CN103451097B - In-vitro cell fusion system and method for increasing bone marrow transplantation success rate - Google Patents
In-vitro cell fusion system and method for increasing bone marrow transplantation success rate Download PDFInfo
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- CN103451097B CN103451097B CN201310362301.4A CN201310362301A CN103451097B CN 103451097 B CN103451097 B CN 103451097B CN 201310362301 A CN201310362301 A CN 201310362301A CN 103451097 B CN103451097 B CN 103451097B
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Abstract
The invention relates to an in-vitro cell fusion system and method for increasing the bone marrow transplantation success rate. The system comprises an upper vessel, an electrode disc, a lower vessel and a cell fusion instrument, wherein an insulated pipeline is coiled on the electrode disc, an even number of metallic wires are uniformly embedded inside the insulated pipeline, half of the metallic wires are connected with the positive pole of the cell fusion instrument, and the other half of the metallic wires are connected with the negative pole of the cell fusion instrument; the upper vessel is connected to the inlet end of the insulated pipeline through an upper pipeline, and the outlet end of the insulated pipeline is connected to the lower vessel through a lower pipeline. According to the system and the method, disclosed by the invention, autologous cells and donor cells are fused through the in-vitro cell fusion system after the autologous cells are collected from a receptor, the cell fusion is promoted through adjusting the field intensity, and the aim of purification is achieved through killing tumor cells in the autologous cells; the system disclosed by the invention can carry out in-vitro cell fusion uninterruptedly and can meet the requirements for large cell number in clinical treatment; the system disclosed by the invention is simple in structure, low in construction cost and convenient in disinfection, and can be made into disposable products so as to facilitate the use in clinical treatment.
Description
Technical field
The present invention relates to medicine technology field, particularly a kind of cell in vitro emerging system and method.
Background technology
Bone marrow transplantation is the one of organ transplantation: input in patient body, to replace the methods for the treatment of of pathology marrow by normal marrow cell by vein.In order to treat hemopoietic function exception, immunodeficiency, Malignancy and some other malignant tumour, particularly in leukemia treating, curative effect is obvious, a lot of patient is had to reach long-term remission, owing to being difficult to find the on all four donor-recipient of HLA in crowd, therefore, except the organ transplantation of enzygotic twins, all can there is rejection in other allohisto compatibilities or organ transplantation.For improving transplanting succeed rate, alleviating or delaying graft-rejection, except protecting from infection, major measure is the monitoring of tissue matching and the forward and backward immunosuppressive therapy of transplanting and every immunology and histological indices before transplanting.Autologous bone marrow cell transplantation is a kind for the treatment of means, but containing massive tumor cell in the autogenous cell gathered, therefore recurrence rate is high.Have report showed cell to merge and can reduce rejection, this phenomenon is confirmed in test.Having document to show, suitable electric current can kill tumour cell and normal tissue cell is harmless.We imagine the rejection after the method minimizing bone marrow transplantation utilizing cell in vitro to merge, utilize the effect of electric current during cytogamy to kill the tumour cell of self simultaneously, but, there is no jumbo cell fusion electrode cup in the world at present, cell in vitro is merged in clinical treatment, is subject to great restriction, therefore, be necessary that a kind of jumbo cell in vitro emerging system of design is to meet the demand of clinical treatment in fact.
Summary of the invention
The object of the present invention is to provide a kind of the cell in vitro emerging system and the method that improve bone marrow transplantation success ratio, to overcome above-mentioned the deficiencies in the prior art.
To achieve these goals, the present invention adopts following technical scheme:
Improve a cell in vitro emerging system for bone marrow transplantation success ratio, comprise upper container, electrode plate, lower container and cell fusion apparatus; In electrode plate, coiling has isolated pipe, and isolated pipe inner homogeneous implants even number one metal wire, and half wire connects the positive pole of cell fusion apparatus, the negative pole of an other half-connection cell fusion apparatus; Upper container connects the inlet end of isolated pipe by upper pipeline, and the exit end of isolated pipe connects lower container by lower pipeline.
