JP4626725B2 - Collagen for cell culture - Google Patents

Description

  The present invention relates to a nerve regeneration inducing tube for reconnecting peripheral nerves cut or excised in an accident or surgery by utilizing the elongation of nerve cells. More specifically, the present invention uses collagen as a scaffold for nerve regeneration, which is used to fix the extending direction of a cut or excised nerve tissue and to join the cut sites without being disturbed by surrounding tissues. The present invention relates to a nerve regeneration induction tube.

  In many cases, peripheral nerve damage due to accidents cannot be repaired. In addition, there are many clinical cases in which peripheral nerves have to be excised with general surgery. For peripheral nerve damage, autologous nerve transplantation was the only measure other than direct anastomosis. However, the results were never satisfactory, the recovery of perception and motor ability was poor, and there were also sequelae due to erroneous control. In addition, there are many patients who suffer from not only aftereffects such as pain and sensory deficits, but also abnormal perception of the affected area, particularly pain.

  Attempts to regenerate nerves by connecting gaps in peripheral nerves using joint tubes made of artificial materials have been actively conducted since the early 1980s. However, research into bonded channels with non-absorbable synthetic artificial materials has failed. In order to solve this problem, it is necessary to prevent the invasion of connective tissue from the outside during regeneration of the nerve bundle, the exchange of substances inside and outside the channel or the formation of capillaries in the channel wall, Consideration must be given to the fact that a substance that can serve as a scaffold suitable for Schwann cell growth is necessary, and that the material used is decomposed and absorbed after regeneration. Considering these conditions, research on artificial nerve junction tubes using biodegradable and absorbable materials has been conducted since then.

  Regarding the regeneration of peripheral nerves, attempts have been made to extend the reproducible stump distance using silicone tubes since the introduction of the silicone tube model in 1982. However, the wall of the silicone tube cannot pass through nutrients, so there are problems such as insufficient supply of nutrients to nerve axons. Capillaries cannot be formed in the silicone, and the silicone tube Satisfactory nerve regeneration has not been obtained using Furthermore, even if the nerve can be regenerated, there is a problem that the silicone tube, which is a foreign substance, must be removed by re-operation or the like.

  On the other hand, regeneration of peripheral nerves using a tube made of a biodegradable polymer instead of a silicone tube has been attempted. If a nerve regeneration tube made of a biodegradable polymer is used, the nerve regeneration tube is gradually decomposed and absorbed in vivo by hydrolysis or the action of an enzyme after the nerve is regenerated. There is no need to take it out.

As a nerve regeneration tube composed of such a biodegradable polymer, for example, Patent Document 1 discloses a nerve regeneration assisting material composed of a bundle of collagen fibers coated with laminin and fibronectin. Patent Document 2 is composed of a biodegradable absorbent material tube and a collagen body having a cavity penetrating the tube along the tube substantially parallel to the axis of the biodegradable absorbable material. An artificial neural tube filled with a matrix gel containing is disclosed. Patent Document 3 discloses a tube of biodegradable absorbable material and an artificial neural tube in which a collagen fiber bundle covered with laminin is inserted into the lumen of the tube substantially parallel to the axis of the tube. Patent Document 4 discloses a nerve reconstruction base material having a structure in which fibers made of a bioabsorbable material are bundled. Patent Document 5 discloses a support such as a sponge, tube, or coil made of collagen. Patent Document 6 discloses a support comprising a sponge-like fine matrix made of a biodegradable material or a bioabsorbable material, and a linear biological tissue guide route or organ guide route. Furthermore, Patent Document 7 discloses a nerve regeneration tube including a sponge made of a biodegradable polymer material and a reinforcing material made of a biodegradable polymer having a longer decomposition and absorption period than that of the sponge, the inner surface of which is made of a sponge. Yes.
All of these nerve regeneration tubes use collagen as a scaffold for nerve regeneration, but collagen adhesion, cell proliferation ability and differentiation inducing ability of collagen are not sufficient.

