CN101851579A

CN101851579A - Novel electric field bioreactor for researching wound healing

Info

Publication number: CN101851579A
Application number: CN201010168125A
Authority: CN
Inventors: 吴江; 杨刚; 何学令; 郭应强; 陈槐卿
Original assignee: 吴江
Current assignee: Sichuan University
Priority date: 2010-05-10
Filing date: 2010-05-10
Publication date: 2010-10-06

Abstract

The invention discloses a novel electric field bioreactor for researching wound healing, and relates to a bioreactor device. The bioreactor comprises a cell culture chamber, two cell culture medium ponds, two agar salt bridges and two Steinberg solution ponds, wherein the two cell culture medium ponds are positioned at the two ends of the cell culture chamber sealed by a culture chamber cover; a gap is formed between the culture chamber cover and the bottom of the cell culture chamber to form an oblong cavity; the cavity is communicated with the cell culture medium ponds at the two ends; an electric field test probe is inserted into the two cell culture medium ponds respectively; the two cell culture medium ponds are connected with one Steinberg solution pond through one agar salt bridge respectively; a carbon electrode is inserted into the two Steinberg solution ponds respectively; and the wires of the carbon electrodes are connected with an electric field source. The electric field bioreactor device has the advantages of stable and uniform electric field, nontoxic and harmless characteristic, real-time observation, simple and convenient operation and capability of acting on cultured cells for a long time.

Description

A kind of novel electric field bioreactor that is used for researching wound healing

Technical field

The present invention relates to a kind of bioreactor device, relate in particular to a kind of novel electric field bioreactor that is used for researching wound healing.

Background technology

After epithelium is injured, can produce an endogenous electric field, this electric field pair cell healing plays a part very important.Study the reaction of cell, thereby further understand the mechanism of cell healing biological DC electric field.But in actually operating, the investigator is difficult to directly with wound as research object, so can carry out cytology research by electric field in the simulated tissue or organ culture.The experimental installation of existing a kind of galvanotaxis culturing room is such: the tissue culture ware of standard, and the inside is full of silicone grease, two long strip shape glass partition 1cm, is fixed on the culture dish bottom in parallel to each other; The groove that cell places glass cover noted earlier to form also has a glass cover to be fixed on the silicone grease top and goes up across whole groove; The aqueous paper slip of bag is placed in the plate to keep humidity; Two 10cm are long, the insides are full of the brinish Glass tubing that contains 2% agar, i.e. salt bridge, and the hole of passing respectively on the glass cover is electrically connected with culturing room; The Ag/AgCl electrode all is connected with direct supply in the beaker.One end of Glass tubing places the nutrient solution of cell, and the other end places the salt solution of beaker.By this device can observation of cell to the reaction of electric field.The galvanotaxis culturing room of another kind of using electric field: the large container control that the electric field experiment is made up of two resin glass culture dish, two culture dish connect by a throat, and platinum wire electrode places the opposite end of container respectively.Be full of the gel of the cell culture fluid composition of dissolving 2% agar in the container, the connecting passage top that is full of nutrient solution is fixed and be installed in to the glass cover and the agar that have attached cell.Glass cover is provided with the glass vision panel that can observe connecting passage.But there is following shortcoming in these devices:

One, the electric field at cell culture chamber two ends is not a parallel electric field, be the equal of an electric field, salt bridge connects cell culture medium and power supply, and an end of salt bridge tube forms point source, therefore the middle portion of culture dish only being arranged is parallel electric field, and other parts are not the electric field of a homogeneous.When two electrodes directly inserted nutrient agar, the electric field in the cell culture chamber neither the homogeneous electric field.In addition, electrode directly inserts nutrient agar, and joule heating concentrates on the two poles of the earth, can not be distributed in real time, and the time has been grown the connection that will change between electrode and the nutrient agar, causes the electric field instability in the cell culture medium, and direction of an electric field is inconsistent;

Two, thereby all culturing room's glass cover is fixed by silicone grease and is formed a fixedly geometric space, and silicone grease directly contacts with cell, and this will influence the cytoactive of substratum the inside;

Three, salt bridge is enclosed in the Glass tubing, and occupation space is very big, is unfavorable for real-time cell observation;

Four, long electrical field stimulation can change the pH value, temperature and the chemical densities that connect the liquid level between cell culture chamber and the salt bridge;

Five, Ag/AgCl electrode product pair cell is deleterious, can enter in the cell culture chamber by salt bridge in test, influences cytoactive.

