JPH03502044A - Cell generation method - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Cell generation method field of invention The present invention relates to methods of stimulating and/or proliferating cells. Than Specifically, the present invention provides methods for using electrically conductive polymeric materials and electrical current or voltage. This invention relates to a method for culturing cells using the present invention.

Background of the invention Electrical stimulation of plant and animal cells improves their proliferative properties and motility.

It has been known for a long time that it can cause changes in the production of biologically active molecules. . They even devised a device that continuously passed a small amount of electrical current through the fracture site to stimulate healing. has been done.

Recently, Collagen Mad IJx fibroblasts have been used to prepare fibroblasts and collagen. It has been shown that things can be stimulated to proliferate by passing an electric current through the medium in which they are placed. It was. Other researchers have shown that cells grown on evaporated gold electrodes can be used to monitor cell proliferation. It was shown that it can be used as a biosensor.

How cells respond to light such as ultraviolet light and lasers.

Proteins similar in function to retinal proteins move electrons across cell membranes. It is considered that it includes.

Mechanically deforming cells can generate electric fields that can change cell function. Ru.

In the past, electrical currents were applied either through the medium or support of the cell culture system, or through individual cells. can only be imparted indirectly by a "patch-clamp" system to the cells. Ta. Obviously, the one-individual-cell method is not practical, and other methods can be used to apply the current. The equipment required for this is cumbersome and difficult to use.

The current and electromagnetic field are 1 if no mechanical pressure is applied.

May affect cell proliferation and synthetic reactions. In particular, I am interested in electrical stimulation of bone formation. It is increasing.

It is quoted here for reference. American Surgeon, 198 “In vivo response to mechanical and electrical stimulation In the report by Raphael C., Lee et al. titled "Comparison of Cellular Responses of A sinusoidal alternating voltage is applied through the membrane support at a current density of between 1.000 nanoamps. By flowing the stream, the Chrondrocytes (Chrondrocytes) It is stated that qualitatively similar changes occurred in biosynthetic activity. The used The Rustin membrane support was not electrically conductive.

Electromechanical pressure generated on the plasma membrane of suspended cells by Ant et al. discloses that cells deform within a uniform electric field. Measurable A membrane-solution system was used in which no significant current was flowing through the membrane. granted The electric field is.

Although it induces surface charges of opposite sign on each side of the membrane, 2M is not conductive and does not conduct through the membrane. , no measurable current flows.

The proliferation of many anchorage-dependent cells is known to depend on their attachment function and proliferative properties. Ru. Other stimuli such as vibration and carrier types promote cell proliferation.

It is quoted here for reference. U.S. Pat. No. 4,335 to l”olbert et al. No. 215 may be used in connection with the present invention. Continuously grow and collect cells Discloses the vegetative system and growth patches of Ruda.

that sporulation of microorganisms can be promoted by adding chemicals to the culture medium; is also known. No. 5charpf, U.S. Pat. No. 3,672,957, referenced Although it is quoted here for.

Culture conditions and culture media useful in connection with the present invention are disclosed.

It is quoted here for reference. Polymer Journal Vol. 19°Nα1. , pp. 157-172 (1987) Electrochemically from an aqueous solution In an article by Qian et al. entitled ``Prepared polypyrrole'' can be used. Electrically conductive polypyrrole and other electrically conductive polymers are disclosed. It is.

(Margin below) The amine groups of amino acids, peptides, or proteins and the glycosidic hydrocarbons of sugars Based on the interaction with xyl groups (the reaction is well known), aldoses are When heated in ethanol or water with a compound containing 7-amino groups, it becomes solid. The body produces glycosidic amides, which can be treated by cooling or by adding ether. and separate. When acid is present in excess, glycosylamine becomes 1-amino-1-deo Rearranges to xy-2-ketose derivative (Amatoli rearrangement). W amino acids are the most common Among them, lysine and ornithine seem to react best. antibody and binding It has now been discovered that similar reactions can be used to give combinations with polymer molecules. It is. Any molecule containing a terminal amine group, including but not limited to lower alkyl diamines) may also be used. Furthermore, the polymer has carboxyl groups. and bond it at the amine and carboxyl groups. It is also possible to attach antibody-binding molecules to conductive polymers by .

