CN103451097B - In-vitro cell fusion system and method for increasing bone marrow transplantation success rate - Google Patents

Abstract

An in vitro cell fusion system and method for improving the success rate of bone marrow transplantation. The system includes an upper container, an electrode disk, a lower container, and a cell fusion instrument; an insulating pipe is coiled on the electrode disk, and an even number of metal wires are evenly implanted inside the insulating pipe , half of the metal wire is connected to the positive pole of the cell fusion instrument, and the other half is connected to the negative pole of the cell fusion instrument; the upper container is connected to the inlet end of the insulating pipe through the upper pipe, and the outlet end of the insulating pipe is connected to the lower container through the lower pipe. In the system and method of the present invention, the autologous cells are collected from the recipient and fused with the donor cells through an in vitro cell fusion system, and the cell fusion is promoted by adjusting the field strength, and the tumor cells in the autologous cells are killed to achieve the purpose of purification; the present invention The inventive system can continuously carry out in vitro cell fusion, which can meet the demand for large amount of cells in clinical treatment; the inventive system has simple structure, low cost, convenient disinfection, can be made into a disposable product, and is convenient for clinical treatment.

Description

An in vitro cell fusion system and method for improving the success rate of bone marrow transplantation

technical field

The invention relates to the field of medical technology, in particular to an in vitro cell fusion system and method.

Background technique

Bone marrow transplantation is a type of organ transplantation: normal bone marrow cells are injected into the patient's body through veins to replace diseased bone marrow. It is used to treat abnormal hematopoietic function, immune function deficiency, hematological malignancies and some other malignant tumors, especially in the treatment of leukemia. Recipients, therefore, in addition to organ transplants of identical twins, other allogeneic tissue or organ transplants will experience rejection. In order to improve the success rate of transplantation and reduce or delay transplantation rejection, in addition to preventing infection, the main measures are tissue matching before transplantation, immunosuppressive therapy before and after transplantation, and monitoring of various immunological and histological indicators. Autologous bone marrow cell transplantation is a treatment method, but the collected autologous cells contain a large number of tumor cells, so the recurrence rate is high. It has been reported that cell fusion can reduce rejection, and this phenomenon has been confirmed in experiments. Literature has shown that appropriate current can kill tumor cells without damage to normal tissue cells. We imagine using the method of in vitro cell fusion to reduce the rejection reaction after bone marrow transplantation, and at the same time use the effect of current during cell fusion to kill tumor cells. Clinical treatment is greatly limited, therefore, it is necessary to design a large-capacity in vitro cell fusion system to meet the needs of clinical treatment.

Contents of the invention

The purpose of the present invention is to provide an in vitro cell fusion system and method for improving the success rate of bone marrow transplantation, so as to overcome the above-mentioned deficiencies in the prior art.

In order to achieve the above object, the present invention adopts the following technical solutions:

An in vitro cell fusion system to improve the success rate of bone marrow transplantation, including an upper container, an electrode plate, a lower container and a cell fusion instrument; an insulating pipe is coiled on the electrode plate, and an even number of metal wires are evenly implanted inside the insulating pipe, and half of the metal wires are connected The positive pole of the cell fusion instrument, the other half is connected to the negative pole of the cell fusion instrument; the upper container is connected to the inlet end of the insulating pipe through the upper pipe, and the outlet end of the insulating pipe is connected to the lower container through the lower pipe.

A further improvement of the present invention lies in that the electrode disc is horizontally arranged between the upper container above it and the lower container below it.

A further improvement of the present invention is that: the insulating pipe is a plastic pipe with an inner diameter of 0.4-0.6 cm.

The further improvement of the present invention lies in that the metal wire is partly embedded in the inner wall of the insulating pipe, and partly exposed in the lumen of the insulating pipe.

The further improvement of the present invention lies in that the electrode disc is an insulating disc or a square disc.

The further improvement of the present invention is that valves are provided on both the upper pipeline and the lower pipeline.

The further improvement of the present invention lies in that the diameter of the metal wire is 0.1 cm.

A further improvement of the present invention is that: the even number of metal wires is 2, 4 or 6.

The further improvement of the present invention is: when the donor bone marrow cells and the recipient bone marrow cells are fused in the insulating pipeline, the cell fusion instrument sets the pulse amplitude to 40-200V voltage, and the pulse width is 10-60us; the pulse interval of the cell fusion instrument is controlled , so that the cells in the insulating pipe flow out after 2 to 5 pulses.

