RU2560567C2 - Method of incorporating target molecules into cells - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry. Disclosed is a method of incorporating target molecules into cells. The method includes fixing an array of working cells on a culture substrate in a culture medium and incorporating target molecules into the array of working cells by piercing cell membrane. The target molecules enter the working cells through pores formed in the cell membranes using an array of needles.

EFFECT: invention improves the efficiency of the method.

20 cl, 2 dwg

Description

The invention relates to biotechnology, mainly to manipulations with prokaryotic, eukaryotic and plant cells at the micro and nanoscale in order to modify the genetic apparatus by transferring biological and synthetic molecules into the cell.

A known method of introducing retroviral vectors into blastoderm cells, including the introduction of gene constructs with a syringe to a depth of 2-3 cm near the germinal disk [1].

This method has limited performance and is not applicable in microbiology due to the lack of controls for the precise movement of the syringe.

A known device and method for transferring molecules into cells using electrical energy, including the use of electrical pulses supplied to the electrodes, the amplitude of which is calculated depending on the distance between the electrodes and the nature of the tissues in such a way as to create an electric field between the electrodes, which ensures the penetration of active beginning in tissue and cells [2] .

The disadvantage of this method is the complexity of the process of introducing molecules into the cells.

There is also known a method of introducing target molecules into cells, including attaching working cells to a culture substrate in a nutrient medium, as well as introducing target molecules into working cells by piercing their cell membranes sequentially with one needle [3].

This method is selected as a prototype of the proposed solution.

The disadvantage of this method is the low productivity of the process associated with the individual operation of a single needle.

The technical result of the invention is to increase the productivity of the method.

The specified technical result consists in the fact that in the method of introducing target molecules into cells, including attaching to a culture substrate in a nutrient medium, at least one working cell, introducing target molecules into at least one working cell by puncture of the cell membrane, an array of working cells is fixed on the culture substrate with at least one needle, and the target molecules enter the working cells through the pores created in the cell membranes using the array of needles.

There are options in which culture plastic, or optically treated glass, or silicon is used as the culture substrate.

There are also variants in which a monolayer of adherent working cells is formed on the culture substrate, or the culture substrate is modified with antibodies to which the working cells are fixed, or fragments of the antibody binding layer are applied to the culture substrate, onto which antibodies specific for a particular type of cells are first fixed, and then working cells.

There are also options in which grooves or dimples are formed on the culture substrate into which the working cells are fixed.

There are also options in which working cells are fixed to the culture substrate using an external magnetic field and by means of particles of a ferromagnet captured by the working cells, or by antibodies attached to it using an external magnetic field and ferromagnetic particles that have entered into a covalent bond with antibodies.

There are also options in which an array of microchips or an array of micropipettes is used as an array of needles.

There is also an option in which the depth of penetration of the needles into the cells is carried out using height limiters.

There is also an option in which the puncture of cells is carried out under the influence of ultrasound.

There are also options in which the target molecules get into the working cells from the nutrient medium when using microchips occurs after they are extracted from the working cells or is carried out from a solution with the target molecules located in micropipettes and this occurs while they are in the working cells.

There is also an option in which the needle array is periodically cleaned and sterilized.

There are also options in which a one-coordinate ruler of working cells is used as an array of working cells, and a one-coordinate ruler of needles is used as an array of needles, or a two-coordinate matrix of working cells is used as an array of working cells, and a two-coordinate needle matrix is used as an array of needles.

There are also options in which the number of needles corresponds to or more than the number of working cells on the culture substrate, wherein the area of the array of needles is equal to the area of the array of needles of working cells.

There is also an option in which more than one puncture is made in each working cell.

There is also an option in which the number of needles is less than the number of working cells on the culture substrate.

Figure 1 shows a diagram of a puncture of an array of working cells with an array of needles.

Figure 2 shows a diagram of a puncture of an array of working cells oriented along three coordinates (X, Y, Z) with an array of needles.

Implementation of the proposed method is as follows.

