Method for precise transfer of microcells

Abstract

The present invention provides a method for precise transfer of cells for reducing the loss of the number of cells generated in the transfer, and for precisely transferring an extremely small amount of cells.

Description

Technical Field

[0001] This invention belongs to the field of biological detection, specifically relating to a method for precise cell transfer and a composition for the method. Background Technology

[0002] In cell experiments, transferring a certain amount of cells from one container to another is a very common procedure. When obtaining small amounts of cells, a serial dilution method is currently commonly used. For cell quantities greater than 10... 3 This method is applicable in cases of (one thousand) cells, and even if a small number of cells adhere to the pipette tip, it will not cause significant errors.

[0003] However, when the amount of cells to be transferred is smaller, for example, less than 10... 3 Especially when taking fewer than 10 cells, the exact number of cells cannot be determined because some cells are adsorbed onto the inner wall of the pipette tip. This adsorption limits the transfer method, and even with repeated attempts, the actual number of cells transferred will still have a significant error.

[0004] Currently, there are products on the market that have undergone surface treatment to reduce the adsorption of pipette tips (e.g., hydrophobic, low-adsorption pipette tips obtained through plasma surface treatment). However, such products are primarily designed for better liquid transfer and still retain some adsorption to cells. They are not suitable for situations requiring high precision in cell count and involving extremely small cell quantities. Examples of such situations include single-cell sequencing, single-cell isolation, capture, single-cell intracellular nucleic acid analysis, detection of residual primitive stem cells in terminally differentiated cells, and assisted reproductive technologies. Therefore, there is still a need in the field for a simple and universal method for accurately transferring small or trace amounts of cells. Summary of the Invention

[0005] To address the problem of inaccurate cell counting and cell loss caused by cell adhesion to transfer tools during the transfer of small quantities of cells, the inventors conducted in-depth experiments and discovered that by immersing the cell transfer component (e.g., a pipette tip) with a cell-mild surfactant before picking up the cells, no cells remain in the cell transfer component after dispensing. In other words, the cells to be transferred can be completely transferred without being left in the cell transfer component due to adhesion, thus achieving precise cell transfer.

[0006] This invention relates to a method for transferring small numbers of cells, particularly trace amounts of cells, and a wetting agent composition for use in said method. The wetting agent composition contains a surfactant for wetting or impregnating cell transfer components before cell movement.

[0007] Therefore, the present invention provides the following aspects.

[0008] Firstly, a cell transfer method, including...

[0009] 1) Pre-treat the cell transfer component with a surfactant; and 2) use the pre-treated cell transfer component from 1) to aspirate and transfer cells.

[0010] The pretreatment involves impregnating the cell transfer component and then emptying it. The impregnation includes repeatedly aspirating and releasing surfactant from the cell transfer component several times.

[0011] Secondly, in the above method, the number of cells transferred is preferably less than 10. 3 10 per time, more preferably less than 10 2 Each cell is a common passaged cell line or primary cell.

[0012] The number of cells transferred is preferably less than 10. 3 10 per time, more preferably less than 10 2 Item / time.

[0013] Thirdly, a wetting agent composition for cell transfer comprising a surfactant.

[0014] The surfactant may be a nonionic surfactant, more preferably selected from Triton-X, Tween, and even more preferably Triton-X100 or Tween20. The wetting agent composition may contain, for example, 0.1% to 0.05% of a Triton-X or Tween compound, preferably 0.01% Triton-X100 or 0.01% Tween20.

[0015] Fourthly, a cell transfer component kit includes a cell transfer component and the wetting agent composition described in the third aspect. The cell transfer component has an open end and a negative pressure end.

[0016] The cell transfer component can be a disposable sterile pipette tip (with or without a filter), syringe needle, glass pipette, polyethylene pipette, slender microneedles, etc. Preferably, the cell transfer component is a food-grade PP pipette tip, with a preferred size of 200–10 μL. The tip opening diameter can be 10–20 μm.

[0017] Preferably, the wetting agent composition of the third aspect is prepared in advance and individually packaged in a sterile tube, more preferably the sterile tube has a portion that allows the open end of the cell transfer member to extend into it.

