the portion unit, is avoided. Thus, in particular, liquids can be safely sucked out of the reaction vessel without damaging the portioning unit. In a preferred embodiment, the magnetic particles do not interact with nucleic acids. Interaction of the magnetic particles with nucleic acids could interfere with the isolation of nucleic acids such as DNA from the cell or cell organelle in the serving 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 aspect, the invention relates to a portion unit of a gelatinous carrier material having at least one biological cell which is incorporated into the carrier material embedded, or at least one Zellorganell embedded in the carrier material, obtainable by a method according to the invention. In a third aspect, the invention relates to the use of a portion unit according to the invention for isolating a nucleic acid. The nucleic acid is protected against 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 may 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 herein refers to DNA fragments longer than 1000 kilobase pairs in length.The DNA is immobilized in the portion unit during isolation and shear protected, thereby making the portion unit particularly useful for isolating high molecular weight DNA DNA is required for a variety of molecular biology procedures, in particular for so-called "molecular combing", which is used to identify DNA rearrangements in the diagnosis of cancer.

To isolate high molecular weight DNA from the at least one cell embedded in the carrier material, the portion unit can be dispensed, for example, into a destination vessel. The target vessel preferably contains a buffer solution. In a next step, the cell is digested or lysed. The lysis of Cell takes place within the serving 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 constant, predetermined temperature. The temperature is for example 37 ° C. The lysis step is performed, for example, overnight, that is, for about 16 hours.

Washing of the serving unit follows to remove the lysed cell material, cell debris, enzymes and other contaminants that might interfere with further use of the DNA from the serving unit. When washing the portion unit, carefully mix and move the portion unit with the wash solution to ensure efficient washing of the portion unit without damaging or destroying the portion unit. 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. For example, to move the portioning unit in the washing solution, the target vessel may be gently turned upside down and back up again one or more times. Subsequently, the washing solution is removed. The washing of the portion unit can be repeated several times, for example, repeated 5 to 50 times. Preferably, the washing of the portion unit is repeated 20 to 50 times, more preferably 50 times.

After washing, the carrier material of the portion unit is removed. For this purpose, for example, the carrier material can be brought back into a flowable state and / or degraded. This step is often temperature-dependent and is therefore preferably carried out at a constant, predetermined temperature. The degradation is preferably achieved by enzymatic digestion of the support material. Agarose can be degraded, for example, by incubation with the enzyme beta-agarase, preferably at 42 ° C. The isolated high molecular weight DNA is now available for further use. All steps of the 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 aspect, the invention relates to the use of a portion unit according to the invention for isolating a protein, preferably a protein having 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 not only provides nucleic acids, but also proteins protection against shear forces. As a result, the portion unit is also suitable for the isolation of proteins and protein complexes.

In a further aspect, the invention relates to the use of a portioning unit according to the invention for the cultivation or cocultivation of cells. Cocultivation of cells co-cultivates at least two different types of cells, for example, cancer cells and fibroblasts.

Due to the three-dimensional environment of the cells in the portion unit, the portion unit is particularly suitable for the three-dimensional cultivation or cocultivation of cells. For this use, the support material is chosen so that it is in a solidified state under the reaction condition in which the cells are to be cultured or cocultured. Human cells and other mammalian cells are typically cultured at 37 ° C (body temperature) such that the support material is preferably in a solidified state at 37 ° C. The support material is preferably at lower temperatures, for example at about 4 ° C, as a flowable mass. Suitable support materials for three-dimensional cultivation or cocultivation of cells are known. For example, hydrogels of structural proteins such as collagen or gelatin-methacrylate can be used as the carrier material. The carrier material is also referred to in this context as a matrix and is preferably similar in composition to the naturally occurring, complex extracellular environment of cells in tissues. In a preferred embodiment, the cells cultured or co-cultured in the serving unit release a compound which can diffuse through the carrier material and thus escape from the serving unit. For example, the cells can secrete proteins that inhibit the growth and / or proliferation of cancer cells. In this case, the unit dose can be used to treat cancer.

In another aspect, the use of a serving unit according to the invention for releasing a compound into the environment of the serving unit is disclosed. The portion unit is preferably surrounded by a fluid, for example a medium, into which the compound is released from the portion unit. For example, as described above, the compound is released from the cell embedded in the substrate and diffuses outward through the substrate. In another embodiment, lysis of the cell embedded in the support material or of the cell organelle embedded in the support material is performed. During lysis, the membrane surrounding the cell or cell organelle is dissolved so that the contents of the cell or cell organelle are released in the carrier material and diffused 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 portion unit can be controlled. This allows a controlled release of the compound into the environment of the portion unit, for example a particularly slow release.

For rapid release of the contents of the cell or of the cell organelle into the surroundings of the portion unit, the carrier material of the portion unit can be degraded during or after the lysis of the cell or of the cell organelle and / or brought into a flowable state, for example by exposing the portion unit to the first reaction condition becomes. In another aspect, the use of a serving unit according to the invention for releasing the cell or cell organelle into the environment of the serving 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 the portion unit of the first reaction condition is suspended.