My baby is L! The present invention utilizes 14 qi energy to culture cells and promote cell growth. provide a way to promote Electrical energy is supplied to the support on which the cells are cultured and to the cells. Polymers that form on the surface and are present around or ingested by the cell. The conductive polymer latex is applied to the sampled conductive polymer latex. More specifically, this method , by using a conductive medium and applying electrical energy. Concerning promoting breeding. In this cell growth, microorganisms form spores. Spore germination is included.

According to an embodiment of the invention, the cells are immobilized on a support and cultured in a nutrient medium. In the present invention, 9 is grown attached to a conductive film or gel. This conductive film A current of about 0.5 to about 1 mA is passed through the film or gel.

According to another embodiment of the invention, the germination of the spores is caused by an electrically conductive material in contact with these spores. Facilitated by passing an electrical charge through a gel or polymeric material.

According to yet another embodiment of the invention, an electric charge is passed through the conductive medium in which the cells are grown. or by passing an electric charge through the medium in which the cells are cultured. growth is promoted.

Then the cells change.

According to another embodiment of the invention, the growth of cells is carried out using a conductive polymer. by surrounding it with latex and passing an electric charge from this latex to the cell membrane. will be promoted.

According to yet another embodiment of the invention, the conductive polymer latex particles An electric current is applied to stimulate these cells.

According to yet another embodiment of the invention, the conductive polymer is.

Attached to antibody molecules by direct polymerization or linking groups and oriented on the surface of cell membranes or spread across the cell membrane.

In yet other embodiments, the ingested polymer and/or conductive polymer A battery structure is formed using a combination of cells with attached antibodies.

(L-Swift Tami θ) Juho-ku n-sucking Cells that can be used in the methods of the invention can be animal, plant or microbial. animal details The animals include mammals, birds and amphibians.

Among the cells that can be propagated according to the present invention are various mammalian cells that can be cultured in vitro. cells, especially tumor cells and hybridomas. Microorganisms include bacteria, fungi, and Including viruses. Cells of fungal origin include Aspergillus and Rhizopus.

Bacterial microorganisms include Bacillus and Clostridium species. The method of the invention is particularly suitable for Escherichia coli and yeast used to produce chemical compounds by biotechnological methods It is effective for culturing.

That is, not only naturally occurring microorganisms and cells, but also transformation, DNA insertion, Microorganisms and cells modified by genetic engineering techniques such as transduction and fusion may also be used.

The present invention is particularly useful for human diploid cells. The present invention is applicable to mammals, birds and It is applicable to all types of animal cells, including amphibian and amphibian cells. in this way, Primary cells derived from embryonic, adult, or tumor cells and established cell lines primary cells can be used. Typical cell examples include the primary cells of the rhesus monkey kidney. cells, baby hamster kidney cells, pig kidney cells, mouse embryonic fibroblasts, Normal human lung embryonic fibroblasts, HeLa cells, chick primary and secondary amoblasts , as well as various cells transformed with 5V-40 or polyoma virus. There are cells.

Once the cells have grown properly, they can be harvested and further processed to the desired size by various means. Processed to produce a product.

For example, human diploid foreskin fibroblasts cultured by the method of the present invention have a vascular shape. processed for the production of plasminogen activator, plasminogen activator, and interferon can be done. Angiogenic factors can be separated from the growth medium or from the cells. plus Minogen activator is produced during a period of maturation after the cells reach their maximum concentration. , recovered from serum-free maintenance medium. Interferon affects mature fibroblasts. and its production increases due to the high concentration of cells.

In cells, DNA replication is effectively suppressed and terminated, but metabolism continues for a relatively long time. used in the process. The cells are.

Continue expression of genes except blocked genes related to DNA replication. Thin When cells are transformed with exogenous genes, these genes are expressed. to give the desired product.

By interfering with DNA replication, nutrients make it easier for microbiological reactions to take place. used more efficiently. Inhibition of DNA replication can be achieved using chemical inhibitors, temperature-sensitive mutants , or mutants lacking intermediates in the biosynthetic pathway to DNA replication. This can be achieved by a variety of means, and proliferation is promoted by the methods of the invention.

