cell electrofusion

Summary

The advantages of cell electrofusion are (1) high fusion rate, (2) non-toxicity, (3) easy operation, and (4) high viability of the fused cells in further culture. The cell fusion of free cells of animals and protoplasts of plant microorganisms after wall removal by electrical stimulation is another advancement in cell engineering tools.

Operation method

Cellular electrofusion technology

Principle

The prepared free cells or protoplasm, placed in the electrofusion chamber, under the action of high-frequency alternating current electric field, the cells are polarized and become dipoles, resulting in interconnection between the cells, if the applied high-frequency alternating current voltage (i.e., sinusoidal p-p value) is too high or the effect of time is too long, then the cells are connected to the beads, the above conditions should be controlled appropriately, as far as possible, so that the two cells are in the point of contact, and then apply a square-wave Then the square wave pulse is applied to stimulate the two cells, due to the instantaneous effect of the electric pulse, the plasma membrane of the two cells at the point of contact can be broken, at this time the lipid molecules of the plasma membrane are reorganized, and at the same time, due to the role of the tension on the surface of the cell, the fusion of the two cells will be completed gradually.

Materials and Instruments

Animal free cells Plant protoplasts
Mannitol Sodium oxide Fibrinol Isolase Snail enzyme CaCl2 MES (2-N-morpholinoethanesulfonic acid) Potassium dextran sulfate
JCF-I型细胞电融合仪 细胞融合池 普通离心机 倒置显微镜(或研究用显微镜) 刻度吸管 不锈钢网(200目) 镊子 解剖刀 解剖剪 注射器 毛细吸智 刻度吸蕾

Move

I. Preparation of plant protoplasts

1, take young leaves or fully expanded cotyledons, first wash with water and absorb the water on the surface, and then put into a small beaker to cut the leaves into pieces.

2, the pieces into 1.5% cellulase, 0.3% dissociative enzyme, 0.5% snail enzyme, 0.6 mol / L mannitol, 50 mmol / _LCaCl2-2H2O, MES 3 mmol / L, potassium dextran sulfate 0.3%, pH 5.6 of the enzyme solution, placed in the dark at 25 ℃ free 5h (or 15 ℃ over the liquid), the end of the free time, you can microscopic examination of protoplasts At the end of the freeing time, the protoplasts can be examined microscopically. If most of the protoplasts have not been released from the tissues, the freeing time in the enzyme solution should be increased. If most of the protoplasts have not been released from the tissue, it is necessary to increase the freeing time in the enzyme solution. Newly shipped yeast can generally free the protoplasts within 5h, but due to the quality of the drug or storage time is too long, resulting in the decline of enzyme vitality, it is necessary to appropriately prolong the enzyme digestion time.

3、Protoplasts from alcoholysis are filtered through a stainless steel mesh to remove the tissues that have not been enzymatically dissolved, and after filtration, the residual protoplasts on the mesh are rinsed with 0.6M sucrose, and then centrifuged at 200r/min for 5min.

4, The supernatant was aspirated with a syringe with a long needle, and then 1-1.5 ml of 0.2 mol/L _CaCl2-2H2O was added to mix the protoplasts.

5、Use a fine-tipped dropper or a syringe with a long needle to slowly inject 20% sucrose liquid into the bottom of the tube, and then centrifuged at 300r/min for 10min, at this time, you can see a layer of milky band at the interface between the upper and lower two liquids, which is pure protoplasts, and its broken parts and other impurities are settled to the bottom of the centrifugal tube, and then sucked off the impurities and sucrose liquid and protoplasts at the bottom of the tube by using a syringe with a long needle or a capillary suction arm, respectively. Sucrose solution at the bottom of the tube and _CaCl2 solution in the upper layer of protoplasts were sucked off with a long needle syringe or capillary pipette arm.

6. Add 2 ml of 0.2 mol/L _CaCl2-2H2O solution to the centrifuge tube, mix well, and then centrifuge at 300 r/min for 5 min to remove the supernatant, and dilute with 0.6 mol/L mannitol solution after measuring and adjusting to contain ^5×104 protoplasts per ml.

II. Preparation of animal cells

Use a sterilized syringe to inhale lml of Alsver solution (glucose 2.05g, sodium rafate 0.88, NaCl 0.42g, plus weighted evaporated water to 100ml), and then take 0.5-1ml of blood from the subwing vein of chickens and put it into a graduated centrifuge tube after removing it and then add 2-3ml of Alsver solution to make the total amount of 4-5ml, mix and seal it, and put it in a refrigerator at 4℃ for use within one week. For the experiment, take lml of stored chicken blood cells, add 4ml of 85% saline, mix well and then centrifuge at 1200r/min for 5min, after removing the supernatant, and then mix well with 0.6mol/L mannitol solution (or 0.6mol/L sucrose solution) and then washed twice by the above centrifugation conditions. diluted and adjusted to contain ^2×105 per milliliter.

