Patent Application
20210047385
The present invention relates to a culture material that contains a scaffold material for culturing nerve cells and a therapeutic agent for nerve damage using the same.
Currently, there is a need to repair a damaged central nerve. To realize this, it is necessary to culture a nerve cell, maintain survival of the nerve cell in vivo, and promote neurite extension of the nerve cell in vivo, thereby reconstructing a neural circuit.
Among nerves, the central nervous system including brain and spinal cord is believed not to be repaired spontaneously when damaged. The reasons for this are that the nerve cell in the central nervous system does not easily divide and proliferate and that body's response to the damage causes formation of hard and inflexible fibrous tissue called glial scar at the damaged site and a nerve fiber cannot extend beyond the glial scar. Another example of the reasons is that factors that inhibit neurite extension (for example, Nogo, MAG, OMgp, and Sema3A) exist in the living body and the action of these factors inhibits neurite extension.
Unlike other type of cells, a nerve cell is composed of a cell body and an axon extending from the cell body, and cells accompanying these. Therefore, culturing the nerve cells requires promoting extension of the axon or the like in addition to maintaining survival of the nerve cell.
However, a serum-containing medium conventionally used for cell culture is not sufficient to promote growth of a fragile cell such as a nerve cell, a neuroblast, or a neural stem cell. Furthermore, the serum-containing medium had the following problem: the medium remarkably promotes growth of a non-nerve cell, resulting in a very high percentage of the non-nerve cells of the total cultured cells, which was disadvantageous. Thus, a culture medium for culturing nerve cells that contains at least 2 mg of a Knitz type protease inhibitor per one liter of medium has been proposed (Patent Literature 1).
Patent Literature 2 discloses a nerve regeneration guide made by shaping a composition that contains a bioabsorbable polymer such as polylactic acid and collagen into a plate, a thread-like, or a network structure. In Patent Literature 2, the nerve regeneration guide was implanted into a part where a rat sciatic nerve was excised and the nerve was taken out after a period of time, and regeneration of the sciatic nerve was confirmed visually.
Furthermore, Patent Literature 3 discloses the following: a scaffold material for implantation produced by bonding a needle-like magnetic substance to one end of a fibrous structure composed of a biodegradable polymer selected from the group consisting of polyglycolic acid, polylactic acid, and a glycolic acid/lactic acid copolymer; or a scaffold material for implantation produced by inserting the needle-like magnetic substance into the lumen of the fibrous structure composed of the biodegradable polymer.
Patent Literature 3 has demonstrated, by using a BBB (Basso, Beattie, Bresnahan) score (score for rating motor paralysis), that restoration of movement was seen in a rat with spinal cord injury when the above-mentioned scaffold structure was implanted in the rat.
As shown in the above-mentioned literatures, the nerve cell needs to extend a projection such as an axon, and therefore, culturing (including growth in the body) thereof requires a scaffold that has bioaffinity or biodegradability.
As is described in Patent Literature 2, collagen is a material that has bioaffinity and is also readily available. It is known that there are many types of collagen. Collagen has a triple helical structure composed of a chains. Patent Literature 4 describes low adhesive collagen (Low Adhesive Scaffold Collagen, hereinafter referred to as “LASCol”) that was produced by cleaving these a chains at the end thereof by using a specified enzyme. LASCol is known as a scaffold material for culturing cells (Patent Literature 4).
When a scaffold using LASCol is utilized instead of a scaffold using conventional collagen, cells to be cultured form an aggregate (spheroid), and thus, the cells to be cultured can be cultured in a three-dimensional form, which is more similar to in vivo state (Patent Literature 4). Such LASCol is also effective in promoting differentiation of stem cells (Patent Literature 5).
Patent Literature 6 discloses a therapeutic agent for central nerve injury that uses TGF-β1, which is a growth factor.
However, it was unknown whether the above-mentioned substance such as collagen or LASCol was effective for maintenance of survival of the nerve cell and neurite extension. A form thereof that may be easily administered to an affected part (for example, injured spinal cord) for repairing a damaged central nerve has also been unknown.
The present inventors have found that LASCol was effective for maintenance of survival of a nerve cell and extension of an axon, thereby completing the present invention.
More specifically, a nerve cell culture material according to the present invention contains LASCol. A therapeutic agent for nerve damage according to the present invention also contains LASCol.