[0018] Fifthly, the use of a composition in reducing surface adsorption of cell transfer components. The composition comprises a nonionic surfactant.

[0019] The method of this invention enables the precise transfer of extremely small amounts of cells, facilitating single-cell sequencing (single-cell isolation and capture), analysis of nucleic acid content within single cells, and detection of residual amounts of primitive stem cells in terminally differentiated cells.

[0020] The method of the present invention is used to transfer a liquid (e.g., culture) medium containing cells using air pressure, such that after the liquid is discharged from the component, the cells are discharged as much as possible without remaining in the component. It should be noted that the method of the present invention is not intended to control or retain cells in the cell transfer component in any way, or to control them at a specific location within the component.

[0021] It should be noted that the purpose of the method of the present invention is to reduce the error in the number of cells caused by adhesion during transfer, so as to achieve thorough transfer while protecting the integrity of the cells.

[0022] This method is applicable to 10. 3 Less than 10 2 This method is particularly suitable for transferring 1 to 10 cells. The focus of this invention is not on reducing volumetric error of the transferred liquid or reducing adhesion of non-cellular liquid portions of the analyte to the pipette tip.

[0023] The advantages of the method of the present invention are at least as follows.

[0024] High efficiency: The method of the present invention improves the efficiency of transferring cells in trace quantities, and can achieve complete transfer even when the number of cells is small.

[0025] Material saving: The method of the present invention greatly reduces the loss of cells due to cell adhesion or wall adhesion, and improves the utilization rate of biological materials or analytical samples.

[0026] Practicality: The method of this invention is simple to operate and convenient to implement. It does not require complex design or processing of cell transfer equipment and components, and is compatible with most current micropipette tips, needles, microtubes, etc. The raw materials used are commonly used laboratory reagents, which do not need to be purchased separately, thus demonstrating high practicality.

[0027] Wide applicability: The method of this invention is applicable to biological samples with cellular structures, and there are no restrictions on the type of cells or the type of cell transfer components. The method has no effect on the transferred cells, is unlikely to cause cell rupture, and does not affect cell viability. The method of this invention can be combined with detection systems for cell transfer, analysis, separation, classification, and micromanipulation.

[0028] Pollution-free: The raw materials used in the method of this invention are commonly used reagents in laboratories, which are non-toxic and harmless to experimental operators, and the operation process does not generate any pollution. Used pipette tips can be disposed of or recycled according to normal experimental waste standards. Attached Figure Description

[0029] Figure 1 These are microscopic images of residual cells inside the pipette tip after cell transfer following wetting with different types of surfactants.

[0030] Figure 2 Microscopic images of residual cells in the pipette tip after cell transfer following infiltration with different concentrations of Triton X-100. Invention Details

[0032] definition

[0033] surfactants

[0034] The term "surfactant" refers to a substance used to reduce the surface tension between two liquids, between a gas and a liquid, or between a liquid and a solid. Surfactants can be classified into cationic surfactants, anionic surfactants, nonionic surfactants, and amphoteric surfactants.

[0035] The term "nonionic surfactant" refers to a surfactant with an uncharged polar group. Nonionic surfactants do not dissociate in water. Their hydrophilic groups are typically polyols such as glycerol, polyethylene glycol, and sorbitol, while their lipophilic groups are typically long-chain fatty acids or long-chain fatty alcohols, as well as alkyl or aryl groups.

[0036] Nonionic surfactants encompass a wide range of compounds with diverse types and structures, such as long-chain fatty alcohol polyoxyethylene ethers, alkylphenol polyoxyethylene ethers, fatty acid polyoxyethylene esters, polyoxyethylene alkylamines, polyoxyethylene alkylamides, and polyethers, as well as poly(oxyethylene) block copolymers. Specific examples of commonly used surfactants in biological laboratories can be listed below. X-series surfactants, polysorbate surfactants, etc.