The nutrient medium used will depend on the microorganisms or cells involved and the desired product. Determined by For example, a nutrient medium can be applied to specific microorganisms or cells.

In addition to nutrients, other substances may also be used to support proliferation and/or cell differentiation. may be included. For example, enzymes, hormones, lymphocytes, toxins or immunoglobulins. natural products such as excreted proteins such as brine, or exboxylated, hydrolyzed Roxylation, esterification (e.g. acetate ester, phosphate ester, uronic acid ester) (or sulfate esters), reduction, methylation or etherification using sugars, etc. Addition of non-proteinaceous organic compounds, which occurs by transformation of substrates such as It can be done.

(Margin below) 1 bottle using standard fermentation techniques and physiological germinants. According to the invention: 1 via an electrically conductive support according to the invention.

Spore-forming microorganisms can be cultivated by passing electrical energy through them. .

Physiological sprouting materials that can be used to practice the invention include sugars, amino acids and and nucleosides. Examples of sugars include glufus, maltose, There is lactose, and sucrose. Examples of amino acids include tyrosine, L-alanine, DL-valine, DL-cysteine, methionine, gluta Mic acid, L-arginine, L-phenylalanine, L-leucine.

L-tryptophan, aspartic acid, glycine, lysine, L-isoleucine These include histidine, serine, threonine, and proline. of nucleosides Examples include inosine, guanosine, and adenosine. Glucon L -Alanine.

and inosine are preferred.

Some of these physiological germinating materials turn into one germinating spore.

More specific and one spore to germinate reduces the time for two germination phases.

For example, using glucose or L-alanine or a combination of these By * B, 5ubti10 and B, starothermo hfl spores germinate faster. In the case of spores, physiological germination By using the material inosine, one germination time is shortened.

The amount of physiological sprouting material used in accordance with the present invention is determined by It depends to some extent on the spores. The effective concentration of physiological sprouting material is 103 per 109 spores.

At least 0,001 mg. In most cases, less than 0.001mg It was found that the time required for the 9 germination phase could not be sufficiently reduced with a small amount. It was issued. Generally, the preferred range is.

From about 0.01 mg to about 10 mg per 103 to 109 spores. physiology Even if the target germination material is used in an amount exceeding 10 mg9, for example, 25 mg1, usually, There is no corresponding advantage. 2 for L-alanine, e.g. 103 to about 1 The amount is about 2 to about 5+ cg per 09 pieces.

Particularly preferred. For glucose and inosine, 103 to about 109 spores About 0.05 to 0.4 mg per serving is particularly preferred.

Electrically conductive materials that can be used in the present invention include natural and synthetic polymers. These polymers include gums, are commonly used in cell culture, and are Is it conductive?

or electrically conductive by mixing or inserting other polymers or materials. being sexualized. The above-mentioned article by Qtan et al. describes such materials and how they can be A method of rendering conductive is disclosed. One way to prepare conductive polymers is + As stated by Qlan et al., acetylene, virol, or thiophene by directly polymerizing the particles in a flexible matrix. polyacetylene, po Molecular complexes with lipyrole, polythiophene, polystyrene, etc. Polyvinyl chloride, polyvinyl alcohol, polyvinyl acetate, polyvinyl butyler polyethylene glycol, gelatin, collagen.

Guar gum, elastin, glycoprotein, carotenoid, hemin, diazobenzyl Prepared using oxymethyl, nitrocellulose, paper, etc.

The following example illustrates one invention. Without departing from the spirit and scope of the invention, It will be apparent to those skilled in the art that many variations in materials and methods are possible.

Example 1: Formation of a conductive polymer film of polypyrrole and cellulose Prepared by dissolving approximately 5.4 al of virol in 200 ml of O, lOM NaCl A cellulose dialysis membrane, which is a polysaccharide matrix, is suspended in the solution. Hanging The turbidity period (seconds to days) and solution temperature (OxC to 20xC) are subsequently does not significantly affect the formation of conductive polymer films. 4.6g persulfur 200ml of ammonium acid ((NH4)2s20s) 0.10M NaCl An oxidizing solution is prepared by dissolving it in Then, containing monomer and matrix This oxidizing solution is added to the above solution. In a few seconds (less than 1 minute), The color of the liquid changes from transparent to light blue and from light blue to black. At this point, the matric Remove the water and wash with copious amounts of 0.10M NaC+. The cleaning process uses conductive polymers Remove excess conductive polymer from the film. Loosely attached poly pillow Further remove the electrolyte by gently wiping the conductive polymer film. Ku. Air dry the resulting material. Conductivity is +0.1S/cl to I57cm Until then. This polymer.