Summary of the invention

At the problem of above-mentioned prior art existence, but the object of the invention is to provide a kind of electric field stable uniform, nontoxic ground long duration of action in culturing cell, and can electric field bioreactor device real-time monitored, simple to operation.

Technical scheme of the present invention is: a kind of novel electric field bioreactor that is used for researching wound healing, comprise a cell culture chamber, two cell culture medium ponds, two agar bridges and two Staenberg solution (steinberg solution, literal translation is Staenberg solution) pond, described two cell culture medium ponds are positioned at by the cell culture chamber two ends of cultivating the chamber cap sealing, interbody spacer one gap forms a rectangular parallelepiped cavity at the bottom of cultivating chamber cap and cell culture chamber, this cavity is communicated with the cell culture medium pond at two ends, be inserted with electrical field test probe in the two cell culture medium ponds respectively, described two cell culture medium ponds are connected with a Staenberg solution pool by an agar bridge respectively, the two ends of described agar bridge are immersed respectively in cell culture medium pond and the Staenberg solution pool, and the agar bridge two ends are the rectangular plate that parallels, the also parallel symmetry of agar bridge termination rectangular plate at cell culture chamber two ends, also be inserted with a carbon dioxide process carbon electrode in the two Staenberg solution pools respectively, the carbon dioxide process carbon electrode lead connects electric field source.

As preferably, comprise base plate and cover plate that a resin glass is made, the cover plate fixed interval is in the base plate top, base plate is provided with five grooves and constitutes a cell culture chamber, two cell culture medium ponds and two Staenberg solution pools, and the groove that is communicated with cell culture medium pond and Staenberg solution pool is full of the salt agar gel and constitutes described agar bridge.

Further, cultivate interbody spacer 300 μ m at the bottom of chamber cap and the septal cell culturing room described in above-mentioned each scheme.

The invention has the beneficial effects as follows: by the observation of cell cultures, electric field distribution is calculated and test, the real-time monitoring of cell culture environment such as pH value etc., culturing cell migration etc., confirm that this bioreactor device can repeat the research of foreign literature report, the electric field stable uniform, easy and simple to handle, stable performance can be applicable to cells involved to the biomedicine of electric field reaction and the research of biophysics aspect.

Description of drawings

Fig. 1 is the sectional view of the embodiment of the invention 1;

Fig. 2 is the vertical view of Fig. 1;

Fig. 3 is the vertical view of the embodiment of the invention 2;

Fig. 4 is the electric field distribution scaling system;

Fig. 5 is a current density distributing figure;

Fig. 6 is microscope observing cell migration original state;

Fig. 7 is microscope observing cell migration state after 4 hours;

Fig. 8 is microscope observing cell migration state after 8 hours;

Fig. 9 is microscope observing cell migration state after 16 hours;

The track that Figure 10 moves under electric field action for cell (the diagram left side is negative electrode);

Figure 11 is the statistics (X, the parasang of Y-axis are μ m, and the diagram left side is a negative electrode) of cell traveling locus under electric field action.

Embodiment

As one embodiment of the present invention, a kind of novel electric field bioreactor that is used for researching wound healing, comprise a cell culture chamber 4, two cell culture medium ponds 3, two agar bridges 6 and two Staenberg solution pools 2, described two cell culture medium ponds 3 are positioned at by cell culture chamber 4 two ends of cultivating chamber cap 5 sealings, cultivate chamber cap 5 and interbody spacer one gap, 4 end of cell culture chamber and form a rectangular parallelepiped cavity, this cavity is communicated with the cell culture medium pond 3 at two ends, be inserted with electrical field test probe 10 in the two cell culture medium ponds 3 respectively, described two cell culture medium ponds 3 are connected with a Staenberg solution pool 2 by an agar bridge 6 respectively, the two ends of described agar bridge 6 are immersed respectively in cell culture medium pond 3 and the Staenberg solution pool 2, agar bridge 6 forbids that any exchange is arranged between Liang Chi, and agar bridge 6 two ends are the rectangular plate that parallels, the also parallel symmetry of agar bridge 6 termination rectangular plates at cell culture chamber 4 two ends, just as a battery lead plate, so cell culture chamber 4 is exposed in the electric field.Also be inserted with a carbon dioxide process carbon electrode 8 in the two Staenberg solution pools 2 respectively, carbon dioxide process carbon electrode 8 leads connect electric field source 9.Electrode byproduct in the Staenberg solution pool 2 is a carbon, and is harmless to substratum.Described cultivation chamber cap 5 and 4 end of cell culture chamber interbody spacer 300 μ m.

