CN104593250B - One single cell separator flexibly and separation and Culture technology - Google Patents

Abstract

A flexible single-cell separator, including but not limited to a micropipette, a screw cap for a culture bottle, a leather case, and a tapered tip, the micropipette includes a fine end of the micropipette and a thick end of the micropipette, and the thick end of the micropipette Connect the bottom of the tapered suction head, the tip of the tapered suction head is slender, one end of the leather sheath is fixed on the thick end of the micropipette, the other end of the leather sheath is fixed with the screw cap of the culture bottle Next, the thick end of the micropipette is nested in the screw cap of the culture bottle. In addition, the present invention also proposes a single cell isolation and culture method, which uses a mixture of old culture solution and fresh culture solution for culturing and expanding the isolated single cell. The beneficial effect of the present invention is that the operation is simple and the cost is low; the second is that the operation can be performed in the case of conventional visualization; the third is that the separation of cells is purposeful; the fourth is that a small amount of cell liquid can be transferred, which is convenient for effectively separating the target cells after dilution; It is low in physical and chemical damage to cells.

Description

A Flexible Single Cell Separator and Separation and Culture Technology

technical field

The invention belongs to the field of biological cell engineering, and more specifically relates to a flexible single cell separator and separation and culture technology.

Background technique

In biological research, cells are the most important research carrier and object. Most cytological research requires functional modification and functional research on cells. In order to achieve research accuracy and functional consistency, monoclonal cells are considered The most effective cell form can ensure the consistency of the cell population and the stability and reliability of the results to the greatest possible extent. In the field of genetic research, monoclonal cells are the most commonly used research tools.

Isolation of monoclonal cells is an essential link in the study of biological cells. In the study of gene function, the isolation of monoclonal cells is particularly important.

The existing monoclonal cell separation technologies mainly include flow cytometry and dilution. The former mainly uses modern flow cytometers for automatic single cell sorting, but this sorting technology causes some unknown effects on the cells due to the addition of some chemical substances. Influence, affecting the growth of cells after separation. At the same time, the requirements for equipment are relatively high, and it is easy to infect bacteria; In the culture plate, single cells are cultured after being confirmed under a microscope. This method has the advantages of fast speed, low cost, and simple operation, but it cannot be positioned and separated, and it is still difficult to isolate the desired target cells.

Contents of the invention

In order to solve the problems still existing in the above-mentioned prior art, the present invention proposes a flexible single cell separator and a separation and culture method.

A flexible single-cell separator, including but not limited to a micropipette, a screw cap for a culture bottle, a leather case, and a tapered tip, the micropipette includes a fine end of the micropipette and a thick end of the micropipette, and the thick end of the micropipette Connect the bottom of the conical suction tip, the tip of the conical suction tip is slender, one end of the leather sheath is fixed on the thick end of the micropipette, the other end of the leather sheath is fixed to the screw cap of the culture bottle Next, the thick end of the micropipette is nested in the screw cap of the culture bottle.

Preferably, the thick end of the micropipette is connected to one end of the holster to serve as a fixing and storage space.

Preferably, the thin end of the micropipette is used for absorbing cells or cell clusters.

Preferably, the thin end of the micropipette can be heated at a high temperature and then drawn to produce a finer tube.

Preferably, the diameter of the fine end of the micropipette is less than 3 mm.

Preferably, the diameter of the screw cap of the culture bottle can be designed according to the bottle mouth specifications of cell culture bottles T25, T75, T175, etc., so as to ensure that the screw cap of the culture bottle can be tightened on the culture bottle.

Preferably, the screw cap of the culture bottle must be connected to the mouth of the culture bottle during use, so as to ensure that the inside of the culture bottle is sealed and sterile in the subsequent operation steps.

Preferably, the leather case is made of materials including but not limited to plastics and rubber.

Preferably, the leather sheath has a good ability to stretch and stretch, ensuring that the front part of the micropipette can move to most positions of the culture bottle.

Preferably, the tapered suction head has a long tapered structure, the bottom of the tapered suction head is connected to the thick end of the micropipette, and the tip of the tapered suction head is elongated, which can absorb a very small amount of liquid.

Preferably, the picking of single cells can be performed under an ordinary inverted microscope or a fluorescent microscope.

Preferably, the picking of the single cells is divided into picking of adherent cells and suspension cells, and the adherent cells can be physically touched to make the cells fall off and sucked into the fine end of the micropipette; the picking of suspension cells can be Carry out directly under the microscope, if necessary, a certain amount of dilution can be carried out before picking.

In addition, the present invention also proposes a single cell isolation and culture method.

A method for separating and culturing single cells, using a mixture of old culture fluid and fresh culture fluid that have cultured the cells to cultivate the isolated single cells to achieve the effect of cell expansion.

Preferably, the proportion of old culture solution in the mixed solution is not less than 5%.

