CN219670526U - Single cell capturing culture dish - Google Patents

Abstract

The utility model provides a single-cell capturing culture dish which comprises a dish body, wherein a cell sample adding groove and a single-cell capturing groove are arranged in the dish body, the bottom surface of the single-cell capturing groove is a concave surface, and a single-cell capturing blind hole for accommodating single cells is formed in the lowest position of the bottom surface of the single-cell capturing groove. The single-cell capturing culture dish is simple in structure and convenient to operate, can effectively avoid the conditions of mixing a plurality of cells, losing single cells, being polluted and the like, and can remarkably improve the efficiency of manual single-cell capturing.

Description

Single cell capturing culture dish

Technical Field

The utility model relates to the technical field of medical equipment, in particular to a single-cell capturing culture dish.

Background

Single cell sequencing technology is a technology that performs sequencing analysis at the single cell level on the genome, transcriptome, and epigenomic levels. Traditional sequencing is performed on a multicellular basis, and in fact, the average of signals in a large number of cells is obtained, losing information on cell heterogeneity (cell-to-cell differences). The single-cell sequencing technology can detect heterogeneous information which cannot be obtained by sequencing of a mixed sample, so that the problem is well solved. Single cell sequencing technology has been continuously developed for the last decade since the first time in 2009. In particular, single cell sequencing has emerged in recent years as a trigger for development and popularity. In 2013, journal of Science has sequenced single cells as one of six areas of greatest annual concern.

Single cell sequencing first requires the acquisition of single cell samples, i.e., single cell capture techniques. Currently common single cell capture techniques include: limiting dilution method, flow sorting method, laser cutting method, micromanipulation method and microfluidic chip method. These methods each have their advantages and disadvantages, but generally rely on relatively expensive equipment and kits.

In single cell sequencing studies of human gametes (sperm or oocytes) and early embryos, often only tens to hundreds of single cell samples can be obtained at a time, as the samples are at a premium. High throughput single cell capture techniques require a higher initial cell mass (at least 10 ⁴ Individual cells) can only be captured by manual sorting. The manual sorting has high requirements for operators, and the operation process is finished in a common culture dish, so that a plurality of cells are mixed, and single cells are lost or polluted easily.

Thus, there is a strong need for a simple, efficient and inexpensive single cell capture culture dish to assist operators in efficiently and manually capturing single cell samples in the study of human gametes and early embryo.

Disclosure of Invention

Based on this, it is necessary to provide a single cell capturing culture dish which has a simple structure, is convenient to operate, and can improve the efficiency of manual sorting capture.

The utility model provides a single-cell capturing culture dish which comprises a dish body, wherein a cell sample adding groove and a single-cell capturing groove are arranged in the dish body, the bottom surface of the single-cell capturing groove is a concave surface, and a single-cell capturing blind hole for accommodating single cells is formed in the lowest position of the bottom surface of the single-cell capturing groove.

In any embodiment, the inner diameter of the single cell capturing blind hole is 1.5 to 5 times of the diameter of the single cell to be captured.

In any embodiment, the depth of the single cell capturing blind hole is 1.5-5 times of the diameter of the single cell to be captured.

In any embodiment, the bottom of the single cell capturing groove is cambered or conical.

In any embodiment, the upper edge of the single-cell capture well is flush with the inner bottom surface of the dish in the region other than the single-cell capture well.

In any embodiment, the number of single cell trapping vessels in the vessel is a plurality.

In any embodiment, the upper edge of the side wall of the cell loading well is higher than the inner bottom surface of the dish body and lower than the upper edge of the outer side wall of the dish body.

In any embodiment, a cell washing tank is further arranged in the dish body, and the cell washing tank is positioned between the cell loading tank and the single cell capturing tank.

In any embodiment, the dish body, the cell loading well, the single cell capturing blind hole and the cell washing well are in an integrated structure.

In any embodiment, the dish further comprises a dish cover, wherein the dish cover can be covered on the dish body.