[0037] X series: X-series nonionic surfactants are prepared by reacting octylphenol with ethylene oxide. They belong to the alkyl aryl polyether alcohol family and have the following general structural formula:

[0038]

[0039] In the formula, x represents the average number of ethylene oxide units in the ether side chain.

[0040] The X series includes the following products (arranged in ascending order of polyethylene oxide chain length).

[0041] Triton X-100 x＝9-10 Triton X-405 (70% active) x＝40

[0042] Triton X-100 refers to polyethylene glycol octylphenyl ether (T-octylphenoxypolyethoxyethan ol).

[0043] The term "polysorbate" refers to compounds or surfactants: polysorbates (PS) are a family of amphiphilic, nonionic surfactants derived from fatty acid-esterified ethoxylated dehydrated sorbitol or isosorbide (derived from sorbitol).

[0044] The term "Tween-like" compounds or surfactants refer to polyoxyethylene sorbitan fatty acid esters, commercially known as Tween. Corresponding to the Span naming convention, various examples include Tween 20 (polysorbate 20), Tween 40 (polysorbate 40), Tween 60 (polysorbate 60), Tween 65 (polysorbate 65), Tween 80 (polysorbate 80), and Tween 85 (polysorbate 85).

[0045] Cell transfer components

[0046] The term "cell transfer device" as used herein includes biologically applicable devices for transferring cells. Examples include biomedical pipettes, tapering pipette tips, micropipette tips, syringe needles, capillary glass pipettes, etc. The cell transfer device usable in this invention needs to have an open end for aspirating cells and a negative pressure end capable of providing negative pressure.

[0047] The term "pipette tip" generally refers to a plastic biological tip, including straight tips, curved tips, tips with dividers, tips with filters, flexible coatable tips, and metering tips. The pipette tips used in this application are those whose inner walls are smooth, specifically the part (absorption section) capable of aspirating and dispensing cell-containing liquid.

[0048] biological methods

[0049] The term "single-cell sequencing" refers to the process of isolating individual cells, capturing their transcripts, generating sequencing libraries, and mapping the transcripts in the sequencing libraries to individual cells. This allows for the extraction, amplification, and high-throughput sequencing analysis of the genome or transcriptome at the single-cell level.

[0050] In this article, the term "micromanipulation" refers to experimental operations performed under microscopic devices such as microscopes, which are close to the cellular scale.

[0051] Cellular materials

[0052] The number of cells transferred in a single transaction can be less than 10. 3 10 to 10 cells, preferably 10 to 10 2 One to ten cells, more preferably. The transferred cells can be passaged cell lines or primary cells. It should be noted that the transferred cells are not limited to being in the form of single cells (discrete cells) or cell aggregates, nor are they required to be discharged at a time as a single cell (1 cell / discharge).

[0053] Detectable cell samples include: cultured cells, tissues, and organs; cell suspensions after enzymatic digestion; body fluids containing cells such as urine, blood, and tissue fluid; or cells in commonly used isotonic cell solutions (e.g., physiological saline, various cell culture media, PBS, PBST, etc.).

[0054] use

[0055] The method and wetting agent composition of this invention can be widely used in experiments requiring high precision in cell number and involving small cell quantities. Examples of such experiments include single-cell sequencing (single-cell isolation and capture), analysis of nucleic acid content within single cells, detection of residual primitive stem cells in terminally differentiated cells, single-cell transcriptomics analysis, proteomics analysis, pronuclear injection, and isolation of viral subtypes.

[0056] This invention includes the following:

[0057] 1. A cell transfer method, the method comprising the following steps:

[0058] 1) Pretreatment of cell transfer components with surfactants;

[0059] 2) Use the cell transfer component pretreated in 1) to aspirate and transfer cells.

[0060] 2. The method of item 1 above, wherein step 1) is performed 1 minute before cell aspiration, for example 10 seconds to 1 minute, preferably 10 to 30 seconds before aspiration.

[0061] 3. The method of item 1 or 2 above, wherein the pretreatment is to infiltrate the cell transfer component and finally empty it, wherein the infiltration includes repeatedly aspirating and dispensing the surfactant from the cell transfer component more than 3 times, preferably 3 to 5 times; wherein the emptying is to ensure that there is no visible liquid in the cell transfer component; wherein the aspiration and dispensing is performed at the maximum volume set by the pipette.