The present invention further improves and is: electrode plate is horizontally placed between the upper container of the side of being located thereon and the lower container below it.
The present invention further improves and is: the plastics tubing of described isolated pipe to be internal diameter be 0.4-0.6cm.
The present invention further improves and is: wire portion embeds isolated pipe inwall, and part is exposed in isolated pipe tube chamber.
The present invention further improves and is: electrode plate is dielectric disc or square plate.
The present invention further improves and is: upper pipeline and lower pipeline are equipped with valve.
The present invention further improves and is: diameter wiry is 0.1cm.
The present invention further improves and is: described even number one metal wire is 2,4 or 6.
The present invention further improves and is: donor bone marrow cell and receptor bone myelocyte merge when isolated pipe endomixis, and it is 40-200V voltage that cell fusion apparatus arranges pulse height, and pulse width is 10-60us; Control the recurrent interval of cell fusion apparatus, isolated pipe inner cell is flowed out after 2 ~ 5 pulses.
The cell in vitro emerging system improving bone marrow transplantation success ratio is adopted to carry out the method for cytogamy, comprise the following steps: the valve on upper and lower pipeline is opened simultaneously, allow the donor bone marrow cell in upper container and receptor bone myelocyte flow in the isolated pipe of electrode plate along upper pipeline; Control the recurrent interval of cell fusion apparatus, isolated pipe inner cell is flowed out after 2 ~ 5 pulses; Wherein, the pulse height that cell fusion apparatus exports is 40-200V voltage, and pulse width is 10-60us.
The present invention further improves and is: isolated pipe inner cell flows out after 3 pulses, and the length of isolated pipe is 15-50cm.
Relative to prior art, the present invention has following beneficial effect:
The invention provides a kind of the cell in vitro emerging system and the method that improve bone marrow transplantation success ratio, after autogenous cell is gathered in acceptor body and donorcells merged by cell in vitro emerging system, by regulating, field intensity is short to be reached into cytogamy, and the tumour cell killed in autogenous cell reaches the object of purification; Present system continually can carry out cell in vitro fusion, can meet the heavy demand of clinical treatment; Present system structure is simple, cheap, and sterilization is convenient, can make disposable product, facilitate clinical treatment to use.
After current bone marrow transplantation, immunological rejection and relapse after transplantation are that to affect bone marrow transplantation successfully crucial, can reduce immunological rejection and relapse after transplantation after bone marrow transplantation by cell in vitro emerging system.Affecting bone marrow transplantation another key successful is in addition marrow distribution type, and the present invention attempts the method for bone marrow transplantation by cytogamy, and reduce immunological rejection, reach and do not need distribution type object, bottom line can reduce half-matched bone marrow transplantation immunological rejection.
Accompanying drawing explanation
Fig. 1 is the structural representation of present system;
Fig. 2 is the result schematic diagram of three groups of simultaneous tests in interior tumor cell proliferation test;
Fig. 3 is mouse survival comparison of test results figure in body;
Fig. 4 (a) is electric shock killing tumor cell result schematic diagram, and Fig. 4 (b) is the schematic diagram of normal marrow cell after emerging system merges;
Fig. 5 is the schematic cross-section of plastics tubing.
Embodiment
Refer to shown in Fig. 1, a kind of cell in vitro emerging system improving bone marrow transplantation success ratio of the present invention, comprises upper container 1, electrode plate 2, lower container 3 and cell fusion apparatus.
Electrode plate 2 is dielectric disc or square plate, and electrode plate 2 is horizontally disposed with, and it coils internal diameter is the plastics tubing of 0.4-0.6cm, and wire 21, two wire 21 upper end that inside of plastic tubes implants two diameter 0.1cm connects cell fusion apparatus the two poles of the earth.Two one metal wires 21 in plastics tubing do not contact.Wire 21 is partially submerged into plastic pipe inner wall, and part is exposed in plastics tubing tube chamber.
Upper container 1 is by the inlet end of upper pipeline connection electrode dish 2 plastics tubing, and the exit end of plastics tubing connects lower container 3 by lower pipeline; Upper pipeline and lower pipeline are equipped with valve.Electrode plate 2 is horizontally placed between the upper container 1 of the side of being located thereon and the lower container 3 below it.