JP-A-5-237139 WO98 / 22155 WO99 / 63908 JP 2000-325463 A JP 2001-70436 A JP 2002-320630 A Japanese Patent Laid-Open No. 2003-19196

  The present invention was devised in view of the current state of the prior art, and its purpose is to induce nerve regeneration using collagen excellent in nerve cell adhesion, cell proliferation ability and differentiation inducing ability as a scaffold for nerve regeneration. To provide a tube.

  As a result of studying a method for producing collagen used as a scaffold for nerve regeneration in order to achieve such an object, the present inventor inevitably mixed during washing and salting out when producing collagen from raw materials such as pig skin. It has been found that sodium chloride has an adverse effect on nerve cell regeneration, and that the use of collagen with a reduced concentration of sodium chloride improves nerve cell proliferation and neurite outgrowth. The invention has been completed.

  That is, the present invention is carried out by isoelectric precipitation at a pH of 7.0 or more and 9.5 or less in a nerve regeneration-inducing tube using collagen as a scaffold for nerve regeneration, and a sodium chloride content concentration of 2.0% in a dry state. % Nerve regeneration-inducing tube, characterized in that it uses collagen purified to a concentration of 0.1% or less, preferably 0.1 to 1.5% by weight.

  In a preferred embodiment of the nerve regeneration-inducing tube of the present invention, the collagen is purified by isoelectric precipitation at a pH of 8.0 or more and 9.0 or less, and the nerve regeneration-inducing tube is formed on a tubular body made of a biodegradable polymer. The biodegradable polymer is selected from the group consisting of polyglycolic acid, polylactic acid, and lactic acid-caprolactone copolymer, and the tubular body has an inner diameter of 0.1. -20 mm, outer diameter 0.15-25 mm, length 1.0-150 mm.

  The present invention uses a purified collagen in which the concentration of sodium chloride, which is inevitably mixed in the collagen production process, is reduced to 2% by weight or less, thereby allowing adhesion of nerve cells, cell proliferation ability and differentiation inducing ability. It is possible to provide an excellent nerve regeneration induction tube.

It is a microscope picture which shows the mode of the cell in the collagen gel of the example of this invention. It is a microscope picture which shows the mode of the cell in the collagen gel of a comparative example. 4 is a graph of absorbance (relative value) measured in Experiment 1. 5 is a graph of absorbance (relative value) measured in Experiment 2. Details of collagen coats 1 to 6 used in Experiment 3 are shown. 5 is a graph of absorbance (relative value) measured in Experiment 3. 5 is a graph of absorbance (relative value) measured in Experiment 3. It is a microscope picture which shows the mode of cell differentiation of the cell cultured on the collagen (pH5.5) coat plate of Experiment 4. It is a microscope picture which shows the mode of cell differentiation of the cell cultured on the collagen (pH8.5) coat plate of Experiment 4. It is a microscope picture which shows the mode of cell differentiation of the cell cultured on the collagen (pH 10.2) coat plate of Experiment 4. 4 is a graph of cell differentiation rate after 4 days of culture measured in Experiment 4. FIG. 6 is a graph of cell differentiation rate after 11 days of culture measured in Experiment 4. FIG.

  The nerve regeneration-inducing tube of the present invention is characterized by using collagen purified as a scaffold for nerve regeneration so that the sodium chloride-containing concentration mixed in the collagen production process is 2% by weight or less in a dry state.

  Collagen plays a role as a substrate for various cells, and therefore, when applied to a living body as a medical material, it has a good affinity with tissue and has been conventionally used as a scaffold for the growth of nerve cells.

  Conventional collagen used as a scaffold for nerve regeneration is generally made from pork skin collected and frozen at a meat inspection station, neutral protease is added to this, heated, and washed repeatedly with sodium chloride solution. After dehydration, wash with isopropanol and acetone, dry under reduced pressure to make a degreased chip, add this degreased chip into acetic acid solution, adjust pH with hydrochloric acid, add and decompose pepsin, sodium hydroxide solution Adjust to high pH (virus inactivation step 1), adjust to low pH with hydrochloric acid (virus inactivation step 2), adjust to pH 2-3 with sodium hydroxide and filter, add sodium chloride solution Salt out, concentrate by centrifugation, add and dissolve this concentrate in purified water, add sodium chloride solution again to salt out, concentrate by centrifugation, and freeze-dry. Thus it is produced.