[0062] 4. The method of any one of items 1-3 above, wherein the surfactant includes a nonionic surfactant selected from Triton-X and Tween classes.

[0063] Triton-X class includes Triton X-114, Triton X-100, and Triton X-405;

[0064] The Tween series includes Tween20 (polysorbate 20), Tween40 (polysorbate 40), Tween60 (polysorbate 60), Tween65 (polysorbate 65), Tween80 (polysorbate 80) and Tween85 (polysorbate 85);

[0065] Triton X-100 is preferred as a Triton-X class compliant product.

[0066] Tween20 is the preferred choice for the Tween class.

[0067] 5. In any of the methods described above, the number of cells transferred is preferably less than 10. 3 10 per time, more preferably less than 10 2 One per use, preferably 10 to 1.

[0068] 6. In any of the above methods, the volume of surfactant aspirated in a single operation can be set according to the volume range of the pipette, with the maximum aspiration and dispensing volume; preferably, the number of aspiration and dispensing operations is 3 to 5 times.

[0069] 7. The method of any one of the above, wherein the cell transfer component has an open end and a negative pressure end, and the cell transfer component is selected from disposable sterile pipette tips (with or without a filter), syringe needles, glass pipettes, polyethylene pipettes, slender microneedles, etc.

[0070] Preferably, the cell transfer component is a disposable sterile pipette tip; the pipette tip is preferably 200-10 μL in size and has an opening diameter of 10-20 μm.

[0071] 8. The method of any of the above, wherein the cells are common passaged cell lines and primary cells, such as 293T, B16F10, HCT116 and human pluripotent stem cells, etc.

[0072] 9. A wetting composition for cell transfer comprising a surfactant.

[0073] The surfactant is a nonionic surfactant, more preferably selected from Triton-X, Tween, and even more preferably Triton-X100 or Tween20. The wetting agent composition may contain, for example, 0.1% to 0.05% of a Triton-X or Tween compound, preferably 0.01% Triton-X100 or 0.01% Tween20.

[0074] 10. A cell transfer component kit comprising a cell transfer component and the wetting agent composition described in item 9.

[0075] 11. The kit of claim 10, wherein the wetting agent composition of the third aspect is pre-prepared and individually packaged in sterile tubes, each tube containing 500 to 1000 μL of the wetting agent composition.

[0076] 12. The kit of item 11, wherein the sterile tube is configured as an aspiration end capable of infiltrating the cell transfer component.

[0077] 13. Use of compositions containing nonionic surfactants in reducing surface adsorption of cell transfer components.

[0078] 14. The use described in item 9, wherein the nonionic surfactant is selected from the Triton-X class and the Tween class.

[0079] 15. The use described in item 9, wherein the concentration of the nonionic surfactant is 0.01% to 0.05%.

[0080] 16. Use of a surfactant in the preparation of a wetting agent composition for microcell transfer, said surfactant being a nonionic surfactant, more preferably selected from Triton-X, Tween, and even more preferably Triton-X100 or Tween20. The wetting agent composition may contain, for example, 0.1% to 0.05% of a Triton-X or Tween compound, preferably 0.01% Triton-X100 or 0.01% Tween20, wherein the microcell transfer is less than 10 cells per transfer. 3 10 cells, more preferably less than 10 2 Each wash label, preferably 10 to 1 cell. Detailed Implementation

[0081] In one specific embodiment, the method of the present invention includes the following steps:

[0082] 1) Prepare a solution containing Triton or Tween compounds in cell culture medium or PBS as a wetting agent composition, wherein the concentration of Triton compounds can be 0.01-0.05% and the concentration of Tween compounds can be 0.01-0.05%;

[0083] 2) Prepare physiological saline, sterile PBS, or cell culture medium containing the cells to be transferred;

[0084] 3) Attach the pipette tip to the pipette, and pipette into the wetting agent composition in 1) 3 to 5 times. On the last time, empty the remaining liquid from the pipette tip.