As preferably, embodiment 1 as depicted in figs. 1 and 2, comprise base plate 1 and cover plate 7 that a resin glass is made, cover plate 7 fixed interval are in base plate 1 top, base plate 1 is provided with five grooves and constitutes a cell culture chamber 4, two cell culture medium ponds 3 and two Staenberg solution pools 2, and the groove that is communicated with cell culture medium pond 3 and Staenberg solution pool 2 is full of the salt agar gel and constitutes described agar bridge 6.

Another embodiment 2, as shown in Figure 3, for economy system shared space in the cell thermostat container, agar bridge 6 can also be bent into the L type, thereby reduce the length of base plate 1, in order to keep normal carbonic acid gas and to discharge water electrolytic gas, cover plate 7 is fixed on from the place of base plate 1 high 3mm by 8 plastic screws, and all the other are identical with embodiment 1.

The electric field simulation result is shown in Figure 4 and 5.Obviously, the EF in the cell culture chamber is uniform parallel field (as shown in Figure 4), and electric current distribution as shown in Figure 5.Find that easily the contact of close agar bridge end has radiation current.But the sense of current unanimity between bridge end and the substratum.Here it is, and the salt bridge termination cuts into rectangular plate to obtain the reason of parallel EF.In order to confirm the function of this novel galvanotaxis bio-reactor, human body epithelial cell sequence Hy926 was cultivated 16 hours, then in online timing microscope (Olympus CCD microscopic system) observation of cell migration down.Write down the cell migration track and it is handled by Image-pro plus5.0 (IPP5.0) analysis software.There is the cell directional migration in the experimental result demonstration, and cell has the galvanotaxis behavior (seeing Fig. 6 to Figure 10) to negative electrode.Come the computation migration distance by IPP5.0, by Microsoft EXCEL 2000 gained cell migration data are converted to scatter diagram (as shown in figure 11) then, the left side of Figure 10 and Figure 11 is a negative electrode, and obviously, most cells is to cathodic migration.

Claims

1. novel electric field bioreactor that is used for researching wound healing, it is characterized in that: comprise a cell culture chamber, two cell culture medium ponds, two agar bridges and two Staenberg solution pools, described two cell culture medium ponds are positioned at by the cell culture chamber two ends of cultivating the chamber cap sealing, interbody spacer one gap forms a rectangular parallelepiped cavity at the bottom of cultivating chamber cap and cell culture chamber, this cavity is communicated with the cell culture medium pond at two ends, be inserted with electrical field test probe in the two cell culture medium ponds respectively, described two cell culture medium ponds are connected with a Staenberg solution pool by an agar bridge respectively, the two ends of described agar bridge are immersed respectively in cell culture medium pond and the Staenberg solution pool, and the agar bridge two ends are the rectangular plate that parallels, the also parallel symmetry of agar bridge termination rectangular plate at cell culture chamber two ends, also be inserted with a carbon dioxide process carbon electrode in the two Staenberg solution pools respectively, the carbon dioxide process carbon electrode lead connects electric field source.

2. a kind of novel electric field bioreactor that is used for researching wound healing according to claim 1, it is characterized in that: comprise base plate and cover plate that a resin glass is made, the cover plate fixed interval is in the base plate top, base plate is provided with five grooves and constitutes a cell culture chamber, two cell culture medium ponds and two Staenberg solution pools, and the groove that is communicated with cell culture medium pond and Staenberg solution pool is full of the salt agar gel and constitutes described agar bridge.

3. a kind of novel electric field bioreactor that is used for researching wound healing according to claim 1 and 2 is characterized in that: interbody spacer 300 μ m at the bottom of described cultivation chamber cap and the septal cell culturing room.