Thus, since conventionally used collagen includes washing with a sodium chloride solution and salting out with a sodium chloride solution in the production process, the sodium chloride concentration of the collagen used is 4% by weight, including a commercially available product. That was all. The present inventor considered that the concentration of sodium chloride in collagen affects the survival and growth of nerve cells, and if this concentration is too high, the cell membrane is destroyed by osmotic pressure. Therefore, when collagen purified so as to reduce the concentration of sodium chloride was used in the nerve regeneration induction tube, this nerve regeneration induction tube exhibited extremely superior cell adhesion and cell proliferation ability than those using conventional collagen. I found out. Based on this finding, the present invention was processed to reduce the concentration of sodium chloride in a dry state as a scaffold for nerve regeneration-inducing tubes to 2.0% by weight or less, preferably 0.1 to 1.5% by weight. Use collagen. This sodium chloride concentration is measured by atomic absorption photometry (ashing). In order to prevent cell membrane destruction by lowering the osmotic pressure, the sodium chloride concentration should be low, but it is considered that the lower limit is about 0.1% by weight in terms of technical aspects and collagen stability. The treatment for reducing the salt concentration includes isoelectric point precipitation (concentration) as described later, and a method by dialysis, and any known method can be used in the present invention.
In addition, the measurement of sodium chloride concentration by atomic absorption spectrophotometry takes 1 to 4 g of a sample in a quartz beaker, gradually raises the temperature on an electric heater and carbonizes it, and finally takes 6 to 8 hours in a muffle furnace. Ashing (500 ° C.), the residue is redissolved with a 10% by weight aqueous hydrochloric acid solution, diluted to a final concentration of 1% by weight, and measured by flame atomic absorption spectrometry using acetylene-air. The measurement wavelength at this time is 589.6 nm.

  The collagen used in the nerve regeneration-inducing tube of the present invention can be produced by any conventionally known method. For example, the conventional collagen commercially available for medical use described above is used as a starting material, and this collagen is cooled at 2 to 10 ° C. Is dissolved in distilled water for injection, adjusted to pH 6.0 or more and less than 10.0 with sodium hydroxide solution, subjected to isoelectric precipitation, centrifuged, the supernatant discarded, and the precipitate freeze-dried Can be manufactured. It has been found that by using collagen having such an isoelectric point range, extremely excellent cell differentiation inducing ability is exhibited, and the present invention has finally been completed. The reason why the differentiation-inducing ability of cells is improved by using collagen having an isoelectric point of pH 6.0 or more and less than 10.0 as a scaffold for nerve regeneration has not been elucidated in detail, but is less than pH 6.0 and 10 It is possible that factors that have a low affinity with cells are contained in the fraction that precipitates at 0.0 or more, and conversely, collagen that precipitates at pH 6.0 or more and less than 10.0 is particularly compatible with cells. It is possible that the property is high. Alternatively, unpurified collagen is composed of a type I collagen and a type III collagen in a ratio of approximately 7: 3, but the influence of the change in the composition ratio of the type I and type III is also considered. In the present invention, a more preferable isoelectric point range is pH 7.0 or more and 9.5 or less, and further preferably pH 8.0 or more and 9.0 or less.

  The nerve regeneration-inducing tube of the present invention can be produced according to a conventionally known method. For example, it is formed by coating a tubular body made of a biodegradable polymer with collagen and filling the inside of the tubular body with collagen. can do. The size of the tubular body is generally 0.1 to 20 mm in inner diameter, 0.15 to 25 mm in outer diameter, and 1.0 to 150 mm in length, although it depends on the part of the nerve to be regenerated and the required strength. Actually, it is preferable to prepare in advance a nerve regeneration guide tube made of a large number of types of tubular bodies in view of time constraints and production costs.