[0085] 4) Use the pipette tip obtained in 3) to draw up the cell-containing liquid from 2) and release the liquid into the target container.

[0086] In one embodiment, the surfactant that can be used is a surfactant with mild cell-lytic activity, such as a nonionic surfactant.

[0087] Examples of nonionic surfactants include: polyethylene glycol p-1,1,3,3-tetramethylbutylphenyl ether, polyethylene glycol octylphenyl ether (Triton X-100), tert-octylphenol polyoxyethylene ether (POE(3)tert-octylphenol, Triton X-114), nonylphenol polyoxyethylene ether (Triton X-405, 70% active ingredient), Tween 20, etc. Triton X and Tween types of nonionic surfactants are preferred, and Triton X-100 and Tween 20 are more preferred.

[0088] In one embodiment, the concentration range of the nonionic surfactant may be 0.01% to 0.05%.

[0089] In one embodiment, the volume of cell fluid transferred in each group is 200–10 μL, preferably 50–10 μL.

[0090] In one embodiment, the nonionic surfactant can be pre-prepared as a stock solution, which is then diluted for use immediately upon application.

[0091] In one embodiment, the pipette tip is a plastic biological tip, which may include, for example, straight tips, bent tips, tips with partitions, tips with filters, flexible and coatable tips, metering tips, and various other tips whose inner walls are smooth in the part (absorption section) that can aspirate and dispense cell-containing liquid.

[0092] In one embodiment, this method can be used in combination with pipette tips that have undergone low-adsorption treatment, such as commercially available low-adsorption pipette tips, siliconized pipette tips, plasma-treated pipette tips, etc.

[0093] In one implementation, the transferred cells can be less than 10. 3 10 to 10 cells, preferably 10 to 10 2 One cell, more preferably 1 to 10 cells.

[0094] Example

[0095] To further illustrate the present invention, the following embodiments are provided. These embodiments are only used to illustrate the implementation of the present invention and are not intended to limit the scope of the present invention.

[0096] Example 1. Comparison of the treatment method of the present invention with that of a low-adsorption nozzle.

[0097] In this embodiment, commercially available low-adsorption pipette tips (Axygen, catalog number TF10-2-CS) were used as a control, and Axygen pipette tips (catalog number: T-300-RS) were used as standard tips. Both types of tips were in the standard white 10μL size.

[0098] Materials: 293T cells.

[0099] The experimental procedure is as follows.

[0100] 1. Prepare 0.01% Triton X-100 and 0.01% Tween 20 solutions separately using PBS;

[0101] 2. Collect and count the cells, then dissolve them in PBS / culture medium to obtain a cell density of 102. 5 / mL of cell solution;

[0102] 3. Divide the cells into four groups, and treat each group with a pipette tip as follows:

[0103] 1) Conventional pipette tip group (negative control group): Cells were directly aspirated and transferred using conventional pipette tips;

[0104] 2) Standard pipette tip + 0.01% Triton group: Using a standard pipette tip, the cells were immersed in 0.01% Triton solution and aspirated and discharged 3 times, then aspirated and transferred.

[0105] 3) Standard pipette tip + 0.01% Tween group: Using a standard pipette tip, soak the sample in 0.01% Tween solution and aspirate and remove the sample 3 times, then aspirate and transfer the cells;

[0106] 4) Low-adsorption pipette tip group (positive control group): cells were directly aspirated and transferred using a low-adsorption pipette tip; the volume of cell solution transferred per transfer in each group was 10 μL.

[0107] After each pipette tip is attached to the pipette, it is aspirated three times in the surfactant wetting solution, and the last time the remaining surfactant in the tip is drained.

[0108] 4. After draining the liquid containing cells, observe the pipette tip and the transferred cells under a microscope, and take photos to record the cell residue on the inner wall of the pipette tip after cell transfer.