During as donor bone marrow cell and the fusion of receptor bone myelocyte, it is 40-200V voltage that cell fusion apparatus arranges pulse height, and pulse width is 10-60us, by upper, valve on lower pipeline is opened simultaneously, allow the donor bone marrow cell in upper container 1 and receptor bone myelocyte slowly flow in the plastics tubing of electrode plate 2 along upper pipeline and (control the recurrent interval of cell fusion apparatus, plastics tubing inner cell in electrode plate 2 is allowed to flow out after 2 ~ 5 pulses (preferably 3 pulses)), such cell fusion apparatus can regulate the spacing of each pulse, the cell (in preferred electrode dish 2, the length of plastics tubing is 15-50cm) in 33x15-50cm electric can be melted as continuous 99 pulses, then proceed to merge from new manipulating cells fusion instrument again.Present system continually can carry out work, solves a difficult problem for Large Copacity cytogamy.
Refer to shown in Fig. 5, preferably, the present invention is a kind of to improve in the cell in vitro emerging system of bone marrow transplantation success ratio, inside of plastic tubes implants the wire 21 of six roots of sensation diameter 0.1cm, on the inwall of the embedding plastics tubing at 60 degree, six roots of sensation wire 21 interval, three adjacent one metal wires 21 connect the positive pole of cell fusion apparatus, the negative pole of three adjacent in addition connection cell fusion apparatus.Six roots of sensation wire 21 is set and can further increases cytogamy effect.
The present invention is a kind of cell in vitro fusion method improving bone marrow transplantation success ratio, comprises the following steps:
1) upper container 1, electrode plate 2, lower container 3 and electro' asion instrument is connected;
2) upper container 1 is put into by after the acceptor of extraction and donorcells mixing;
3) valve on upper and lower pipeline is opened simultaneously, and open cell fusion apparatus, regulate cell fusion apparatus to export pulse parameter: pulse height is 40-200V voltage, and pulse width is 10-60us;
4) acceptor in upper container and donorcells flow down in the plastics tubing in electrode plate 2 and merge, and plastics tubing inner recipient and stem cell donator flow out after 3 or more pulses.
Interior tumor cell proliferation test: melanoma cell B16 and donor bone marrow cell are carried out injecting after cytogamy (B16+ medullary cell+fusion) in Mice Body through the inventive method, and done two groups of controlled trials simultaneously, one group is injected melanoma cell (B16F1) 5x10
5implant C57BL/6 mouse (6-8 age in week) subcutaneous (B16), another group injects melanoma cell (B16) 5x10
5with donor bone marrow cell 5x10
5implant C57BL/6 mouse (6-8 age in week) subcutaneous (B16+ medullary cell).Refer to shown in Fig. 2, after experimentation on animals showed cell merges, the ability of medullary cell Tumor suppression growth strengthens greatly; Do not become knurl after melanoma cell B16 merges with donor bone marrow cell in body or become knurl ability to reduce.
Mouse survival test in body: refer to shown in Fig. 3, first group (in figure B16+BM cell(fusion)): by fused cell (melanoma cell (B16) 5x10
5with with dog medullary cell 5x10
5) to implant C57BL/6 mouse subcutaneous; And do two groups of controlled trials simultaneously, one group (in figure B16) by tumour cell with 5x10
5implant mouse (3-4 age in week) subcutaneous; Another group (in figure B16+BM cell) injects non-fused cell (melanoma cell (B16) 5x10
5with dog medullary cell 5x10
5) to implant C57BL/6 mouse subcutaneous.Can find that the mouse survival rate after cytogamy is apparently higher than other two groups from Fig. 3, not merge group survival rate minimum.Dog and mouse are heterogenous animals, but after two kinds of cytogamy, immunological rejection reduces, and survival rate improves, and melanoma cell B16 and dog medullary cell do not merge group, in body, strong immunological rejection occurs, adds that the impact of tumour cell itself causes survival rate to reduce.