  Examples of the biodegradable polymer constituting the tubular body include polyglycolic acid, polylactic acid, lactic acid-caprolactone polymer, glycolic acid-caprolactone copolymer, polydioxanone, and glycolic acid-trimethylene carboxylic acid. From the viewpoint of easy availability and handling, it is preferable to use polyglycolic acid, polylactic acid, lactic acid-caprolactone copolymer, particularly polyglycolic acid. A biodegradable polymer may be used independently and may be used in mixture of 2 or more types.

  As the tubular body, a product obtained by molding the biodegradable polymer into a porous tubular body may be used, or a tubular body obtained by bundling a plurality of ultrafine fibers of the biodegradable polymer. It may be used. What is necessary is just to adjust suitably the pore diameter and porosity of a porous body or a stitch (mesh) according to the intended use and intensity | strength.

  Moreover, it is preferable that the diameter of the ultrafine fiber which consists of a biodegradable polymer is 1-50 micrometers. If the fiber diameter is too small, the fiber gap becomes dense, so that it is difficult for collagen to permeate or the flexibility of the tubular body may be reduced. On the other hand, if the fiber diameter is too large, the amount of collagen retained decreases, and the nerve growth rate may not increase or the strength of the tubular body may be insufficient. More preferably, the diameter of the ultrafine fiber is 3 to 40 μm, and more preferably 6 to 30 μm.

  In order to form a tubular body, it is preferable that 5 to 60 ultrafine fibers made of a biodegradable polymer having the above fiber diameter are bundled and alternately knitted as warps and wefts. If the number of ultrafine fibers bundled is too small, the strength of the tubular body may be insufficient, or a sufficient amount of collagen may not be secured. Conversely, if the number of ultrafine fibers bundled is too large, it may not be possible to create a thin tubular body or secure the flexibility of the tubular body. More preferably, the number of ultrafine fibers is 10 to 50, and more preferably 20 to 40.

  When forming the tubular body by alternately knitting the ultrafine fiber bundle, the mesh pore diameter is preferably about 5 to 300 μm, more preferably 10 to 200 μm. When the pore size of the mesh is too small, the proliferation of cells and tissues may be inhibited due to the invasion of capillaries and the decrease in water permeability. If it exceeds about 300 μm, the tissue may enter excessively and the growth of cells and tissues may be inhibited.

  In the present invention, the outer surface of the tubular body is coated by applying a collagen solution a plurality of times by a method known to those skilled in the art, and the interior (lumen) of the tubular body is filled with collagen. At this time, the collagen solution may contain laminin, heparan sulfate proteoglycan, entactin and growth factor. As growth factors, EGF (epidermal growth factor), βFGF (fibroblast growth factor), NGF (nerve growth factor), PDGF (platelet-derived growth factor), IGF-1 (insulin-like growth factor), TGF-β ( Transforming growth factors). Moreover, it is preferable to apply the collagen solution a plurality of times so that the collagen solution is completely dried every time it is applied once using a brush or a brush in the form of a hydrochloric acid solution, and then applied the next time.

  The tubular body coated and filled with collagen is preferably subjected to freezing, lyophilization, and crosslinking treatment to crosslink the collagen. Freezing is preferably performed at −10 to −196 ° C., more preferably at −20 to −80 ° C. for 3 to 48 hours. By freezing, fine ice is formed between collagen molecules, and the collagen solution undergoes phase separation and becomes sponge. Next, the frozen collagen solution is lyophilized under vacuum at an initial temperature of −40 to −80 ° C. for about 12 to 48 hours. By freeze-drying, fine ice between collagen molecules is vaporized and the collagen sponge is refined. Examples of the crosslinking method include γ-ray crosslinking, ultraviolet crosslinking, electron beam crosslinking, thermal dehydration crosslinking, glutaraldehyde crosslinking, epoxy crosslinking, and water-soluble carbodiimide crosslinking, but it is easy to control the degree of crosslinking and perform crosslinking treatment. Thermal dehydration crosslinking that does not affect the living body is preferred. The thermal dehydration crosslinking treatment is performed at a temperature of about 105 to 150 ° C., more preferably about 120 to 150 ° C., more preferably about 140 ° C. under a vacuum, for example, about 6 to 24 hours, more preferably about 6 to 12 hours. More preferably, it is performed for about 12 hours. If the crosslinking temperature is too high, the strength of the biodegradable absorbent material may be reduced. Further, if the crosslinking temperature is too low, there is a possibility that sufficient crosslinking reaction does not occur.