According to the single-cell capturing culture dish, a cell sample adding groove and a single-cell capturing groove are formed in the culture dish body, the bottom surface of the single-cell capturing groove is a concave surface, and a single-cell capturing blind hole is formed in the lowest position of the bottom surface of the single-cell capturing groove. When in use, the culture medium containing the cells to be captured is added into a cell sample adding tank, single cells are selected and put into a single cell capturing tank after washing; because the bottom surface of the single cell capturing groove is concave and is provided with a single cell capturing blind hole, single cells can sink into the single cell capturing blind hole; single cells can be transferred from single cell capture blind holes into PCR tubes for amplification and sequencing using a pipette. The single-cell capturing culture dish is simple in structure and convenient to operate, can effectively avoid the conditions of mixing a plurality of cells, losing single cells, being polluted and the like, and can remarkably improve the efficiency of manual single-cell sorting and capturing.

Drawings

FIG. 1 is a schematic top view of a single cell capture culture dish according to an embodiment of the utility model;

fig. 2 is a cross-sectional view taken along the A-A plane in fig. 1.

Reference numerals illustrate:

1. single cell capture culture dish; 11. a dish body; 12. a cell loading groove; 13. a single cell capture tank; 14. single cell capturing blind hole; 15. a cell washing tank; 16. and (5) a dish cover.

Detailed Description

The following detailed description of the present utility model will provide further details in order to make the above-mentioned objects, features and advantages of the present utility model more comprehensible. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present utility model. The present utility model may be embodied in many other forms than described herein and similarly modified by those skilled in the art without departing from the spirit of the utility model, whereby the utility model is not limited to the specific embodiments disclosed below.

Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature. In the description of the present utility model, the meaning of "plurality" means at least two, for example, two, three, etc., unless specifically defined otherwise.

In the present utility model, unless explicitly specified and limited otherwise, the terms "mounted," "connected," "secured," and the like are to be construed broadly, and may be, for example, fixedly connected, detachably connected, or integrally formed; can be mechanically or electrically connected; either directly or indirectly, through intermediaries, or both, may be in communication with each other or in interaction with each other, unless expressly defined otherwise. The specific meaning of the above terms in the present utility model can be understood by those of ordinary skill in the art according to the specific circumstances.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this utility model belongs. The terminology used herein in the description of the utility model is for the purpose of describing particular embodiments only and is not intended to be limiting of the utility model. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.

Referring to fig. 1 and 2, an embodiment of the present utility model provides a single-cell capturing culture dish 1, where the single-cell capturing culture dish 1 includes a dish body 11, a cell sample adding groove 12 and a single-cell capturing groove 13 are disposed in the dish body 11, the bottom surface of the single-cell capturing groove 13 is a concave surface, and a single-cell capturing blind hole 14 for accommodating single cells is disposed at the lowest position of the bottom surface of the single-cell capturing groove 13.

The single-cell capturing culture dish 1 is provided with the cell sample adding groove 12 and the single-cell capturing groove 13 in the dish body 11, so that the bottom surface of the single-cell capturing groove 13 is a concave surface, and a single-cell capturing blind hole 14 is arranged at the lowest position of the bottom surface of the single-cell capturing groove 13; when the single cell capturing culture dish 1 is used for manually sorting single cells, a culture medium containing cells to be captured is added into the cell loading tank 12, and an operator can select single cells from the cell loading tank 12 by using a micro-operation needle and put the single cells into the single cell capturing tank 13 after washing; due to the special design of the single cell capturing groove 13, the bottom surface of the single cell capturing groove 13 is downwards sunken and provided with the single cell capturing blind hole 14, and single cells in the single cell capturing groove 13 sink into the single cell capturing blind hole 14 under the action of gravity, so that the single cells can be conveniently and rapidly locked in the single cell capturing blind hole 14; single cells can be transferred from single cell capture blind hole 14 to the cell lysate of the PCR tube for further amplification and sequencing using a conventional micropipette. The single-cell capturing culture dish 1 has a simple structure and is convenient to operate, the conditions of mixing of a plurality of cells, single-cell loss, pollution and the like can be effectively avoided, the efficiency of manual single-cell capturing can be remarkably improved, and the single-cell sequencing research of human gametes and early embryos can be facilitated.