Suitable for the growth of animal cells and spore-forming microorganisms.

Example 2: Polyaniline and cellulose electrically conductive borimam earth blood trident Prepared by dissolving approximately 20+al of aniline in 300ml of 1.0M HCI A cellulose dialysis membrane, which is a polysaccharide matrix, is suspended in the solution. Hanging The turbidity time (several seconds to 12 hours) and solution temperature (0 to 20 It does not significantly affect the formation of the conductive polymer film that takes place. 200a+1 1. 11.5g ammonium persulfate (N) 14)2S20 in OM HCI An oxidizing solution is prepared by dissolving B. This oxidizing solution is then added to the Add to the above solution containing the polymer and matrix. for a few seconds (from 1 minute) Over a short period of time, the color of this solution changes from clear to light blue-green to green. . At this point, remove the cellulose matrix and wash with copious amounts of 1.0M HCI. Purify. This cleaning step removes excess conductive polymer from the conductive polymer film. - is removed. Mild (adhered polyaniline and electrolyte make this film mild) Further removal is achieved by wiping quickly. Dry the resulting material. the As for conductivity.

0.1 to I S7cm. The stability of this material can be increased upon exposure to base. child Polymers have been used to grow fungal microorganisms.

Example 3: Graft removal of polypyrrole/polyvinyl alcohol Polyvinyl alcohol (PVA) with an average molecular fi of 88,000 (Aldric hH8,966-a) Dissolve 1.2g in 200ml of 0.1M NaCl. The mixture was allowed to dissolve and then heated to 6SxC. Once all the PVA granules have dissolved, The liquid was cooled to room temperature. Then, while stirring, add io, 8 to I virol, added to this solution. After the virol was dissolved, the solution was left overnight at 4χC. . 200ml of 0.2M ammonium persulfate cooled to 4xC was added with stirring. , was slowly added to the above PVA/virol solution. The color of the solution ranges from light line color to dark green. The color turned dark brown over time, and the mixture was left stirring at room temperature for 15 to 30 minutes. Then, This solution was filtered using glass wool. For the dark colored filtrate.

Contains polypyrrole/polyvinyl alcohol (graft) polymer in solution form. there was. This filtrate (50 val) was then added to 19 acrylonitrile in a large dish. It was gradually poured into Trill (solvent depth 1-1.5 cm) and left to stand for 1-2 hours. . By casting the polymer onto this organic solvent, a thin film of polymer can be formed. - (black) was obtained. Discard the solvent and place the black film in a 100u+1 a Washed three times with crylonitrile and dried.

Even if low molecular weight PVA (molecular weight 2000) was similarly polymerized, similar results could be obtained. It was done. Two pointed probes approximately 1 cm in length are inserted into the polymer sample to create a resistance. When measured, it was 0.08 to O, J, and Ω.

Similarly, polystyrene polypyrrole, polyvinyl butyral virol, Conductive polyamides including polyethylene virol and polypropylene virol mer can be prepared.

Example 4: DEAE-Sephadex attached to polypyrrole 4g of DEAE-cellulose (Cellex”, manufactured by BioRad), Added to 200+ol of O, 1M NaCl. 10-. Add virol from 8 to I After stirring gently for 1 hour and 1.5 hours, the mixture was left at 4xC overnight.

Rapidly add 200 ml of 0.1 M NaCl solution containing 0.2 M ammonium persulfate. was added to the Ce1leχ-monomer solution while stirring. The obtained solution is The mixture was further stirred at room temperature for 2 hours. The solution is then filtered and the precipitate is reduced to 0. Washed several times with 1M NaCl and then with water. The resulting black powder was air-dried. Ta. The resistance of a 1 cm piece of this polymer was 0.25-0.3 Ω.