The present invention can provide a method for culturing nerve cells by using the above-mentioned nerve cell culture material and can also provide a method for treating nerve damage by using the above-mentioned therapeutic agent for nerve damage.
A component (LASCol) contained in the nerve cell culture material and the therapeutic agent for nerve damage according to the present invention is nontoxic and has a high bioaffinity.
Furthermore, when the pH of the therapeutic agent for nerve damage according to the present invention is adjusted and the temperature thereof is raised, the form of LASCol therein changes from a liquid state to a gel state. Therefore, LASCol can be injected as liquid and thus can be administered more easily to an affected part (for example, injured spinal cord), which makes treatment using LASCol less invasive. Furthermore, LASCol tends to stay in the affected part after injected into the body. Consequently, the frequency of administration required while the nerve cells grow can be reduced, which makes a patient's burden light.
What enables such administration is presumed to be the following properties of the component (LASCol) contained therein: LASCol is less viscous even at a high concentration and has a slower fiber formation rate compared with conventional collagen. These properties are presumably due to “the structure of LASCol resulting from a specified enzymatic treatment that truncates the end of an α chain (a telopeptide region likely to cause allergic reaction) while maintaining a triple helical structure.”
Hereinafter, a nerve cell culture material and a therapeutic agent for nerve damage according to the present invention will be described with reference to figures and Examples.
The following description is merely illustrative of an embodiment and an example of the present invention and the present invention is not limited to the following description. The following description may be modified without departing from the spirit of the invention.
LASCol that is used as a material for the nerve cell culture material and the therapeutic agent for nerve damage according to the present invention contains a degradation product of collagen or atelocollagen. Alternatively, LASCol may be used alone. Adhesiveness of collagen to cells has been weakened in the degradation product, and thus, the degradation product has the property of becoming low adhesive.
LASCol can be obtained by degrading collagen or atelocollagen enzymatically. The peptide sequence of LASCol varies depending on a degradation condition. In other words, a different type of LASCol can be obtained by using a different degradation condition.
The characteristic of LASCol that can be used in the present invention is that LASCol consists of a combination of a chains in which a chemical bond between Y1 and Y2 is cleaved in an amino-terminal amino acid sequence including a triple helical domain of collagen or atelocollagen, the sequence being shown below (A).
(where G represents glycine, and Y1 to Y9 each represent an optional amino acid)
The triple helical domain of collagen is known to have a succession of -G-X—Y— sequences (where G represents glycine, and X and Y each represent an optional amino acid). In the above-mentioned sequence, “G” in “—Y3-G-Y4-Y5-” represents glycine on the N-terminal side of the triple helical domain. As can be seen from the above-mentioned sequence, the cleavage of the chemical bond between Y1 and Y2 is cleavage that was carried out outside of the triple helical domain. As described below, a different degradation condition leads to cleavage inside of the triple helical domain. One of the LASCols used in the present invention is LASCol in which cleavage has occurred outside of the triple helical domain. Hereinafter, this LASCol is referred to as LASCol-A.
LASCol, which is used in the nerve cell culture material and the therapeutic agent for nerve damage according to the present invention, can be favorably used particularly for maintenance of survival of a nerve cell or neurite extension. As shown in Examples described below, LASCol-A has a very poor ability to culture a cell other than the nerve cell. However, LASCol-A has an ability to maintain survival of the nerve cell and promote extension of a nerve fiber.
It is known that the following LASCol is obtained under a certain degradation condition. Such LASCol consists of a combination of α chains in which a chemical bond between X1 and X2, a chemical bond between X2 and G, a chemical bond between G and X3, a chemical bond between X4 and G, or a chemical bond between X6 and G is cleaved in an amino-terminal amino acid sequence including a triple helical domain of collagen or atelocollagen, the sequence being shown below (B).
(where G represents glycine, and X1 to X6 each represent an optional amino acid)
This is referred to as LASCol-B. In LASCol-B, cleavage has occurred inside of the triple helical domain. In SEQ ID NO: 2, G in “-G-X1—X2-G-” is glycine on the N-terminal side of the triple helical domain. Needless to say, there may be other LASCols that contain other peptides. Among currently known LASCols, LASCol-A is most favorable from the viewpoint of maintenance of survival of the nerve cell and occurrence of neurite extension. However, other LASCols are not excluded.