An array of working cells 2 is fixed on the culture substrate 1 (Figure 1) located in a nutrient medium. After that, an array of working cells is punctured with an array of needles 3. The sizes of the working cells have a wide range of values, mainly from 200 μm in eggs to 20 μm in somatic cells. There are also working cells, the sizes of which are beyond this range. Lymphocytes have a size of 7-9 microns, monocytes - 12-20 microns. Given this factor, the non-flatness of the culture substrate (hereinafter “the substrate”) should be quite high. Using a culture plastic, such as polystyrene, polycarbonate or polyvinyl chloride, 10 × 10 mm in size as a substrate, using mechanical polishing methods, it is possible to increase the non-flatness of its surface to less than 1 μm. In this case, it is quite effective to use working cells with sizes exceeding 20 microns. If we use optically treated glass as a culture substrate, the non-flatness of which in the same areas can be less than 0.1 μm, then it can work with cells having sizes less than 20 μm. It should be noted that other materials non-toxic to living cells, such as mica or silicon, can be used as materials for the culture supports. For methods of preparing the surfaces of various materials and their cleaning, see [4, 5].

It should be noted that the bottom of the Petri dish can be used as a culture substrate if it meets the non-flatness criteria described above.

Next, the culture substrate is placed in a Petri dish with a nutrient medium. There are several options for fixing arrays of working cells on culture substrates.

In the first embodiment, adherent cells are used. The surface of the substrate is first sterilized in an autoclave or a dry oven. After that, the substrate is placed in a Petri dish, into which a nutrient medium with working cells is poured. Depending on the type of cells, their monolayer is formed on the surface of the substrate within three to four hours (average adhesion time). There are also cells (fibroplasts) that adhere to the surface of the culture substrate in 20 minutes.

In the second embodiment, monoclonal antibodies specific for a particular type of cell are immobilized on a culture substrate (the substrate is modified with antibodies). This process can take place by treating the substrate with a substance that provides covalent bonding with antibodies. The distance between the antibodies attached to the substrate is determined by the density of antibodies in the solution, the amount of this solution and the area over which antibodies are formed. For example, at an antibody concentration of 0.05 - 0.2 μg / ml, a solution amount of 50 μl and a substrate size of 10 × 10 mm, the distance between the antibodies will be in the range of 10-100 μm. There are two possible uses for antibody-modified substrates. In one embodiment, the antibody support is dried at room temperature for subsequent use; in another, it is used without drying. Next, a substrate with antibodies is placed in a Petri dish and the nutrient medium with working cells is poured. In this case, the working cells are fixed on the substrate. Working cells can be located in a dense layer, as well as at some distance from each other. This distance is determined by the distance between the antibodies that are primarily fixed on the surface of the substrate. The number of working cells introduced into the Petri dish can be 20% higher than the number of antibodies modified on the substrate. This process is described in more detail in [6].

In the third embodiment, fragments of the binding layer are applied onto the substrate using a printer in increments of, for example, two times the size of the working cells. For printer operation, see [7]. Further, antibodies are immobilized (fixed) from these antibody fragments on these fragments, and then, as in the previous paragraph, working cells are fixed on the antibodies.

In a fourth embodiment, grooves 5 are formed on a culture, for example silicon, substrate 4 (FIG. 2), having, for example, a rectangular or triangular shape. These grooves can be formed by traditional photolithographic methods [4, 5]. The width of the grooves A should correspond to the size of the cells, and the depth B should be at least 40% of these sizes. The distance between the grooves should be about the size of the cells. Then, on the bottom of the grooves 5, using a printer [7], fragments of the antibody binding layer 6 are formed, on which antibodies 7 and then the working cells are fixed 8. The binding layer 6 can cover the entire bottom of the grooves 5 in the Y coordinate, then the antibodies 7 and the working cells 8 will be located in the grooves 5 tightly to each other.

In the fifth embodiment, wells 5 are formed on the substrate 4 by photolithography methods [4, 5], the dimensions of which A in the plane of the substrate 4 correspond to the sizes of the working cells 8, and the depth B is at least 40% of these sizes. In these wells, a binding layer 6 is formed in a similar manner, on which antibodies 7 and then working cells are fixed 8. (Conventionally, the fourth and fifth options are considered in one Fig. 2, since the grooves in section are depicted in the same way as the holes.)