[0109] The results showed that after the conventional pipette tip group aspirated and transferred cells, a relatively large number of cells remained in the inner wall. Figure 1 The leftmost column (arrows indicate cells); after aspirating and transferring cells, the low-adsorption pipette tip group had fewer cells remaining on the inner wall than the conventional pipette tip group, but still retained a certain amount of cells. Figure 1 (As indicated by the arrow in the far right column). In contrast, conventional pipette tips soaked in 0.01% Triton X-100 or 0.01% Tween 20 solutions had no cells remaining on the inner wall. Figure 1 (The two middle columns). In addition, microscopic observation confirmed that the transferred cells were morphologically intact.

[0110] The above observations show that the cell transfer method of the present invention can provide better results than commercially available low-adsorption treatment nozzles.

[0111] Furthermore, it is expected that the cell transfer method of the present invention can be used in combination with commercially available low-adsorption treatment nozzles.

[0112] Example 2. Comparison of treatment effects of different concentrations of surfactant

[0113] In this embodiment, the concentration gradient of Triton was used to wet the pipette tip, further determining the optimal concentration of Triton for use.

[0114] Material

[0115] Cell type: 293T cells.

[0116] Wetting solution: 1%, 0.1%, 0.01%, and 0.005% Triton X-100 solutions prepared with PBS.

[0117] Grouped into six groups in total:

[0118] (1) Conventional gun head assembly

[0119] (2) Standard nozzle + 1% group

[0120] (3) Standard nozzle + 0.1% group,

[0121] (4) Standard nozzle + 0.05% group,

[0122] (5) Standard nozzle + 0.01% group, and

[0123] (6) Standard nozzle + 0.005% group.

[0124] The experiments were conducted using the same procedures as in Example 1, except for a different concentration of Triton X-100 and a different cell type. The results are shown below. Figure 2 .

[0125] The results showed that, in the conventional control group, significant cell residue was visible on the inner wall of the pipette tip. Figure 2 (Top left small image, arrows indicate cells); a small amount of cell residue was also observed in the standard pipette tip +0.005% group; ideal transfer results were obtained in the standard pipette tip +1%, standard pipette tip +0.1%, standard pipette tip +0.05%, and standard pipette tip +0.01% groups, with no cell residue. Figure 2 The morphology of the transferred cells was confirmed to be intact both inside and outside the neck through microscopic observation.

[0126] Considering that the volume of Triton remaining in the pipette tip is significantly less than the volume of cell-containing liquid being aspirated, i.e., when cells are aspirated, the final concentration of Triton X-100 in the pipette tip is much lower than the concentration used for infiltration, the Triton concentration used for infiltration in the methods and compositions of the present invention is recommended to be 0.1%-0.05%, preferably 0.01%-0.05%.

Claims

1. A cell transfer method, the method comprising the following steps: 1) Pretreatment of cell transfer components with surfactants; 2) Using the pretreated cell transfer component from 1), aspirate and transfer cells. The pretreatment is performed by immersing and emptying the cell transfer device. The immersion includes repeatedly aspirating a surfactant with the cell transfer device more than three times. The surfactant is selected from Triton-X type surfactants with a concentration of 0.1%-0.05% or Tween type surfactants with a concentration of 0.1%-0.05%. The number of cells transferred is less than or equal to 10. 3 Item / time.

2. The method according to claim 1, wherein step 1) is performed 1 minute before aspirating the cells.

3. The method of claim 2, wherein step 1) is performed 10 seconds to 1 minute before aspirating the cells.

4. The method of claim 3, wherein step 1) is performed 10 to 30 seconds before aspirating the cells.

5. The method according to claim 1, wherein the number of cells transferred is less than 10. 2 Item / time.

6. The method according to any one of claims 1-5, wherein the Tween type is selected from Tween20, Tween40, Tween60, Tween65, Tween80 and Tween85; The Triton-X class is selected from Triton X-114, Triton X-100 and Triton X-405.

7. The method according to claim 1, wherein the cell transfer component has an open end and a negative pressure end, and the cell transfer component is a disposable sterile pipette tip.

8. The method according to claim 7, wherein the pipette tip has a capacity of 10-200 μL.

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