Furthermore, the nerve cell culture material and the therapeutic agent for nerve damage may contain a growth factor for the nerve cell.
The LASCol used for the nerve cell culture material and the therapeutic agent for nerve damage according to the present invention can be stored as a solution under an acidic condition. The LASCol turns into a gel state when pH and a concentration thereof are adjusted and a temperature thereof is raised to body temperature. Gelling suppresses diffusion of LASCol in the body, and LASCol exerts the effect of culturing the nerve cells in the affected part for a long period of time. In the present invention, culturing of nerve cells also includes, for example, allowing the nerve cell to survive in a form close to that in vivo (the nerve cell can survive well) and to extend an axon (neurite) thereof.
The elastic modulus of gelled LASCol is proportional to the concentration of LASCol in the solution, pH, and temperature. In Examples described below, an embodiment is illustrated in which the pH and concentration of LASCol are adjusted to prepare liquid LASCol, and the liquid LASCol is sucked into a syringe and administered by injection into the affected part, thereby allowing the LASCol to turn into gel in the affected part. However, the LASCol used as the nerve cell culture material and the therapeutic agent for nerve damage according to the present invention may be shaped into a film form or a sponge form and be implanted in the affected part. In this context, the film form or the sponge form refers to LASCol that was processed into a specified shape (also referred to as a shaped form).
As described below, it can be stated that the LASCol used in the present invention turns into gel when a concentration thereof is 3.5 mg/ml (20 Pa in terms of “practical elastic modulus” described below) or more. Therefore, when the concentration of the LASCol used as the nerve cell culture material and the therapeutic agent for nerve damage is 3.5 mg/ml or higher, the LASCol can stay in the body and regenerate the nerve cell when administered into the body.
Findings about a method for producing LASCol are almost the same for both LASCol-B and LASCol-A. Thus, findings common to both are described simply as findings about LASCol. In the following description, “degradation product” means LASCol.
Collagen or atelocollagen as a material for LASCol is not limited to any particular one and may be any well-known collagen or atelocollagen.
Examples of the collagen include collagens of mammals (for example, a cow, a pig, a rabbit, a human, a rat, or a mouse), birds (for example, a chicken), fishes (for example, a shark, a carp, an eel, a tuna [for example, a yellowfin tuna], a tilapia, a sea bream, or a salmon), or reptiles (for example, a soft-shelled turtle).
Examples of the collagen used in the present invention include collagen derived from, for example, a dermis, a tendon, a bone, or a fascia of any of the above-mentioned mammals or the above-mentioned birds, collagen derived from, for example, a skin or a scale of any of the above-mentioned fishes, and collagen derived from, for example, a dermis, a tendon, or a bone of any of the above-mentioned reptiles.
Examples of the atelocollagen used for producing LASCol include atelocollagen that is produced by treating collagen of any of the above-mentioned mammals, birds, fishes, or reptiles with a protease (for example, pepsin), wherein a telopeptide has been partially removed from the amino terminus and/or the carboxyl terminus of the collagen molecule.
Among the above examples, collagen or atelocollagen of a chicken, a pig, a cow, a human, or a rat can be preferably used. More preferably, collagen or atelocollagen of a pig, a cow, or a human can be used as the material for LASCol.
Furthermore, the collagen or atelocollagen of a fish can be used as the material for LASCol. Using a fish allows for obtaining the material easily and safely in a large quantity and providing a degradation product of collagen or atelocollagen (LASCol) that is virus-free and safer to humans.
When collagen or atelocollagen of a fish is used as the material for LASCol, it is preferable to use collagen or atelocollagen of a shark, a carp, an eel, a tuna (for example, a yellowfin tuna), a tilapia, a sea bream, or a salmon; and it is more preferable to use collagen or atelocollagen of a tuna, a tilapia, a sea bream, or a salmon.
When atelocollagen is used as the material for LASCol, it is preferable to use atelocollagen that has a heat denaturation temperature of preferably 15° C. or higher, and more preferably 20° C. or higher. For example, when the atelocollagen of a fish is used as the material for the degradation product, it is preferable to use the atelocollagen of a tuna (for example, a yellowfin tuna), a tilapia, a carp, or the like, since such atelocollagen has a heat denaturation temperature of not lower than 25° C.