  An experiment demonstrating the superiority of the effect of the collagen of the present invention is shown below.

(Measurement of sodium chloride concentration)
Measurement of sodium chloride concentration by atomic absorption spectrophotometry takes 1 to 4 g of sample in a quartz beaker, gradually raises the temperature on an electric heater and carbonizes it, and finally ashes in a muffle furnace over 6 to 8 hours. (500 ° C). The residue is redissolved with a 10% by weight aqueous hydrochloric acid solution, diluted to a final concentration of 1% by weight, and measured by flame atomic absorption spectrometry using acetylene-air. The measurement wavelength is 589.6 nm.

Experiment 1: Collagen gel culture experiment Purpose of this experiment Normally, two-dimensional culture on the bottom of a well plate is fundamental in cell culture experiments. However, when three-dimensional culture is performed, it is said that the behavior of cells during two-dimensional culture is greatly different, and if the ability to regenerate nerves is evaluated, three-dimensional culture is considered to be a system that is closer to reality. . Therefore, the purpose of this experiment was to perform three-dimensional culture with a collagen gel and check whether the behavior of cultured cells differs depending on the type of collagen.

2. Collagen used in this experiment (1) Collagen of Comparative Example “NMP Collagen PS” manufactured by Nippon Ham Co., Ltd. was used as collagen of Comparative Example. The collagen of this comparative example is produced by performing a degreasing process and a purification process using pig skin as a starting material, and the degreasing process includes a repeated washing step with a sodium chloride solution. Includes a salting-out step with sodium chloride. The collagen of this comparative example contained 4.0% by weight of sodium chloride in a dry state as measured by atomic absorption spectrophotometry (ashing).
(2) Collagen of the Invention Example A part of the collagen of the above Comparative Example was used as a starting material and purified by isoelectric precipitation at a pH of 8 or more and less than 9, to prepare a collagen of the invention example. This collagen of the present invention contained 1.0% by weight of sodium chloride in a dry state as measured by atomic absorption spectrophotometry (ashing).

3. Preparation of Collagen Gel Medium The two types of collagen prepared above were each dissolved in hydrochloric acid according to a conventional method to prepare a 0.5 wt% collagen-hydrochloric acid solution. Among these, 300 μl of the collagen solution of the present invention was added to 8 wells of a 24-well microplate (manufactured by IWAKI), and 300 μl of the collagen solution of the comparative example was added to 8 wells of the same plate. Thereafter, the plate was allowed to stand at 37 ° C. for 30 minutes in an incubator.

4). Collagen gel culture of PC12 cells (1) PC12 cells (cells derived from rat adrenal pheochromocytoma manufactured by Dainippon Pharmaceutical Laboratories) were previously cultured in DMEM medium to passage number 6, and the cells were collected by centrifugation, The number of cells was adjusted to 1 × 10 ⁶ cells in 15 ml of DMEM medium and suspended, and 15 μl of 50 μg / ml NGF (nerve growth factor, manufactured by R & D systems Inc., phosphate buffer solution) was added to prepare a culture solution. .
The DMEM medium is RPMI 1640 liquid medium (Dainippon Pharmaceutical Laboratory Products, glutamic acid-free and sodium bicarbonate included), 500 ml of fetal calf serum (Dainippon Pharmaceutical Laboratories), and horse serum (Dainippon Pharmaceutical Laboratories). ) 5 ml of 50 ml, 200 mM glutamine solution (manufactured by Dainippon Pharmaceutical Laboratory Products, 29.23 mg / ml) is added and mixed.
(2) 300 μl of the prepared culture solution was dropped into wells of a collagen gel medium prepared in advance.
(3) The well plate was cultured in an incubator (37 ° C., CO ₂ concentration 5.0%) for 4 days.