Example 5: Polyphosphazene/polypyrrole graft polymer - Hexachlorocyclotriphosphazene (nitrided chloride) obtained from Aldrich When phosphorus trimer) (NPCl2)3 is heated at 250xC, the polymer amount of dichlorophosphazene polymer was produced.

This non-crosslinked polymer was then dissolved in a suitable organic solvent; further dissolved polypyrrole.

A highly reactive dichlorophosphazene polymer by reacting with A graft polymer was obtained from

However, other routes are also possible. React the above trimer with polypyrrole or by dissolving the above trimer in a solvent containing virol. It is a route. Here, virol replaces the chloride ion of the trimer. next process By adding an oxidizing agent such as ammonium persulfate to this solution, It is the polymerization of virol to polyphosphazene trimer. poly pillow Once the polymer has polymerized into a trimer, the trimer can be heated to form a non-crosslinked polyphosphor. Zen polypyrrole polymers can be formed.

If polyvinyl alcohol is used instead of the above trimer, polyvinyl alcohol A polypyrrole-polypyrrole copolymer is obtained.

(Margin below) Example 6: Attachment of polypyrrole to microparticle cellulase! 10g! particle cellulose 20 powder (Whatman, cc41.ca, t 84061-050) 0 mj2 monomer solution (10, W of pyrrole 10.1M NaCl) at 4xC. Suspended overnight. Next, the suspension of cellulose powder was gently stirred for 2 hours. After 0. IM　While stirring 0.2 ammonium persulfate 200- in NaCl Added dropwise.

Stirring was continued for 1 h. The powder was then washed 3 times with NaCl (0.1 M) and the supernatant was Further washing was performed with water until chloride ions were removed. Prepare an aqueous suspension of cellulose. ) It was cast into 17 dishes (square and circular) and left overnight to dry.

Two pimps attached to a multimeter cast cellulose resistor Measured with a lobe. The average resistance of this material is determined by placing the probe approximately 1-2 cm apart. The resistance was 0.1 to 0.7 Ω.

Example 7: Method for Attaching Polypyrrole to Collagen Soluble collagen (4■) (type ■.

acid soluble, NCLC-3511) in 2rn1 acetic acid (diluted 1:1000) ) at 4xC for 4.8 hours.9 then 23. 1 o'clock on OOORPM Centrifuged for a while. 1/6 volume of 0.1N Na0i1 was added to the supernatant (one sample of supernatant was mixed with 166 μl of Na0)1). A white precipitate was obtained. Mixture 15 Centrifuged at 0 ORPM for 20 minutes. Next, the formed white pellet was j! of virol (0.1 M virol in 0,IM NaCl) solution overnight at 4xC. suspended. Next, 0.1M ammonium persulfate solution (0.IM in NaCl) Add dropwise to the collagen/virol solution, swirl, and 4. Leave to incubate for 1 hour at xC. The resulting black suspension was heated to 150 ORP. Centrifuged at M for 20 minutes. Discard the supernatant and wash the pellet 3 times with 0.1M NaCl. It was then washed several times with distilled water. Add 1 ton of acetic acid (1 part) to a black pellet. Add 1000).

The mixture was left at 4xC overnight. The suspension was centrifuged for 30 minutes.

Unbound polypyrrole and collagen are removed into the supernatant. kolar Dissolve the gen/polypyrrole complex in ethanol solution and spray it on the glass surface. By doing so, collagen/polypyrrole was made into a thin film. evaporate organic solvent At the same time, biomolecules were left on the 2 surfaces.

The effect of cell proliferation was observed using BHK fibroblasts according to the method of (1973). I guessed it.

Polystyrene-virol blood prepared in accordance with the present invention with a diameter of 5. 105 cells of BHKC13 were inoculated per ground. Two types of media were compared: Contains IM magnesium acetate in 5BK (saline dipotassium bicarbonate).

Complete medium with serum-free minimal salt buffer and 10% serum.

Four conductive posts to which two platinum wires were fixed were placed on one blood. 0 ．． A constant current of 5μ was applied. Another blood was used as a control.