In the sixth embodiment, nanoparticles of a ferromagnet, such as iron oxide, with a particle size of about 20 nm, are introduced into a Petri dish containing a nutrient medium with working cells. Working cells capture these particles. On average, 20 particles per working cell are needed. A flat magnet with induction of the order of 2 T is placed under the bottom of the Petri dish. As a result, working cells containing particles of a ferromagnet are fixed on the surface of the culture substrate, for example, in the form of a monolayer. The use of ferromagnetic particles to fix the working cells is advisable if the cells are unable to adhere, but capable of capturing particles of a ferromagnet.

In the seventh embodiment, nanoparticles of a ferromagnet coated with a layer providing covalent bonding with antibodies specific for a certain type of cells are introduced into a solution with antibodies, for example, in a test tube. Next, a solution with antibodies with attached particles of a ferromagnet is applied to the culture substrate located in the Petri dish with a layer about 1 mm thick. A magnet with induction of the order of 2 T is placed under the bottom of the Petri dish. Antibodies are attached to the culture substrate. The density of the antibodies on the substrate is determined by their concentration in the solution and the amount of solution on a certain area of the substrate. After this, a nutrient medium is poured into the Petri dish with working cells that bind to antibodies and thus attach to the culture substrate. It should be noted that when using holes in the substrate in some cases, if the sizes of the holes are of the order of 100 μm, by adjusting the magnitude of the magnetic field, it can be achieved that antibodies with magnetic particles attached to them by a ferromagnet and, accordingly, cells are fixed in the holes.

After an array of working cells has been fixed on the culture substrate, their membranes are punctured with an array of needles to penetrate into the resulting holes (pores) of the target molecules. This can happen as follows.

In the first embodiment, the array of needles is made in the form of an array of microchips 9 (Fig. 2), made, for example, on a silicon module 10. This module is mounted on a holder (not shown) and, using, for example, height limiters, puncture the working cells 8 ( for example, in manual mode). As height limiters, you can use the protrusions 11, located on the periphery of the culture substrate 4, or protrusions located on the silicon module outside the zone of the array of microchips (not shown). Having the same height C, the protrusions 11 provide parallelism between the silicon module 10 and the substrate 4. The protrusions on the culture substrate 4 and the silicon module 10 can be formed by photolithography methods. In order to exclude the effect of cells squeezed by protrusions on the puncture depth with a total protrusion area of about 2 × 2 mm, the force with which the silicon module is pressed against the substrate should be about 20 G. Standard silicon modules [8] of size 5 can be used as an array of microchips [8] × 5 mm, where the microchips occupy an area of 2 × 2 mm. Specially made modules with microchips can also be used. The height D of the microchips can be 2-10 microns with angles at the apex of 8-45 degrees, and the distance between them can be, for example, 20-400 microns depending on the distance between the working cells. It should be noted that if there are arrays of needles with certain distances, then the working cells must be placed on the culture substrate in accordance with these distances. The thickness C of the height limiters 11 should be approximately 2 μm less than the total height of the needles D and cells E protruding above the surface of the substrate 4. This will ensure a puncture of the cell membranes to a depth of about 1.5-2 μm, taking into account the deformation of the cells. It should be noted that the depth of puncture for each type of cell can be selected individually, based on the size of the pores, which should be units of microns. The use of protrusions 11 is one of the options for adjusting the depth of punctures in cells 8. In other embodiments, you can use accurate, for example, interferometric, linear sensors of silicon module 10. After piercing the membranes, the spikes 9 are removed from cells 8, and the target molecules penetrate into the cells through the pores 8.

In the second embodiment, the puncture of the working cells is carried out by an array of micropipettes, and the target molecules are introduced by feeding them in solution from the reservoir through micropipettes. Micropipettes can be made using the technology described in detail in [9, 10, 11]. In this case, height limiters - protrusions should also be used. The amount of solution penetrating into each working cell can be in the range of 2-10% of its volume. Knowing the number of working cells and micropipettes, you can calculate the total volume of the solution with the target molecules, which must be introduced into the working cells. This can be solved by introducing a piston solution with a small diameter of less than 1 × 1 mm into the feed system. The amount of movement of the piston is easily translated into the volume of the solution introduced into the cell array. The technology of introducing a solution with target molecules into cells is described in detail in [12].