The above-mentioned arrangement allows for adjusting a denaturation temperature (temperature at which a substance turns into gel) of the nerve cell culture material and the therapeutic agent for nerve damage of this embodiment preferably to 15° C. or higher, and more preferably to 20° C. or higher. Consequently, the above-mentioned arrangement allows for providing a nerve cell culture material and a therapeutic agent for nerve damage that are excellent in stability during storage and stability during use.
Such collagen or atelocollagen may be obtained by a well-known method. For example, collagen-rich tissue of a mammal, a bird, or a fish may be put into an acidic solution with a pH of about 2 to 4, thereby eluting collagen. Furthermore, a protease such as pepsin is added to the eluate to partially remove a telopeptide at the amino terminus and/or carboxyl terminus of the collagen molecule. Furthermore, a salt such as sodium chloride may be added to the eluate to precipitate atelocollagen.
LASCol is obtained by allowing an enzyme to act on collagen or atelocollagen, thereby degrading such material. Alternatively, LASCol can also be obtained by producing a degradation product of collagen or atelocollagen (for example, by chemical synthesis or expression of recombinant protein), wherein the degradation product has an already cleaved chemical bond within the triple helical domain.
Hereinafter, a method for obtaining LASCol by degrading the above-mentioned collagen or atelocollagen with an enzyme (for example, protease) will be described.
The enzyme is not limited to any particular one. For example, a cysteine protease is preferably used.
It is preferable to use, as the cysteine protease, a cysteine protease that contains a larger amount of basic amino acids than the amount of acidic amino acids, or a cysteine protease that is active at a hydrogen ion concentration in the acidic range.
Examples of such a cysteine protease may include actinidain [EC 3.4.22.14], papain [EC 3.4.22.2], ficin [EC 3.4.22.3], bromelain [EC 3.4.22.32], cathepsin B [EC 3.4.22.1], cathepsin L [EC 3.4.22.15], cathepsin S [EC 3.4.22.27], cathepsin K [EC 3.4.22.38], cathepsin H [EC 3.4.22.16], alloline, and a calcium dependent protease. The text in square brackets represents an enzyme code number.
Among these, it is preferable to use actinidain, papain, ficin, cathepsin K, alloline, or bromelain, and it is more preferable to use actinidain, papain, ficin, or cathepsin K.
The above-mentioned enzyme can be obtained by a known method. For example, the enzyme can be obtained by producing the enzyme by chemical synthesis; extracting the enzyme from a cell or tissue of a bacterium, a fungus, or various animals and plants; producing the enzyme by a genetic engineering process; or other methods. Needless to say, a commercially available enzyme can also be used.
When collagen or atelocollagen is cleaved by degrading the same with an enzyme (for example, a protease), the cleaving step can be carried out by, for example, any of the methods (i) to (iii) described below. The following methods (i) to (iii) are merely examples of the cleaving step, and the method for producing LASCol is not limited to these methods (i) to (iii).
LASCol-B can be obtained by the following methods (i) and (ii). LASCol-A and LASCol-B can be obtained by the following method (iii).
(i) A method that includes bringing collagen or atelocollagen into contact with an enzyme in the presence of a high concentration of salt.
(ii) A method that includes bringing collagen or atelocollagen into contact with an enzyme that has been in contact with a high concentration of salt.
(iii) A method that includes bringing collagen or atelocollagen into contact with an enzyme in the presence of a low concentration of salt.
Specific examples of the above-mentioned method (i) may include a method that includes bringing collagen or atelocollagen into contact with an enzyme in an aqueous solution containing a high concentration of salt.
Specific examples of the above-mentioned method (ii) may include a method that includes bringing an enzyme into contact with an aqueous solution containing a high concentration of salt in advance and subsequently bringing collagen or atelocollagen into contact with the enzyme.
Specific examples of the above-mentioned method (iii) may include a method that includes bringing collagen or atelocollagen into contact with an enzyme in an aqueous solution containing a low concentration of salt. The specific composition of the above-mentioned aqueous solution is not particularly limited. For example, water can be used.