A spread is a uniform, polygonal shape that is no longer round and has sharp edges. Measured as a percentage of cells. Cells that simply increase in size and are not rounded are called " "Proliferation" was not recognized.

After 30 minutes, the rising negative surface charge density was measured by dye binding. Chris the surface Equilibrate with violet cationic dye solution (0.1mM in distilled water) and distill Washed with water. Acid-ethanol binds to the fixed negative charge and removes the remaining dye. The sample was eluted using a filter and measured using a colorimetric method.

The percent per 10 Car of cell proliferation measured in the control charge was 0.6. there were. Cell proliferation of electrically stimulated cells was 70.

Fibroblasts, yeast, hybridomas, bacteria and fungi can also be grown using this method. It is possible.

(Margin below) Example 9: Attachment and expansion of HeLa cells to polypyrrole/collagen films breeding Late log phase tleLa cell cultures growing in plastic dishes were (7) EGTA (2mM) and ) in PBS! J7'shi: / (0,05%) One recovery was made by exposing the sample to water for 5 minutes. The cells were centrifuged at 200g for 5 minutes. MEM containing 10% fetal bovine serum suspended in it. 1- of this cell suspension was used as the primary, and as a control, a natural cell suspension was used. 35 cylinders of plastic tissue culture containing the gel of Lagen fibers and the gel of Example 7. Initially, 104 cells were obtained per 9 culture plates. humidify these dishes The cells were incubated for 120 minutes at 37×C in a CD□ atmosphere. Biro The gel-containing dish contains a plate connected to a conductive post inserted into the gel. A constant current of 0.5 mA was applied through the latina wire.

The total number of cells was estimated as follows. In other words, gel.

Collagenase in serum-free HEM (Sigma, C-2139, 0.2 mg/m Dissolve it in a sieve and place it in a Coulter electron particle counter. Estimation is made by counting cells with an onic particle counter). established.

The number and percentage (%) of cells detached by exposure to trypsin were determined in two stages. Parallel experiments were carried out according to the method. first.

Wash the dishes with Hank's Balanced Salt Solution and 0.25% trypsin (in PBS) for 10 min. The cells were incubated for a period of time, and the number of detached cells was counted. Next, apply the gel to collagenase lyse by exposure and determine the number of remaining cells and remaining adherent cells. was determined as above. Total cell number was estimated by this second method.

That is, the number of cells detached by trypsin and the number of cells remaining attached to the gel. Estimated by adding the number of cells.

This means that the total number of cells obtained by simply digesting the gel with collagenase is 10 There was always agreement within a range of %.

In electrically stimulated dishes, cell attachment is rapid and extensive. Approximately 95 % of cells attached and proliferated. In the control, adhesion was approximately 70%. Ta.

Example 10: Bacillus Stearothermophilus spores Control slant cultures of child B-Stearothermophilus .

Transfer to 100 volumes of nutrient medium in a 500 ml Erlenmeyer flask. This frame Place the lasco on a rotary shaker for 24 hours at 300 RPM. Sterilize Add 0.5 of the contents of the flask to a 100 x 15 mm plate of nutrient agar. Inoculate ~1.0d and hold at 55xC for 7 days. During this period, microorganisms form spores. do. What is the density of spores?

10e' pieces/ml in solution. Bacillus spores as described above. Prepared. However, according to the method of Example 3, polypyrrole-polyvinyl alcohol 100 x 15 plates were coated using the 100 x 15 plate. Constantly 0.8A After applying the current and incubating for 2 days, the density of spores in the solution was It was 1010 pieces/d.

Example 11: Polyvinyl alcohol-virol latex virol (Aldri ch Che + n1 cals) was vacuum distilled and cooled at 4xC under an argon atmosphere. Stored in storage. Water was double distilled in an Allvirex apparatus. Ferric chloride (F eC13・61120; B111l), potassium iodide (BDH; ″A nalaR” grade), iodine (BDH) and boric acid (BDH, “Ana 1aR'' grade) was used as is.

Two samples of partially hydrolyzed poly(vinyl acetate) were used as is. ta = (i) apparent relative molecular weight (relative molarllaSs) ], 25. 88% hydrolyzate (BDH) which is OOO (PVA-88); (ii) Polyvinyl alcohol (PVA- 96), 96% hydrolyzate (Aldrich Chemicals).