The process of puncture of the cells may be accompanied by the flow of ultrasound into the puncture zone. This can be done using an ultrasonic device [13].

After about every ten punctures, the needle module should be cleaned and sterilized. Purification is necessary to remove cell debris residues, which can prevent cell membranes from being punctured and enter cells. And sterilization is necessary to prevent infection of the nutrient medium. It should be noted that the optimal number of punctures before sterilization in each case is determined empirically. The needles can be cleaned in running distilled water for 5 minutes, followed by drying at room temperature. After that, it is possible to sterilize in an autoclave - 45 minutes at a temperature of 120 ° C and a pressure of 1.2 atm, or in a dry heat oven for about 2 hours at a temperature of 140-160 ° C.

There are options in which a single-coordinate ruler of working cells is used as an array of working cells, and a single-coordinate ruler of needles is used as an array of needles, or a two-coordinate matrix of working cells is used as an array of working cells, and a two-coordinate needle matrix is used as an array of needles. A single-coordinate ruler of working cells can be formed in one groove or in holes located in one line.

In that case, if the number of needles 9 (Fig. 2) is equal to the number of working cells 8, orderly fixed in the grooves or holes 5 by means of a binding layer 6 and antibodies 7, then it can be oriented

pierce each cell with each needle. The area of the array of needles may be equal to the area of the array of working cells. For this, it is necessary to orient the silicon module 10 relative to the landmarks on the culture substrate 4. This can be done, for example, by using additional grooves or holes 12 (reference marks) located at a distance equal to the length of the square silicon module 10 (for example, four grooves along each side of the module 10). Reference marks 12 can be of the same size as the main ones, if the sizes of the working cells 8 are 100-200 microns. If the grooves or holes 5 for working cells 8 have dimensions of the order of 10 μm, then it is difficult to use the same grooves or holes as reference marks and reference marks 12 must be done in the same photolithography process as the main ones, but larger. Observing in an optical microscope the symmetrical arrangement of the silicon module 10 with the needles 9, and more precisely its edges 13 relative to the reference marks 12, you can orient the module 10 in the X, Y coordinates relative to the working cells 8. In this case, the module 10 with the needles 9 can be located on the three-coordinate (X, Y, Z) micromanipulator [14] (not shown).

There is an option in which the number of needles on the module obviously exceeds the number of working cells on the substrate (for the same areas). In this case, more than one pore can be formed in each cell.

A variant is also used in which the number of needles on the module is less than the number of working cells on the culture substrate with an equal distance between the cells and needles. In this case, the puncture of the membranes can be carried out step by step sequentially throughout the entire field of the substrate with the working cells. The module with needles should be located on the micromanipulator (see, for example, [14]), and alignment will occur along the reference holes located over the entire area with the working cells.

After the target molecules are introduced into the working cells, further actions with them can occur in the same Petri dish without removing them from the substrate, for example, for the expression of recombinant proteins, for the production of human proteins in yeast or plant working cells.

In another embodiment, the working cells with the target molecules are removed from the culture substrate. After that, the working cells are transferred to another culture dish for cultivation in order to obtain proteins, as well as the use of these cells in regenerative and replacement cell therapy.

To determine the effectiveness of this method of introducing target molecules into cells, the HEP-2 human epidermoid carcinoma cell line (cell size 8-15 μm) with the potential of spontaneous adherence to plastic was used as working cells.

The aim of the experiment was to determine the effectiveness of the method of introducing target molecules into cells and the effect of the method used, in particular, the procedure for puncturing cell membranes using an array of needles, on the viability (survival) of cells.