The method for cell fusion using an in vitro cell fusion system that improves the success rate of bone marrow transplantation, comprising the following steps: opening the valves on the upper and lower pipelines at the same time, allowing the donor bone marrow cells and recipient bone marrow cells in the upper container to flow in along the upper pipeline In the insulating pipeline of the electrode plate; control the pulse interval of the cell fusion instrument, so that the cells in the insulating pipeline flow out after 2 to 5 pulses; the pulse amplitude output by the cell fusion instrument is 40-200V, and the pulse width is 10-60us .

The further improvement of the present invention lies in that the cells in the insulating pipeline flow out after 3 pulses, and the length of the insulating pipeline is 15-50 cm.

Compared with the prior art, the present invention has the following beneficial effects:

The invention provides an in vitro cell fusion system and method for improving the success rate of bone marrow transplantation. Autologous cells are collected from a recipient and fused with donor cells through an in vitro cell fusion system. Cell fusion is promoted by adjusting the field strength, and killing The tumor cells in the dead autologous cells can achieve the purpose of purification; the system of the present invention can continuously carry out in vitro cell fusion, which can meet the large demand of clinical treatment; the system of the present invention has simple structure, low cost, convenient disinfection, and can be made into disposable products. It is convenient for clinical treatment.

At present, immune rejection and post-transplantation recurrence after bone marrow transplantation are the key factors affecting the success of bone marrow transplantation. In vitro cell fusion system can reduce immune rejection and post-transplantation recurrence after bone marrow transplantation. In addition, another key to the success of bone marrow transplantation is bone marrow matching. The present invention attempts to reduce immune rejection through cell fusion in bone marrow transplantation, achieve the purpose of not needing matching, and at least reduce the immune rejection of haploidentical bone marrow transplantation.

Description of drawings

Fig. 1 is the structural representation of the system of the present invention;

Figure 2 is a schematic diagram of the results of three groups of comparative tests in the in vivo tumor cell proliferation test;

Fig. 3 is a comparison chart of the mouse survival test results in vivo;

Figure 4(a) is a schematic diagram of the results of electric shock killing tumor cells, and Figure 4(b) is a schematic diagram of normal bone marrow cells fused by the fusion system;

Fig. 5 is a schematic cross-sectional view of a plastic pipe.

Detailed ways

Please refer to FIG. 1 , an in vitro cell fusion system for improving the success rate of bone marrow transplantation according to the present invention includes an upper container 1 , an electrode plate 2 , a lower container 3 and a cell fusion instrument.

The electrode disc 2 is an insulating disc or a square disc. The electrode disc 2 is arranged horizontally, and a plastic tube with an inner diameter of 0.4-0.6 cm is coiled on it. Two metal wires 21 with a diameter of 0.1 cm are implanted inside the plastic tube. The upper ends of the two metal wires 21 are Connect the two poles of the cell fusion instrument. The two wires 21 in the plastic tube are not in contact. The metal wire 21 is partly embedded in the inner wall of the plastic tube, and partly exposed in the lumen of the plastic tube.

The upper container 1 is connected to the inlet end of the plastic pipe of the electrode plate 2 through the upper pipe, and the outlet end of the plastic pipe is connected to the lower container 3 through the lower pipe; both the upper pipe and the lower pipe are provided with valves. The electrode disc 2 is horizontally arranged between the upper container 1 above it and the lower container 3 below it.

For example, when the donor bone marrow cells fuse with the recipient bone marrow cells, the cell fusion instrument sets the pulse amplitude to 40-200V, and the pulse width to 10-60us; Somatic bone marrow cells and recipient bone marrow cells slowly flow into the plastic tube of electrode disc 2 along the upper channel (control the pulse interval of the cell fusion instrument, let the cells in the plastic tube on electrode disc 2 go through 2 to 5 pulses (preferably 3 pulses) Outflow), so that the cell fusion instrument can adjust the interval of each pulse, such as continuous 99 pulses can electrofusion the cells within 33x15-50cm (preferably the length of the plastic tube in electrode disc 2 is 15-50cm), and then operate the cells again The fusion instrument continues to perform fusion. The system of the invention can work continuously, and solves the problem of large-capacity cell fusion.

Please refer to Fig. 5, preferably, in an in vitro cell fusion system for improving the success rate of bone marrow transplantation of the present invention, six metal wires 21 with a diameter of 0.1 cm are implanted inside the plastic tube, and the six metal wires 21 are embedded in plastic at intervals of 60 degrees. On the inner wall of the tube, three adjacent metal wires 21 are connected to the positive pole of the cell fusion instrument, and the other three adjacent metal wires are connected to the negative pole of the cell fusion instrument. Setting six metal wires 21 can further increase the effect of cell fusion.