In some of these embodiments, the inner diameter of the single cell capture blind hole 14 is 1.5 to 5 times the diameter of the single cell to be captured. In this way, the single cells placed in the single cell capturing groove 13 can be better locked in the single cell capturing blind hole 14, and the movement of the single cells in the single cell capturing blind hole 14 can be limited, so that the single cells can be more conveniently transferred from the single cell capturing blind hole 14 to the cell lysate of the PCR tube through the micropipette. It is understood that the inside diameter of the single cell capture blind hole 14 can be, but is not limited to, 1.5 times, 2.0 times, 2.5 times, 3.0 times, 3.5 times, 4.0 times, 4.5 times, 5 times the diameter of the single cell to be captured.

In some of these embodiments, the single cell capture blind hole 14 has a depth of 1.5 to 5 times the diameter of the single cell to be captured. By means of the arrangement, the single cell capturing groove 13 has a proper accommodating depth, single cells placed in the single cell capturing groove 13 can be well accommodated and locked in the single cell capturing blind hole 14, the cells are not easy to automatically deviate from the single cell capturing blind hole 14, and the single cells can be conveniently transferred through the micropipette. It is understood that the depth of the single cell capture blind hole 14 can be, but is not limited to, 1.5 times, 2.0 times, 2.5 times, 3.0 times, 3.5 times, 4.0 times, 4.5 times, 5 times the diameter of the single cell to be captured.

In some of these embodiments, the inner diameter of the single cell capture blind hole 14 is 30 μm to 300 μm and the depth of the single cell capture blind hole 14 is 30 μm to 300 μm, the inner diameter of the single cell capture blind hole 14 being the same as the depth thereof.

In some of these embodiments, the bottom surface of the single-cell trapping groove 13 has an arc shape or a conical shape. The bottom surface of the single-cell capturing groove 13 is provided with a downward concave shape which is in a cambered surface shape or a conical shape, so that single cells placed in the single-cell capturing groove 13 can more conveniently and smoothly enter the single-cell capturing blind hole 14 under the action of gravity. It will be appreciated that the bottom surface of the single-cell trapping groove 13 may have other downward concave shapes other than the arc shape and the conical shape, so long as it is ensured that the single cells automatically enter the single-cell trapping blind hole 14 along the bottom surface of the single-cell trapping groove 13. For example, the bottom surface of the single-cell trapping groove 13 may have an irregularly curved shape.

Referring to FIG. 2, in some embodiments, the upper edge of the single-cell trapping groove 13 is flush with the inner bottom surface of the dish 11 in the other region except for the single-cell trapping groove 13. In other words, the single cell trapping groove 13 and the single cell trapping blind hole 14 are formed by the concave downward of the inner bottom surface of the dish body 11.

In some of these embodiments, the number of single cell trapping vessels 13 in the cuvette body 11 is plural. So set up, can hold a plurality of single cells that select separately by hand in a dish body 11, can first select separately a plurality of single cells and put into different single cell capturing groove 13 respectively, again concentrate the single cell in the single cell capturing blind hole 14 of each single cell capturing groove 13 respectively transfer to the cell lysate of PCR pipe, can simplify the operation flow, improve manual single cell capturing's work efficiency.

In some of these embodiments, the number of single-cell trapping grooves 13 in the dish body 11 is 5 to 20, and the shape of the single-cell trapping groove 13 is circular.

In a specific example, the number of single cell capturing grooves 13 in the dish body 11 is 8, the inner diameter of the single cell capturing groove 13 is 5mm, the bottom surface is in a circular arc shape and the depth of the concave is 0.5mm; a cylindrical single-cell capturing blind hole 14 is formed in the center of the bottom recess of each single-cell capturing groove 13, the inner diameter of the single-cell capturing blind hole 14 is about 1.5 times the diameter of the single cell, and the depth of the single-cell capturing blind hole 14 is the same as the inner diameter thereof.