Materials and methods:the bottom of the plastic Petri dishes with a diameter of 3 cm (JET BIOFIL, China) was used as a culture substrate. HEP-2 working cells were cultured in complete DMEM growth medium until a monolayer was formed. The viability of the working cells was determined by eliminating the erythrosine dye. As the target molecule, the plasmid pCI-neo-GFP (green fluorescent protein) in a nutrient medium solution was used, which makes it possible to evaluate the effectiveness of the method of introducing target molecules into working cells using a fluorescence microscope. The formation of pores in the membranes of the working cells was carried out by puncture of the cell membrane using an array of needles in manual mode.

Technical characteristics of the two-coordinate matrix of the needle array: matrix size - 8000 × 8000 microns; the distance between the needles in the array is 20 μm (i.e., in this experiment, the number of needles in the array is less than the number of working cells located on the culture substrate); the height of the needles is 3.0 μm. To ensure parallel supply of a matrix with an array of needles to the substrate, preservation of the cells, and control of the depth of penetration of the needle into the cell, height limiters are formed located on the periphery of the matrix of needles array with a thickness of 5 μm. Height limiters allow puncture of cell membranes to a depth of 1.5-2.0 μm, taking into account cell deformation.

As a control of the proposed method for introducing target molecules into working cells to assess the viability of working cells and the effectiveness of the method, the most commonly used method of chemical transfer of target molecules to working cells using the standard solution set CytofecteneTM Transfection Reagent Kit (BIO-RAD, USA) according to manufacturer's instructions.

Experiment description: After the monolayer was formed by an array of HEP-2 working cells on a culture substrate (plastic of Petri dishes), target molecules (plasmid pCI-neo-GFP in the amount of 10 μg per cup) were added to the nutrient medium. Next, the membranes of the working cells were punctured using a matrix with an array of needles in manual mode. Previously, in order to prevent infection of the nutrient medium with working cells, the matrices with arrays of needles were sterilized in a dry heat oven for 2 hours at a temperature of 160 ° C.

After making the punctures, the working cells were left on the culture substrate in a nutrient medium for another 1.5 hours for the penetration of target molecules from the solution into the working cells through the pores created using an array of needles. The effectiveness of the method of introducing target molecules into the working cells was evaluated through 24 hours to incorporate the target molecules of the green fluorescent protein pCI-neo-GFP into the working cells using an Axioskop 40 fluorescence microscope (C. Zeiss).

Results: Working cell viability HEP-2 in the experiment on the proposed method for introducing target molecules into cells was on average 94-100%, in the method for transferring target molecules to cells chemically using CytofecteneTM Transfection Reagent Kit, the average viability of working cells was 56-71%. The efficiency of the method of introducing target molecules into HEP-2 working cells in an experiment according to the proposed method, estimated by the inclusion of pCI-neo-GFP target molecules by working cells, averaged 82-91%, while in the method of transferring target molecules to cells using the CytofecteneTM Transfection Reagent Kit solution kit, the effectiveness of a similar indicator was significantly lower and was determined at 28% of the total number of cells.

Distinctive features, namely, that an array of working cells is fixed on the culture substrate, and the target molecules enter the working cells through the pores created in the cell membranes using an array of needles, which increases the productivity of the method. When an array of needles is used, there is no need to combine each needle with each cell and the productivity of the method also increases for this reason. Moreover, the process is simplified by minimizing manual labor when combining needles with cells and increasing its reliability by reducing the likelihood of combination errors. In addition, the individual alignment of the needle with the cell is only possible when using cells having sizes greater than 10 microns. In the proposed solution, the cell size in the direction of reduction can be almost any. Thus, the proposed method has enhanced functionality in comparison with the known.

Distinctive features are that a monolayer of adherent working cells is formed on the culture substrate, or the culture substrate is modified with antibodies to which the working cells are fixed, or fragments of the antibody binding layer are applied to the culture substrate, to which antibodies are first fixed, and then the working cells and also that they use grooves and dimples in which the working cells are fixed, and also that they use a magnetic field to fix the working cells, increase the reliability of fixing the working cells cells and their method of performance respectively.

The use of microchips and micropipettes expands the functionality of the method and its productivity by choosing its own regime for each type of cell.

The use of height limiters increases the likelihood of a guaranteed puncture of each cell when they are intact and, accordingly, the productivity of the method as a whole.