In another aspect, there is disclosed a pipette tip having a cylindrical longitudinal end for introducing fluid, 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 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 low melting Aga rose serving unit with cancer cells embedded in the low melting agarose is prepared. For this purpose, cancer cells derived from a lung tumor of a cancer patient are provided as a suspension at 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. Further, 1.5 g low-melting agarose in powder form is added to 100 ml of an aqueous buffer solution also containing 10 mM Tris, pH 7.2, 20 mM NaCl and 50 mM EDTA and boiled for about 3 minutes to obtain the low-melting agarose to solve. The solution is then cooled to 45 ° C. 100 μΙ of the prewarmed cell suspension are mixed with 100 μΙ of 1, 5% agarose at 45 ° C. The flowable mixture is maintained at a temperature of 45 ° C. 100 μΙ of the mixture are sucked by a pipetting robot with a flow rate of 50 μΙ / 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 μΙ 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 submerged by the pipetting robot for 20 seconds in a pre-cooled buffer solution having a temperature of 4 ° C. As a result, the mixture is solidified, so that the portion unit forms. The portioning unit is blown out of the pipette tip by the pipetting robot into a microreaction vessel containing 500 .mu.l lysis buffer for lysing the cells. Subsequently, the unit portion is used to isolate high molecular weight DNA from the cancer cells embedded in the low melting point agarose. Example 2

A low melting Aga rose serving unit with cell nuclei embedded in the low melting agarose is prepared. For this purpose, nuclei derived from leukemia cells of a leukemia patient are provided as a suspension at 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 nuclear suspension is preheated to 45 ° C for 5 minutes. Further, 1.5 g of low-melting agarose in powder form is placed in 100 ml of an aqueous buffer solution also containing 10 mM Tris, pH 7.2, 20 mM NaCl and 50 mM EDTA and boiled for about 3 minutes to dissolve the low-melting agarose , The solution is then cooled to 45 ° C. 00 μΙ of the preheated nuclear suspension are mixed with 100 μΙ of 1, 5% agarose at 45 ° C. The flowable mixture is maintained at a temperature of 45 ° C. 75 μΙ of the mixture are sucked by a pipetting robot with a flow rate of 50 μΙ / s into a pipette tip with a longitudinal end with an opening for introducing and discharging fluid. The longitudinal end of the pipette tip has the shape of a truncated cone and the opening has an inner diameter of 2 mm. The longitudinal end of the pipette tip is separated from the pipette animal robot for 10 seconds in a pre-cooled buffer solution, which has a temperature of 4 ° C immersed. As a result, the mixture is solidified, so that the portion unit forms. The portioning unit is elastic and is blown out of the pipette tip of the pipetting robot into a microreaction vessel containing 500 μ l lysis buffer for lysing the cell nuclei. As the portioning unit is blown out of the pipette tip, the portioning unit deforms to pass the opening of the pipette tip. After purging, the unit portion returns to its original shape and is used to isolate high molecular weight DNA from the cell nuclei embedded in the low melting agarose.

Example 3

A portion unit of gelatin-methacrylate with cancer cells embedded in the gelatin-methacrylate is prepared. For this purpose, cancer cells derived from a breast tumor of a transgenic mouse are provided as a suspension at a concentration of 2,000,000 cells / ml. The cell suspension is pre-cooled to 4 ° C for 5 minutes. 100 μΙ of the precooled cell suspension are mixed with 100 μΙ 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 μΙ of the mixture is 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 μΙ 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 incubated for 5 minutes at 37 ° C. As a result, the mixture is solidified, so that the portion unit forms. The portion unit is blown into a cell culture dish containing 5 ml of cell culture medium for culturing the cells. Subsequently, the serving unit is used to cultivate the cancer cells embedded in the gelatin-methacrylate. Portion units are prepared with agarose or low-melting agarose as carrier material. The predetermined amount of the mixture, which is introduced into a pipette tip having a longitudinal end with an opening for introducing and discharging fluid, is in each case 100 .mu.Ι. Table 1 gives the minimum size of the inside diameter of the opening of the pipette tip for different concentrations of Aga rose or low melting Aga rose in the mixture. The concentration is given as weight per volume (w / v).

Table 1 :

Minimum size of the inside diameter

Concentration of agarose or

the opening of the pipette tip to the low melting agarose in

Introduction and application of fluid to the mixture (w / v)

(Mm)

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

Claims

claims

Anspruch [en] A method for producing a portion unit from a gelatinous carrier material having 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:

a) providing a flowable, solidifiable mixture comprising the support material and the at least one cell or the at least one cell organelle in a first reaction condition; b) introducing a predetermined amount of the mixture into a pipette tip having a longitudinal end with an opening for insertion and removal fluid, wherein the opening is large enough to expose the portion unit through the opening,

c) solidifying the mixture in the pipette tip by incubating the longitudinal end of the pipette tip in a second reaction condition in which the mixture solidifies to form the portion unit, and

d) discharging the portion unit from the pipette tip.

2. The method of claim 1, wherein step b) is carried out by sucking the predetermined amount of the mixture into the pipette tip with negative pressure.

3. The method according to claim 1 or 2, wherein step b) takes place at a flow rate of 1 μΙ / s to 300 μΙ / s, preferably from 1 μΙ / s to 100 μΙ / s.

4. The method according to any one of claims 1 to 3, wherein the predetermined amount of the mixture is about 5 μΙ to about 300 μΙ, preferably about 100 μΙ.

5. The method according to any one of claims 1 to 4, wherein step d) is carried out by blowing the portion unit from the pipette tip.

6. The method according to any one of claims 1 to 5, wherein the carrier material comprises agarose, preferably low-melting agarose.

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. The method according to any one of claims 1 to 6, wherein the at least one cell organelle is a cell nucleus or an itochondrium, preferably a cell nucleus.

The method of any one of claims 1 to 8, wherein the mixture further comprises magnetic particles which preferably do not interact with nucleic acids.

A method according to claim 9, wherein an average particle size of the magnetic particles is about 10 nm to about 1000 nm, preferably about 50 nm to about 500 nm.

11. Portion unit of a gelatinous 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 one of claims 1 to 10.

12. Use of the portion unit according to claim 11 for isolating a nucleic acid.

13. Use of the portion unit according to claim 11 for the isolation of a protein.

14. Use of the portion unit according to claim 11 for the cultivation or cocultivation of cells.