The suspension polymerization reaction was carried out in a 500 ml flask. First, 0.3g of PVA and 8.83g (0,033 mol) of FeC1,”6H,0 and 1001n1 Dissolved in water. 1 mf (0,014 mol) of purified virol monomer was added to this solution. mer and the mixture was heated at 20xC for 18-14 hours. Stir with a tarer. In all cases.

During this time, a monomer conversion of over 95% was achieved.

Within a few minutes of adding virol to the reaction solution, the color changed from orange to brown-black. This indicated that polymerization had started. However, no precipitate was formed. It was. At the end of this reaction period, the suspension formed was heated to about 1 Centrifuged for hours. This results in a black precipitate and a pale green but transparent upper layer. Qing occurred. Of course, by visible absorption spectroscopy, the supernatant was The results showed that it did not contain any alcohol. Shake the sediment in pure water could be easily redispersed. The dispersion thus formed is allowed to stand still. No tendency to agglomerate was shown. Freeze-drying the aqueous dispersion produces a fine black color. It became powder.

Various other examples and modifications of the above-described embodiments can be realized by reading this specification. without departing from spirit and scope.

It will be clear to those skilled in the art. All such instances and modifications.

Within the scope of the appended claims.

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Claims (19)

[Claims] 1. In a method of stimulating and/or growing cells in a nutrient medium, the cell contacting the cells with an electrically conductive material and an amount that promotes growth and proliferation of the cells; The improvement is to pass a current through the medium or apply a voltage to the medium. How to do it. 2. 2. The method of claim 1, wherein the current is about 0.5 to about 1 mA. 3. 2. The method according to claim 1, wherein the cells are mammalian cells, avian cells, bisexual cells. selected from the group consisting of microbial cells, microorganisms, and plant cells. 4. 2. The method of claim 1, wherein the medium includes a polymeric support. 5. 5. The method of claim 4, wherein the polymeric support is electrically conductive. 6. 5. The method according to claim 4, wherein the polymer support comprises collagen, elastomer, etc. polymers selected from the group consisting of stin, guar gum and gelatin. nothing. 7. 7. The method according to claim 6, wherein the polymer is mixed with a conductive polymer. Conductivity is imparted by applying or combining them. 8. 8. The method according to claim 7, wherein the conductive polymer is polyacetylene, Polypyrrole, polypyrrole cellulose, polyaniline cellulose, polypyrrole From the group consisting of polyvinyl alcohol and polypyrrole collagen selected. 9. 2. The method of claim 1, wherein the method comprises exposing cells to a conductive polymer latex. and passing an electric charge through the latex. 10. 2. The method of claim 1, wherein the conductive medium is a polymer, and the conductive medium is a polymer. The remer is taken up by the cells. 11. 2. The method of claim 1, wherein the conductive medium is a polymer, and the conductive medium is a polymer. The remer is attached to the antibody by a linking group. 12. 12. The method according to claim 11, wherein the linking group is an alkyldiamine. . 13. A device for growing cells, comprising an electrically conductive material and a means for passing an electric current through the material. A device equipped with 14. 14. The apparatus of claim 13, wherein the conductive material is assembled with a conductive polymer. It is a support material that has been combined with other materials. 15. 15. The device of claim 14, wherein the support material is nylon, polysalt. vinyl oxide, polyvinyl alcohol, polyvinyl acetate, polyvinyl butyral, polyvinyl Liethylene glycol, gelatin, collagen, guar gum, elastin, saccharide protein, carotenoid, hemin, diazobenzoyloxymethyl, nitrocellulose selected from the group consisting of: 16. 15. The device of claim 14, wherein the conductive polymer is polyacetylene. selected from the group consisting of polypyrrole, polythiophene, and polystyrene. It will be done. 17. 14. The device of claim 13, wherein the electrically conductive material is attached with growing cells. It can be supported by wearing. 18. 14. The device according to claim 13, wherein the material is grown by cells to be grown. Ingested. 19. 14. The apparatus of claim 13, wherein the electrically conductive material is attached to the cells to be grown. bound to a specific antibody.