Although the specific composition of the above-mentioned salt is not particularly limited, a chloride is preferably used. The chloride is not limited to any particular one. For example, NaCl, KCl, LiCl, or MgCl2 can be used.
Although the concentration of the salt in the above-mentioned aqueous solution containing a high concentration of salt is not particularly limited, a higher concentration is more preferable. For example, the concentration is preferably 200 mM or higher, more preferably 500 mM or higher, still more preferably 1000 mM or higher, even more preferably 1500 mM or higher, and most preferably 2000 mM or higher.
Although the concentration of the salt in the above-mentioned aqueous solution containing a low concentration of salt is not particularly limited, a lower concentration is more preferable. For example, the concentration is preferably 200 mM or lower, more preferably 150 mM or lower, still more preferably 100 mM or lower, even more preferably 50 mM or lower, and most preferably substantially 0 mM.
Although collagen or atelocollagen may be dissolved in the above-mentioned aqueous solution (for example, water) in any amount, by way of example, it is preferable that 1 part by weight of collagen or atelocollagen be dissolved in 1000 parts by weight to 10000 parts by weight of the aqueous solution.
The above-mentioned arrangement enables efficient contact between an enzyme and the collagen or atelocollagen when the enzyme is added to the aqueous solution. Consequently, the collagen or atelocollagen can be degraded efficiently with the enzyme.
Although the enzyme may be added to the aqueous solution in any amount, by way of example, it is preferable that 10 parts by weight to 20 parts by weight of the enzyme be added to 100 parts by weight of the collagen or atelocollagen.
The above-mentioned arrangement, in which the concentration of the enzyme in the aqueous solution is high, enables efficient degradation of the collagen or atelocollagen with the enzyme (for example, a protease).
Furthermore, other conditions (for example, the pH of the aqueous solution, temperature, and a contact time) under which the collagen or atelocollagen is brought into contact with the enzyme in the aqueous solution are not particularly limited and may be selected as appropriate. However, these conditions are preferably within the ranges described below. Preferable ranges of these conditions are illustrated below.
1) The pH of the aqueous solution is preferably 2.0 to 7.0, and more preferably 3.0 to 6.5. For keeping the pH of the aqueous solution within the above-mentioned range, a well-known buffer may be added to the aqueous solution. The above-mentioned pH allows the collagen or atelocollagen to be dissolved in the aqueous solution uniformly, and consequently allows the enzymatic reaction to proceed efficiently.
2) The temperature is not limited to any particular value and may be selected depending on the enzyme to be used. The temperature is, for example, preferably 15° C. to 40° C., and more preferably 20° C. to 35° C.
3) The contact time is not limited to any particular length and may be selected depending on the amount of the enzyme and/or the amount of the collagen or atelocollagen. The contact time is, for example, preferably 1 hour to 60 days, more preferably 1 day to 7 days, and even more preferably 3 days to 7 days.
When necessary, at least one step selected from the group consisting of a step of readjusting the pH, a step of inactivating the enzyme, and a step of removing contaminants may be performed after allowing the collagen or atelocollagen to be in contact with the enzyme in the aqueous solution.
The step of removing contaminants can be carried out by a general method for separating a substance. The step of removing contaminants can be carried out by, for example, dialysis, salting-out, gel filtration chromatography, isoelectric precipitation, ion exchange chromatography, or hydrophobic interaction chromatography.
The nerve cell culture material according to the present invention is used, for example, as follows: firstly a solution containing LASCol is coated onto a culture dish, secondly a culture medium such as D-MEM (Dulbecco's modified Eagle's medium) is added onto the culture dish, and then the nerve cells are seeded thereon.
The therapeutic agent for nerve damage according to the present invention is administered to the affected part after identifying the damaged area after a certain period of time has passed since the nerve was damaged. For example, in the case of spinal cord, the therapeutic agent for nerve damage is administered to the affected part, for example, by injection after the injured part of the spinal cord was identified, for example, by roentgenography not immediately after spinal cord injury but after a certain period of time has passed. In this case, it is desirable that the LASCol contained in the therapeutic agent for nerve damage have an elastic modulus (“practical elastic modulus” described below) not less than a predetermined value. This is because there is a risk that LASCol with a low elastic modulus may not stay in the affected part and flow out therefrom.