The present invention is an in vitro cell fusion method for improving the success rate of bone marrow transplantation, comprising the following steps:

1) Connect the upper container 1, the electrode plate 2, the lower container 3 and the electrofusion instrument;

2) Mix the extracted acceptor and donor cells and put them into the upper container 1;

3) Open the valves on the upper and lower pipelines at the same time, turn on the cell fusion instrument, and adjust the output pulse parameters of the cell fusion instrument: the pulse amplitude is 40-200V voltage, and the pulse width is 10-60us;

4) The acceptor and donor cells in the upper container flow down and fuse in the plastic tube on the electrode plate 2, and the acceptor and donor stem cells in the plastic tube flow out after 3 or more pulses.

In vivo tumor cell proliferation test: Melanoma cell B16 and donor bone marrow cells were fused by the method of the present invention and then injected into mice (B16 + bone marrow cells + fusion), and two groups of control experiments were performed at the same time, one group was injected with melanoma Cells (B16F1) 5x10 ⁵ were implanted into C57BL/6 mice (6-8 weeks old) subcutaneously (B16), and another group was injected with melanoma cells (B16) 5x10 ⁵ and donor bone marrow cells 5x10 ⁵ into C57BL/6 mice Rat (6-8 weeks old) subcutaneously (B16+ bone marrow cells). Please refer to Figure 2. Animal experiments show that the ability of bone marrow cells to inhibit tumor growth is greatly enhanced after cell fusion; melanoma cell B16 and donor bone marrow cells are fused without tumor formation or tumor formation ability is reduced in vivo.

In vivo mouse survival test: Please refer to Figure 3, the first group (B16+BM cell (fusion) in the figure): implant fusion cells (melanoma cells (B16) 5x10 ⁵ and dog bone marrow cells 5x10 ⁵ ) Subcutaneously in C57BL/6 mice; and two groups of control experiments were conducted at the same time, one group (B16 in the figure) implanted tumor cells into mice (3-4 weeks old) subcutaneously at ^5x105 ; the other group (B16+BM in the figure) cell) into unfused cells (melanoma cells (B16) 5x10 ⁵ and dog bone marrow cells 5x10 ⁵ ) and implanted subcutaneously in C57BL/6 mice. It can be seen from Figure 3 that the survival rate of the mice after cell fusion was significantly higher than that of the other two groups, and the survival rate of the non-fusion group was the lowest. Dogs and mice are heterogeneous animals, but after the fusion of the two cells, the immune rejection is reduced and the survival rate is improved. In the unfused group of melanoma cell B16 and dog bone marrow cells, strong immune rejection occurs in vivo, plus the influence of the tumor cells themselves lead to reduced survival rates.

Tumor cell electric shock experiment: Please refer to Figure 4(a) and Figure 4(b), put melanoma cells and normal bone marrow cells into the electrofusion system respectively, give pulse amplitude 200V, pulse width 50ms, 2 pulses, Put it into a 6-well plate, DMEM 3ml, 10% bovine serum, culture in a CO2 incubator for one day, observe under a microscope, most of the tumor cells died, and the shape and number of bone marrow cells were basically normal.

Claims (2)

1. An in vitro cell fusion system that improves the success rate of bone marrow transplantation, is characterized in that it comprises an upper container (1), an electrode plate (2), a lower container (3) and a cell fusion instrument; the electrode plate (2) is coiled with Insulated pipeline, evenly implanted even number of metal wires (21) inside the insulated pipeline, half of the metal wires (21) are connected to the positive pole of the cell fusion instrument, and the other half are connected to the negative pole of the cell fusion instrument; the upper container (1) is connected to the insulating pipeline through the upper pipeline The inlet end of the insulating pipe is connected to the lower container (3) through the lower pipe; The electrode disc (2) is horizontally arranged between the upper container (1) above it and the lower container (3) below it; The metal wire (21) is partly embedded in the inner wall of the insulating pipe, and partly exposed in the lumen of the insulating pipe; There are valves on the upper pipeline and the lower pipeline; The insulating pipe is a plastic pipe with an internal diameter of 0.4-0.6cm; The electrode disc (2) is an insulating disc or a square disc; The even number of metal wires (21) is 6; the six metal wires (21) are embedded on the inner wall of the plastic pipe at intervals of 60 degrees; When the donor bone marrow cells and recipient bone marrow cells are fused in the insulated pipeline, the cell fusion instrument sets the pulse amplitude to 40-200V, and the pulse width to 10-60us; control the pulse interval of the cell fusion instrument so that the cells in the insulated pipeline pass through Flow out after 2 to 5 pulses. 2. An in vitro cell fusion system for improving the success rate of bone marrow transplantation according to claim 1, characterized in that the diameter of the metal wire (21) is 0.1 cm.