5. Observation of cell appearance After 4 days of culture, the appearance of cells in the collagen gel was observed with a microscope, and representative examples were photographed. The results are shown in FIGS.

6). Measurement of the number of viable cells (1) In order to measure the number of viable cells after 4 days of culture, 50 μl of 1 wt% collagenase solution was added dropwise to the wells, and the collagen gel was agitated at 37 ° C. for 30 minutes while gently stirring the wells. Dissolved.
(2) After dissolution of the collagen gel, 50 μl of the MTT assay solution was added to each well and allowed to stand for 30 minutes in an incubator.
(3) After standing for 30 minutes, the absorbance at 450 nm was measured, and for each collagen gel, the average value and the standard deviation were determined from the absorbance values in 8 wells, and the results are shown as a graph in FIG. In the graph of FIG. 3, the absorbance of the collagen of the example of the present invention is expressed as a relative value with the average absorbance of the collagen gel of the comparative example being 100. Absorbance is directly proportional to the number of viable cells.

7). Consideration of this experiment (1) Observation of the state of cells As is clear from the comparison of FIG. 1 and FIG. 2, in the culture using the collagen of the present invention example (FIG. 1), the culture using the collagen of the comparative example (FIG. 2). ) More proliferating cells and remarkable neurite outgrowth.
(2) Measurement of the number of viable cells As is clear from FIG. 3, the absorbance of the culture using the collagen of the example of the present invention is 39% higher on average than the absorbance of the culture using the collagen of the comparative example. There was also a significant difference (p <0.01). Therefore, it can be seen from the results of FIG. 3 that the collagen of the present invention has significantly higher cell proliferation ability than the collagen of the comparative example.
(3) From the above results, it can be said that the collagen of the present invention example is superior in cell proliferation ability and differentiation inducing ability than the conventional collagen of the comparative example.

Experiment 2: Collagen coat adhesion experiment Purpose of this experiment In order to compare the cell adhesiveness of the collagen of the present invention and conventional collagen, the cells floating with an aspirator after culturing with both collagens are aspirated and removed, and only the cells that adhere to the plate are measured, The purpose was to confirm whether there was a significant difference in cell adhesion depending on the type of collagen by comparing the numbers.

2. Preparation of collagen coat (1) The collagen of the example of the present invention used in Experiment 1 and the collagen of the comparative example were diluted with hydrochloric acid so as to be 0.05% by weight, and each of 8 wells of a 24-well microplate (IWAKI) was used. 300 μl of these collagen solutions were added to the wells and allowed to stand in the refrigerator for 1 hour.
(2) After standing for 1 hour, the collagen solution in each well was sucked with an aspirator, and the collagen coat adhering to the well was naturally dried in a clean bench for 1 hour.

3. Collagen-coated culture of PC12 cells (1) PC12 cells (cells derived from rat adrenal pheochromocytoma manufactured by Dainippon Pharmaceutical Laboratories) were previously cultured in DMEM medium to passage number 6, and the cells were collected by centrifugation, The number of cells was adjusted to 5 × 10 ⁶ cells in 25 ml of DMEM medium and suspended, and 25 μl of 50 μg / ml NGF (nerve growth factor, manufactured by R & D systems Inc., phosphate buffer solution) was added to prepare a culture solution. .
(2) 300 μl of the prepared culture solution was added to each well of a collagen-coated well plate prepared in advance.
(3) The well plate was cultured in an incubator (37 ° C., CO ₂ concentration 5.0%) for 5 days.

4). Measurement of the number of adherent cells (1) In order to remove floating cells and cells that did not adhere properly, all the medium was aspirated while tilting the well plate at 85 degrees. At this time, care was taken not to suck adherent cells.
(2) Thereafter, 300 μl of DMEM medium and 30 μl of MTT assay solution were added to each well and allowed to stand for 30 minutes in an incubator.
(3) After standing for 30 minutes, the absorbance at 450 nm was measured, and the average value and standard deviation were determined from the absorbance values in 8 wells for each collagen coat, and are shown as a graph in FIG. In the graph of FIG. 4, the absorbance of the collagen coat of the present invention example is expressed as a relative value with the average absorbance of the collagen coat of the comparative example being 100.