In some embodiments, the side wall of the cell loading well 12 is connected to the inner bottom surface of the dish 11, and the upper edge of the side wall of the cell loading well 12 is higher than the inner bottom surface of the dish 11 and lower than the upper edge of the outer side wall of the dish 11. Because the side wall of the cell sample adding groove 12 is connected with the inner bottom surface of the dish body 11, the upper edge of the side wall of the cell sample adding groove 12 is higher than the inner bottom surface of the dish body 11 and lower than the upper edge of the outer side wall of the dish body 11, and the cell sample adding groove 12 can be formed on the inner bottom surface of the dish body 11 through the surrounding of the side wall.

It will be appreciated that the height of the side wall of the cell loading well 12 in the vertical direction is the height of the cell loading well 12. The height of the cell loading well 12 can be set according to actual needs. The bottom surface of the cell loading well 12 may be flush with the inner bottom surface of the dish 11, or may be higher or lower than the inner bottom surface of the dish 11.

In one specific example, the bottom surface of the cell loading well 12 is planar, avoiding accumulation of multiple single cells within the cell loading well 12, and facilitating manual single cell sorting.

In some of these embodiments, the number of cell loading wells 12 is 1, and the shape of the cell loading well 12 is elliptical.

In one specific example, the cell loading well 12 has a major diameter of 32mm and a minor diameter of 6mm; the cell loading well 12 had a sidewall thickness of 0.5mm and a sidewall height of 0.5mm.

In some embodiments, a cell washing tank 15 is further provided in the dish body 11, and the cell washing tank 15 is located between the cell loading tank 12 and the single cell capturing tank 13. By providing the cell washing tank 15 in the dish body 11, after the single cells are manually sorted out from the cell loading tank 12, the single cells can be put into the cell washing tank 15 to be repeatedly washed, and impurities and non-target cells which may remain on the single cells can be removed.

In some embodiments, the side wall of the cell washing tank 15 is connected to the inner bottom surface of the dish body 11, and the upper edge of the side wall of the cell washing tank 15 is higher than the inner bottom surface of the dish body 11 and lower than the upper edge of the outer side wall of the dish body 11. Thus, the cell washing tank 15 can be formed on the inner bottom surface of the dish body 11 by enclosing the side wall of the cell washing tank 15.

It will be appreciated that the height of the side wall of the cell washing tank 15 in the vertical direction is the height of the cell washing tank 15. The height of the cell washing tank 15 may be set according to actual needs. The bottom surface of the cell washing tank 15 may be flush with the inner bottom surface of the dish body 11, or may be higher or lower than the inner bottom surface of the dish body 11.

In one specific example, the bottom surface of the cell wash tank 15 is planar. Thus, the sorted single cells, impurities and non-target cells can be prevented from being accumulated in the cell washing tank 15, and the single cells can be washed more conveniently.

In some of these embodiments, the number of cell wash tanks 15 is 2 to 5, and the cell wash tanks 15 are circular in shape. Setting the number of the cell washing tanks 15 to 2 to 5 can further improve the efficiency of single cell sorting capture.

In a specific example, the number of the cell washing tanks 15 in the dish body 11 is 2, the outer diameter of the cell washing tanks 15 is 6mm, and the inner diameter is 5mm; the cell washing tank 15 had a sidewall thickness of 0.5mm and a sidewall height of 0.5mm.

In some embodiments, the cell loading well 12, the single cell capturing well 13 and the cell washing well 15 in the dish body 11 are independent, that is, the cell loading well 12, the single cell capturing well 13 and the cell washing well 15 are not communicated, so that a plurality of cells can be better prevented from being mixed.

In some of these embodiments, the single cell capture culture dish 1 is fabricated from a biosafety grade plastic. As examples, the biosafety level plastic may be polypropylene plastic, polystyrene plastic or polycarbonate plastic, preferably polypropylene plastic is used.

In some of these embodiments, the dish body 11, the cell loading well 12, the single cell capture well 13, the single cell capture blind hole 14, and the cell washing well 15 in the single cell capture culture dish 1 are integrally formed. Specifically, the dish body 11, the cell loading well 12, the single-cell capturing well 13, the single-cell capturing blind hole 14 and the cell washing well 15 may be integrally formed into an integral structure by injection molding or the like. This can reduce the production cost of the single cell capture culture dish 1.