The use of ultrasound during puncture of the membranes reduces the deformation of the working cells during punctures, their safety and productivity of the method.

The use of cleaning and sterilization of needles increases the productivity of the method due to a larger number of viable working cells into which target molecules are embedded.

Various options for using the ratio of the number of needles and working cells are also aimed at increasing the productivity of the method.

Literature

1. Patent RU 2303068. 07.20.2007.

2. Application RU 2007102568. 07.27.2008.

3. Markus Montag. Preimplantation genetic diagnosis: Polar body biopsy. Note 140. March 2007.

4. Mazel E.Z. Planar technology of silicon devices. M .: Energy. 1974.384 s.

5. Stepanenko I.P. Fundamentals of Microelectronics. M .: Soviet radio. 1980.423 s.

6. Patent RU 2267787. 01.20.2005.

7. www.blek-medical.ru (Biochips - present and future).

8. Application US2006027525. 02/09/2006.

9. Application KR20070059916. 06/12/2007.

10. www.nascatec.com

11. www.ntmdt.ru

12. Herald Andrulat and Sonja Voss. Intracytoplasmic Sperm Injection (ICSI). - Procedure and Equipment. Note 009. June 2006.

13. www.elpapiezo . ru

14. www.dinamotimal.ru

Claims (20)

1. The method of introducing target molecules into cells, including attaching to a culture substrate in a nutrient medium, at least one working cell, as well as introducing target molecules into at least one working cell by puncture of the cell membrane of at least one a needle, characterized in that an array of working cells is fixed on the culture substrate, and the target molecules enter the working cells through the pores created in the cell membranes using an array of needles. 2. The method according to claim 1, characterized in that the cultural plastic is used as the culture substrate. 3. The method according to claim 1, characterized in that the culture substrate can be made of optically processed glass. 4. The method according to claim 1, characterized in that the culture substrate can be made of silicon. 5. The method according to claim 1, characterized in that fragments of a binding layer forming covalent bonds for antibodies are applied onto the culture substrate, onto which antibodies specific for a particular type of cells are first fixed, and then working cells. 6. The method according to claim 1, characterized in that grooves are formed on the culture substrate into which the working cells are fixed, while the width of the grooves corresponds to the size of the cells, the depth of the grooves is not less than 40% of the cell size, and the distance between the grooves is of the order of the cell size. 7. The method according to claim 1, characterized in that the wells are formed on the culture substrate into which the working cells are fixed, while the sizes of the holes correspond to the sizes of the cells, and the depth of the holes is not less than 40% of the sizes of the cells. 8. The method according to claim 1, characterized in that the working cells are fixed to the culture substrate using an external magnetic field and by means of particles of a ferromagnet captured by the working cells. 9. The method according to claim 1, characterized in that the working cells are fixed on the culture substrate using antibodies attached to it using an external magnetic field and ferromagnetic particles that have entered into a covalent bond with the antibodies. 10. The method according to claim 1, characterized in that as an array of needles using an array of microchips. 11. The method according to claim 1, characterized in that as an array of needles using an array of micropipettes. 12. The method according to claim 1, characterized in that the depth of penetration of the needles into the cells is carried out using height limiters. 13. The method according to claim 1, characterized in that the puncture of the cells is carried out under the influence of ultrasound. 14. The method according to claim 1, characterized in that the target molecules get into the working cells from a solution with the target molecules located in micropipettes, and this happens while they are in the working cells. 15. The method according to claim 1, characterized in that the array of needles is periodically cleaned and sterilized. 16. The method according to claim 1, characterized in that a two-coordinate matrix of working cells is used as an array of working cells, and a two-coordinate matrix of needles is used as an array of needles. 17. The method according to claim 1, characterized in that the number of needles corresponds to the number of working cells on the culture substrate, while the area of the array of needles is equal to the area of the array of working cells. 18. The method according to claim 1, characterized in that the number of needles is greater than the number of working cells on the culture substrate, while the area of the array of needles is equal to the area of the array of working cells. 19. The method according to p. 18, characterized in that in each working cell do more than one puncture. 20. The method according to claim 1, characterized in that the number of needles is less than the number of working cells located on the culture substrate.

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