5. Consideration of this experiment As is clear from FIG. 4, the absorbance representing the number of adherent cells of the collagen coat of the present invention example is 49% higher on average than the absorbance of the collagen coat of the comparative example, and this difference is also statistically significant. Difference (0.01 <p <0.05). The cell adhesion of the scaffold in regenerative medicine is a very important factor, and the results of FIG. 4 show that the collagen of the present invention is more suitable for use as a scaffold for nerve regeneration than conventional collagen.

Experiment 3: Collagen coat culture experiment with varying sodium chloride concentration The purpose of this experiment The purpose of this study was to examine how the difference in the concentration of sodium chloride in collagen affects the survival and proliferation of cells.

2. Preparation of collagen coat (1) Collagen of the present invention used in Experiment 1, sodium chloride added thereto to adjust the sodium chloride concentration to 5% by weight and 10% by weight, Collagen of Comparative Example used in Experiment 1 Then, sodium chloride was added thereto to adjust the sodium chloride concentration to 5 wt% and 10 wt% (see collagen coats 1 to 6 in FIG. 5). Adjust each collagen so that it becomes 0.01 wt% hydrochloric acid solution, use two 24 well microplates (IWAKI), drop each collagen into 4 wells by 300 μl, and leave it in the refrigerator for 1 hour. did.
(2) After standing for 1 hour, the collagen solution in each well was sucked with an aspirator, and the collagen coat adhering to the well was naturally dried in a clean bench for 1 hour.

3. Collagen-coated culture of PC12 cells (1) PC12 cells (cells derived from rat adrenal pheochromocytoma manufactured by Dainippon Pharmaceutical Laboratories) were previously cultured in DMEM medium to passage number 6, and the cells were collected by centrifugation, The number of cells was adjusted to 1 × 10 ⁶ cells in 15 ml of DMEM medium and suspended, and 15 μl of 50 μg / ml NGF (nerve growth factor, manufactured by R & D systems Inc., phosphate buffer solution) was added to prepare a culture solution. .
(2) 300 μl of the prepared culture solution was dropped into each well of a collagen-coated well plate prepared in advance.
(3) The well plate was cultured in an incubator (37 ° C., CO ₂ concentration 5.0%) for 5 days.

4). Measurement of the number of viable cells (1) 30 μl of the MTT assay solution was added to each well and allowed to stand in an incubator for 30 minutes.
(2) After standing for 30 minutes, the absorbance at 450 nm was measured, and the average value and standard deviation were determined from the absorbance values in 8 wells for each collagen coat, and are shown as graphs in FIGS. In the graphs of FIGS. 6 and 7, the absorbance of the collagen coat of the present invention example is expressed as a relative value with the average absorbance of the collagen coat of the comparative example as 100.

5. Discussion of this experiment As apparent from FIGS. 6 and 7, the absorbance tends to increase as the sodium chloride concentration of the collagen decreases, and the collagen of the present invention (collagen coat 1 (sodium chloride concentration of 1% by weight)) is used. The absorbance of the cultured medium was 27% higher on average than that of the culture using the collagen of the comparative example (collagen coat 6 (sodium chloride concentration 10% by weight)), and this difference was statistically significant. (P <0.01). From the results shown in FIGS. 6 and 7, it can be seen that the collagen of the present invention having a low sodium chloride concentration is superior in cell growth ability compared to the collagen of the comparative example.

Experiment 4: Cell differentiation evaluation experiment by neural crest cell culture using collagen having different isoelectric points. The purpose of this experiment was to investigate how much the difference in isoelectric point of collagen has on cell differentiation.