Tumour cell electric shock experiment: refer to shown in Fig. 4 (a) He Fig. 4 (b), melanoma cell and normal marrow cell are put into electro' asion system respectively, give pulse height 200V, pulse width is 50ms, 2 pulses, put into 6 orifice plates, DMEM3ml, 10% bovine serum, cultivates basis of microscopic observation after a day in CO2 incubator, the large portion of tumour cell is dead, and Morphology of Bone Marrow, number are normal.
Claims (2)
1. improve a cell in vitro emerging system for bone marrow transplantation success ratio, it is characterized in that, comprise upper container (1), electrode plate (2), lower container (3) and cell fusion apparatus; The upper coiling of electrode plate (2) has isolated pipe, and isolated pipe inner homogeneous implants even number one metal wire (21), and half wire (21) connects the positive pole of cell fusion apparatus, the negative pole of an other half-connection cell fusion apparatus; Upper container (1) connects the inlet end of isolated pipe by upper pipeline, and the exit end of isolated pipe connects lower container (3) by lower pipeline;
Between the upper container (1) that electrode plate (2) is horizontally placed on the side of being located thereon and the lower container (3) being positioned at below it;
Wire (21) is partially submerged into isolated pipe inwall, and part is exposed in isolated pipe tube chamber;
Upper pipeline and lower pipeline are equipped with valve;
The plastics tubing of described isolated pipe to be internal diameter be 0.4-0.6cm;
Electrode plate (2) is dielectric disc or square plate;
Described even number one metal wire (21) is 6; On the inwall of the embedding plastics tubing at 60 degree, six roots of sensation wire (21) interval;
Donor bone marrow cell and receptor bone myelocyte merge when isolated pipe endomixis, and it is 40-200V voltage that cell fusion apparatus arranges pulse height, and pulse width is 10-60us; Control the recurrent interval of cell fusion apparatus, isolated pipe inner cell is flowed out after 2 ~ 5 pulses.
2. a kind of cell in vitro emerging system improving bone marrow transplantation success ratio according to claim 1, is characterized in that, the diameter of wire (21) is 0.1cm.
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| CN201310362301.4A CN103451097B (en) | 2013-08-19 | 2013-08-19 | In-vitro cell fusion system and method for increasing bone marrow transplantation success rate |
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| CN201310362301.4A CN103451097B (en) | 2013-08-19 | 2013-08-19 | In-vitro cell fusion system and method for increasing bone marrow transplantation success rate |
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| CN103451097B true CN103451097B (en) | 2015-05-27 |
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| US11377652B2 (en) | 2018-07-09 | 2022-07-05 | NanoCav, LLC | Micro flow-through electroporation devices and methods of cell transfection |
| USD1016324S1 (en) | 2020-07-08 | 2024-02-27 | NanoCav, LLC | Biological cell processing chip |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4764473A (en) * | 1983-05-13 | 1988-08-16 | Kerforshungsanlage Julich | Chamber for the treatment of cells in an electrical field |
| CN102174387A (en) * | 2011-01-25 | 2011-09-07 | 重庆大学 | Low-voltage direct-current controlled continuous flow cell electrofusion chip |
| CN102206581A (en) * | 2011-03-28 | 2011-10-05 | 北京航空航天大学 | Cell electrofusion chip |
-
2013
- 2013-08-19 CN CN201310362301.4A patent/CN103451097B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4764473A (en) * | 1983-05-13 | 1988-08-16 | Kerforshungsanlage Julich | Chamber for the treatment of cells in an electrical field |
| CN102174387A (en) * | 2011-01-25 | 2011-09-07 | 重庆大学 | Low-voltage direct-current controlled continuous flow cell electrofusion chip |
| CN102206581A (en) * | 2011-03-28 | 2011-10-05 | 北京航空航天大学 | Cell electrofusion chip |
Non-Patent Citations (1)
| Title |
|---|
| 异体树突状细胞与骨肉瘤细胞融合瘤苗的制备及其抗骨肉瘤主动免疫效应实验的研究;于哲;《中国博士学位论文全文数据库医药卫生科技辑》;20090215;E072-54 * |
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