If the bone marrow or ascites tumor cells in passaged culture are used, they can be washed twice with 0.9% saline, centrifuged at 800-1000r/min for 5min each time, and then washed twice with 0.6mol/L mannitol or sucrose solution at 600-800r/min each time, centrifuged for 5min, and finally adjusted to contain ^1×105 tumor cells per ml with mannitol.

III. Cell electrofusion method

1、Meter 2S structure and use method

The technical parameters of JCF-type cell electrofusion instrument are designed through a large number of fusion experimental data and with reference to the parameter values of Shimadzu (Japan) SSH-type electrofusion device. When the power is turned on, the indicator light is on, and the output switch and sinusoidal output switch are dialed, and the sinusoidal frequency button is selected and pressed (divided into five grades of 0.6, 0.8, 1.3, and 1.5 MHz), and at this time, the output cable is connected with the oscilloscope and the high-frequency sinusoidal waveforms appear on the oscilloscope, and when the sinusoidal amplitude knob is rotated left and right, the p-p value of the sinusoidal voltage is lowered and elevated, and the output cable is connected with the electro-fusion chamber. If the output cable is connected to the electric fusion chamber, the thin chest is connected to each other. At this time and then rotate the sine amplitude (i.e., sinusoidal voltage) knob, then the instrument on the voltmeter pointer with the rise and fall. If the sinusoidal voltage is too high, the cells will be connected faster and beaded, which is not conducive to the fusion of the two cells.

Move the sinusoidal output switch to the "composite output" gear, according to the required power size selection of composite output I or, II gear, I gear for low power, II gear for high power; and then according to the requirements of the experiment, select and press the "pulse width" toggle key you need (points) 1ms, 0.2ms, 0.1ms, 10ms, 1us five grades) and "pulse amplitude" button (50V, 100V, 150V, 200V, 250V five grades), but also equipped with pulse amplitude rotary (fine-tuning), the adjustment range of 0-50V, which can be made to increase the value of each grade by 50V.

Apply pulse stimulation can be divided into "self-control" and "hand-control" two gears, when using the "self-control" output pulse, first move the switch to the self-control gear, and then according to the need to select and press the Pulse Frequency" (divided into 2sl times and 1 time per second, 2 times, 5 times, l0 times a total of five grades), you can automatically send electric pulse; if the switch is moved to the "hand-controlled" file, you need to press the "hand-controlled" button. If you move the switch to "hand control", you need to press the "hand control" button and send a pulse every time you press it. Regardless of the self-control or hand-controlled output, each pulse output is accompanied by a chirping sound, the output and pulse oscilloscope connection, you can observe by the pulse width, amplitude and pulse spacing composed of oscilloscope image, when changing the value of the above relevant parameters, the image also changes: move the switch to the opposite direction, the negative pulse, when the value of the pulse width and amplitude of the pulse is unchanged, the image is the same as the positive pulse waveform, but the opposite aspect of the symmetry of top and bottom. Up and down symmetry. Connect the output of the cable with the two electrodes of the electrofusion chamber and send a certain square wave pulse according to the above requirements, and the cells can be fused after being electrically stimulated.

2、Electrofusion operation

Plant protoplast fusion

Turn on the power supply, close the "output switch", move the switch to the sinusoidal output gear, at this time, according to the experimental needs, select the sinusoidal amount of 1-1.3MHz, pulse width of 0.1ms or 10μs, pulse amplitude of 150V, the pulse frequency of 1 times / s, and press the following buttons in turn each calibration button, and finally adjust the sine voltage (i.e., sine amplitude) knob so that the voltmeter is at 5-10V.

Caveat

The main problems that need to be solved in the existing cell electrofusion methods include: low cell fusion throughput; low survival rate of fused cells; and the cell electrofusion process is not yet able to meet the visualization requirements.The use of microelectrode arrays can not only improve the efficiency of cell fusion, but also improve the throughput of cell fusion, so it has become an inevitable trend in the development of cell fusion process; at the same time, to improve the rate of image data acquisition; the use of more integrated image processing software functions and the use of PDMS, glass and other transparent media to produce fusion chips will not be affected by the intensity of light, which can greatly improve the image quality and understanding of the cell fusion process, and also improve the image quality. This can greatly improve the image quality and provide the necessary preconditions for understanding the subtle process of cell fusion.

Common Problems

Cells must be brought into close contact with each other before fusion with electric pulses, and this method of electrofusion is very useful in the fusion of protoplasm for the production of hybrid plants, the fusion of embryonic cells for the preparation of animal clones, and is especially useful in the production of hybridoma cells for the preparation of monoclonal antibodies. Several laboratories have demonstrated that electrofusion using electric fields is 10 to 100 times more efficient than conventional chemical fusion methods, and more recently, electrofusion of adherent cells has been used to study the kinetic rearrangement of cytoskeletal components and organelles during cell fusion.

Sourced from "Cell Electrofusion and Recent Advances" Chinese Journal of Bioengineering.

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