2. Preparation of Isoelectric Concentrated Collagen Powder (1) MilliQ water was added to 6 g NMP collagen PS to prepare a total 1000 ml 0.6 wt% collagen solution.
(2) Stirring on ice for 1 to 3 days to completely dissolve collagen in water.
(3) 1N NaOH was added dropwise, and 200 ml of collagen solution containing precipitates was collected in a separate container at pH 5.5.
(4) The dropwise addition of 1N NaOH was continued, and similarly 200 ml each was collected in separate containers at pH 8.5 and pH 10.2.
(5) The three obtained samples were transferred to a centrifuge tube and centrifuged at 3,000 rpm for 45 minutes.
(6) The supernatant of each centrifuge tube was discarded, and the precipitate was frozen at −40 ° C. overnight and then treated with a freeze dryer for 2 days.
(7) The obtained samples were sequentially designated as Sample 1 (pH 5.5), Sample 2 (pH 8.5), and Sample 3 (pH 10.2).
In each collagen sample, the sodium chloride concentration was 1.2% by weight.

3. Preparation of Collagen Solution (1) 0.001M HCl was added to 300 mg of each of Samples 1, 2, and 3 to prepare 10 ml of 0.3 wt% collagen solution.
(2) After mixing with Voltex, it was allowed to stand at 4 ° C. overnight to completely dissolve the collagen.
(3) To 1 ml of each 0.3% by weight concentration sample, 9 ml of 0.001M HCl was added and mixed well to prepare a 0.03% by weight collagen solution.
(4) 200 μl of the 0.03% by weight collagen solution was dispensed into each well of a 24-well microplate (manufactured by IWAKI) and left at room temperature (20 ° C.) for 10 minutes.
(5) The collagen solution was aspirated, 1 ml PBS (-) was added, and then aspirated and washed, and washing was repeated again.
(6) It left still in the clean bench as it was and dried.

4). Cell Culture (1) Neural crest-derived pigment cells (made by Kurabo Industries, Code No. KM-4009MP) are used in a dedicated medium (made by Kurabo Industries, Code No. M-254-500 + Code No. S-002-5) at 3.75 × 10. Dilute to ⁴ cells / ml and inoculate 500 μl of each well.
(2) The cells were cultured at 37 ° C. in a CO ₂ concentration of 5.0% for 4 days.
(3) After aspirating the medium, 500 μl of a special medium was newly added, followed by culturing at 37 ° C. in a CO ₂ concentration of 5.0% for 3 days.
(4) After aspirating the medium, 500 μl of a special medium was newly added, and further cultured for 4 days at 37 ° C. in a CO ₂ concentration of 5.0%. Then, the degree of cell differentiation in each well was observed.

5. Consideration of this experiment As is apparent from FIGS. 8 to 10, there is a marked difference in the level of cell differentiation due to the difference in the isoelectric point of collagen, and the differentiation is significantly promoted in sample 2 (pH 8.5). Can be confirmed from observation of cell morphology.
Furthermore, from FIG. 11 and FIG. 12, when the ratio of differentiated cells in all living cells is calculated, it can be confirmed that the cell differentiation rate (%) is higher at pH 8.5 than at pH 5.5 and pH 10.2. .
Cell differentiation rate (%) = (number of differentiated cells / total number of surviving cells) × 100
Generally, in view of the fact that collagen is metabolized and absorbed in the body in about 2 weeks, if it has a cell differentiation rate of 40% or more after 11 days of culture, it is good in the actual nerve regeneration field. The result is considered to be obtained. The cell differentiation rate is more preferably 50% or more, more preferably 60% or more, and even more preferably 70% or more.

  The nerve regeneration-inducing tube of the present invention is extremely useful because it has excellent cell adhesiveness, cell proliferation ability, and cell differentiation-inducing ability, and thus is widely applied in nerve regeneration medicine.

Claims (2)

  A culture medium for cell culture, comprising collagen which has been subjected to isoelectric precipitation at a pH of 7.0 or more and 9.5 or less and which has been treated so that the concentration of sodium chloride is 2.0% by weight or less in a dry state.   It is characterized in that collagen subjected to isoelectric precipitation at pH 7.0 to 9.5 and treated so that the sodium chloride content is 2.0% by weight or less in a dry state is used as a cell culture medium. Cell culture method.