In some of these embodiments, the single cell capture culture dish 1 further comprises a dish cover 16, the dish cover 16 being capable of being snapped onto the dish body 11. The dish cover 16 can be covered on the dish body 11 if necessary to avoid contamination of single cells. Likewise, the capsule 16 may be made of a biosafety grade plastic material, such as polypropylene plastic, polystyrene plastic, or polycarbonate plastic.

In one specific example, the height of the capsule 16 is 10mm, the outer diameter of the capsule 16 is 57mm, the thickness of the side walls and top surface of the capsule 16 is 1mm, and the inner diameter is 55mm. The height of the dish 11 was 14mm, the outer diameter was 53mm, and the bottom thickness was 2mm.

The following describes a specific procedure of use of the single-cell capture culture dish 1 according to one embodiment of the present utility model:

adding a culture medium containing cells to be sorted and captured into a cell sample adding groove 12 of a dish body 11, and covering paraffin oil to ensure that cell samples in the grooves in the dish body 11 are not mixed;

after single cells to be captured are sucked from the cell sample adding tank 12 through the micromanipulation needle, the single cells are repeatedly washed in the cell washing tank 15 to remove impurities and non-target cells which possibly remain;

placing the washed single cells in the cell washing tank 15 into the single cell capturing tank 13 by using a micromanipulation needle, wherein the bottom surface of the single cell capturing tank 13 is concave, a single cell capturing blind hole 14 is formed at the lowest part of the bottom surface of the single cell capturing tank 13, the single cells sink in the single cell capturing blind hole 14 under the action of gravity, and the single cells are difficult to move due to the small aperture;

filling the single cell capturing blind holes 14 of all the single cell capturing grooves 13, confirming again under an inverted microscope, using a micropipette to tightly align the center of a gun head to the single cell capturing blind holes 14 under a split microscope, rapidly sucking single cells and 0.2-0.5 microliter of culture medium into the micropipette, and transferring the single cells into a cell lysate of a PCR tube for further amplification and sequencing;

after the single cell transfer in all the single cell capturing grooves 13 is completed, whether the cell residues exist in all the single cell capturing blind holes 14 or not is confirmed again under an inverted microscope; the above operation is continued to be repeated until all single cell trapping is completed.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples illustrate only a few embodiments of the utility model, which are described in detail and are not to be construed as limiting the scope of the utility model. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the utility model, which are all within the scope of the utility model. The scope of the utility model is therefore intended to be covered by the appended claims, and the description and drawings may be interpreted in accordance with the contents of the claims.

Claims (10)

1. The single-cell capturing culture dish is characterized by comprising a dish body, wherein a cell sample adding groove and a single-cell capturing groove are arranged in the dish body, the bottom surface of the single-cell capturing groove is a concave surface, and a single-cell capturing blind hole for accommodating single cells is formed in the lowest position of the bottom surface of the single-cell capturing groove.

2. The single cell capture culture dish of claim 1, wherein the inner diameter of the single cell capture blind hole is 1.5-5 times the diameter of the single cell to be captured.

3. The single cell capture culture dish of claim 1, wherein the depth of the single cell capture blind hole is 1.5-5 times the diameter of the single cell to be captured.

4. The single cell capture culture dish of claim 1, wherein the bottom of the single cell capture well is cambered or conical.

5. The single cell capture culture dish of claim 1 wherein the upper edge of the single cell capture well is flush with the inner bottom surface of the dish body in areas other than the single cell capture well.

6. The single cell capture culture dish of claim 1 wherein the number of single cell capture wells in the dish is a plurality.

7. The single cell capture culture dish of any one of claims 1 to 6 wherein the upper side wall edge of the cell loading well is higher than the inner bottom surface of the dish body and lower than the upper side wall edge of the outer side wall of the dish body.

8. The single cell capture culture dish of any one of claims 1 to 6 wherein a cell wash tank is also provided within the dish body, the cell wash tank being located between the cell loading tank and the single cell capture tank.

9. The single cell capture culture dish of claim 8 wherein the dish body, the cell loading well, the single cell capture blind hole, and the cell washing well are of an integrally formed structure.

10. The single cell capture culture dish of any one of claims 1 to 6 further comprising a dish cover capable of being snapped onto the dish body.