The basic principle of the present invention is to quickly and simply make microtubes, and the experiment of the microtubes can ensure the use in a sterile environment, to carry out aseptic picking of single cells, to achieve the purpose of rapid single cell separation, so as to perform single cell extraction. The cells are cultured to obtain monoclonal cell lines; in view of the existing separation of high equipment requirements, inability to locate separation, and high requirements for aseptic operation, this single cell separator is designed to quickly separate from conventional culture bottles To the target cells, there is no need to put the microscope into the operating table, and the single cell separation can be accurately fixed and positioned under the inverted microscope and the fluorescent microscope. The equipment only needs a conventional microscope and operating table to complete. It is very convenient to separate a small amount of target single cells in the laboratory to obtain the target cell line due to the high reuse characteristics. At the same time, the isolated cells are cultured in old culture medium to meet the requirements of cell microenvironment and multi-cell population. It is in a constant state as much as possible to ensure cell growth and value-added.

The beneficial effects of the present invention are as follows: one is simple and rapid operation, low condition requirements, low equipment requirements, and low cost; the other is that it can be operated under conventional visualization conditions, mainly under the inverted microscope and fluorescent microscope; the third is separation The cells are purposeful, and can perform single-cell separation of a very small number of cells when the proportion of target cells is extremely low; fourth, it has the ability to transfer a small amount of cell fluid, and can transfer a small amount of cell fluid, which is convenient for effective separation of target cells after dilution; Fifth, it has low physical and chemical damage to cells.

Description of drawings

Figure 1 is a schematic diagram of a flexible single-cell separator.

Explanation of the drawings: 1. The thin end of the micropipette, 2. The thick end of the micropipette, 3. The screw cap of the culture bottle, 4. The leather sheath, 5. The tapered tip.

detailed description

Specific embodiment 1

A flexible single-cell separator, including but not limited to a micropipette, a culture bottle screw cap (3), a leather case (4), a conical suction tip (5), and the micropipette includes a micropipette fine end (1) And micropipette thick end (2), described micropipette thick end (2) connects the bottom of described tapered suction head (5), and described tapered suction pipe (5) tip top is elongated, and described holster (4) One end is fixed on the thick end of the micropipette (2), the other end of the leather sheath (4) is affixed to the screw cap (3) of the culture bottle, and the thick end of the micropipette (2) is embedded in the In the screw cap (3) of the culture bottle, the thick end of the micropipette (2) is connected to one end of the holster (4) to serve as a fixation and storage space, and the thin end (1) of the micropipette is used for cell Or for the absorption of cell clusters, the thin end (1) of the micropipette can be heated at a high temperature and then drawn to produce a finer tube, and the diameter of the thin end (1) of the micropipette is less than 3 mm. The diameter of the screw cap (3) of the culture bottle can be designed according to the bottleneck specifications of cell culture bottles T25, T75, T175, etc., so as to ensure that the screw cap (3) of the culture bottle can be tightened on the culture bottle, and the screw cap of the culture bottle can be tightened. The cover (3) must be connected to the mouth of the culture bottle during use, so as to ensure that the inside of the culture bottle is sealed and aseptic in the subsequent operation steps. The holster (4) is made of materials such as plastic, and the holster (4) has a good stretching and stretching ability to ensure that the front part of the micropipette can move to most positions of the culture bottle. The tapered suction head (5) is a long tapered structure, the bottom of the tapered suction head (5) is connected to the thick end of the micropipette (2), and the tip top of the tapered suction head (5) is elongated, It can absorb very small amount of liquid. Picking of single cells can be performed under an ordinary inverted microscope or a fluorescent microscope. The picking of the single cells is divided into picking of adherent cells and suspension cells. The adherent cells can be physically touched to make the cells fall off and sucked into the fine end of the micropipette; the picking of suspension cells can be directly carried out under a microscope If necessary, a certain amount of dilution can be carried out before picking, and the mixed solution of the old culture solution and the fresh culture solution used to cultivate the cells can be used to cultivate the separated single cells to achieve the effect of cell expansion. The mixed solution The proportion of the old medium in the medium is not less than 5%.

Claims (6)

1. a single cell separator flexibly, it is characterised in that described separator includes Micro pipette, culture bottle spiral cover, leather sheath, taper suction nozzle, described micro pipette includes micro pipette Taper end and micro pipette butt end, described micro pipette butt end connects the bottom of described taper suction nozzle, Top, described taper suction nozzle tip is elongated, and it is thick that described leather sheath one end is fixed on described micro pipette End, the described leather sheath other end is affixed with described culture bottle spiral cover, described micro pipette butt end set It is embedded in described culture bottle spiral cover.

Single cell separator flexibly the most according to claim 1, it is characterised in that After described micro pipette taper end is the most heated, carries out wire drawing process, produce more Fine tubule.

Single cell separator flexibly the most according to claim 1, it is characterised in that Described micro pipette taper end diameter is less than 3 millimeters.

Single cell separator flexibly the most according to claim 1, it is characterised in that Described leather sheath uses plastics, the material of rubber to make.

Single cell separator flexibly the most according to claim 1, it is characterised in that Described leather sheath has the ability well stretching and stretching.

Single cell separator flexibly the most according to claim 1, it is characterised in that Described taper suction nozzle is long pyramidal structure, micro pipette butt end of ining succession bottom described taper suction nozzle, Top, described taper suction nozzle tip is elongated.