The nerve cell culture material or the therapeutic agent for nerve damage according to the present invention is provided, for example, in a dry state (including powder and a shaped form) or a gel state. The expression “using the nerve cell culture material or the therapeutic agent for nerve damage according to the present invention at a predetermined concentration” includes a case where instructions to add a certain amount of solvent to LASCol in a dry state are attached to the product or passed on to the user, and in accordance with the instructions, a favorable concentration of LASCol of the present invention is prepared.
“Administration” as used herein means administering a therapeutic agent to a patient via the affected part. Thus, administration of the therapeutic agent according to the present invention includes not only injection but also insertion of the therapeutic agent into a site incised by incision, application of the therapeutic agent onto the affected part, and the like. Furthermore, the therapeutic agent for nerve damage according to the present invention can be regarded as a method for treating nerve damage by using the therapeutic agent for nerve damage according to the present invention.
Nerve damage to be treated by using the present invention is damage in the central nerve area and the peripheral nerve area; and examples thereof include traumatic injury caused by an accident such as traffic accident, sport accident, and a fall, and damage caused by a disease such as spinal cord tumor and hernia.
50 mM citric acid buffer solutions (pH 3.0) each containing sodium chloride at a concentration of 0 mM or 1500 mM were prepared. Water was used as a solvent of these aqueous solutions.
For activating actinidain, actinidain was dissolved in 50 mM phosphate buffer (pH 6.5) containing 10 mM dithiothreitol and the resultant aqueous solution was left to stand at 25° C. for 90 minutes. Note that actinidain had been purified by a well-known method before use (see, for example, Non-Patent Literature 1).
Next, pig-derived type I collagen was dissolved in the 50 mM citric acid buffer solution containing the salt (pH 3.0). The resultant solution containing the pig-derived type I collagen was brought into contact with the aqueous solution containing actinidain at 20° C. for 10 days or longer to produce a degradation product of type I collagen. Note that the pig-derived type I collagen had been purified by a well-known method (see, for example, Non Patent Literature 1).
The above-mentioned degradation product was subjected to sodium lauryl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to separate the degradation product of type I collagen.
Subsequently, the degradation product of type I collagen was transferred onto a PVDF (polyvinylidene difluoride) membrane by a routine method. Then, an amino acid sequence of the amino terminus of a degradation product of an α1 chain transferred onto the PVDF membrane was determined by the Edman degradation technique.
Note that APRO Science Inc. or Collaborative Laboratory (Analytical tools) of the Faculty of Medicine of Kindai University conducted the actual Edman analysis in accordance with a well-known method, at the request of the present inventors.
Table 1 shows the amino acid sequence of the amino terminus and the vicinity thereof of the degradation products of the α1 chain that were obtained at salt concentrations of 0 mM and 1500 mM.
As shown in Table 1, cleavage occurred outside of a triple helical domain represented by “GPMGPSGPRG⋅ ⋅ ⋅” when the salt concentration was low (0 mM), while cleavage occurred inside of the triple helical domain when the salt concentration was high (1500 mM). In SEQ ID NO: 3, the triple helical domain starts from glycine (G) that is the third amino acid from the left. A solution produced in the case of 0 mM is a LASCol-A solution and a solution produced in the case of 1500 mM is a LASCol-B solution. In the following Examples, the LASCol-A solution was used as the LASCol solution.
In LASCol-A, cleavage also occurs in an a2 chain. In Table 2, SEQ ID NO: 5 represents the amino acid-terminal portion of the α2 chain. In SEQ ID NO: 5, the triple helical domain starts from glycine (G) located at the left end of “⋅ ⋅ ⋅GPMGLMG⋅ ⋅ ⋅.” SEQ ID NO: 6 represents the end of the α2 chain produced at a salt concentration of 0 mM, which is a condition for production of LASCol-A. When compared with SEQ ID NO: 2, SEQ ID NO: 6 corresponds to a sequence resulting from cleavage of a chemical bond between G and X3 in SEQ ID NO: 2.
In other words, in LASCol-A, cleavage in the α1 chain has occurred outside of the triple helical domain, while cleavage in the α2 chain has occurred inside of the triple helical domain. LASCol-A only needs to have either one of cleavages shown in SEQ ID NO: 3 and SEQ ID NO: 6.
These LASCols are stored in an acidic solution in a temperature range from 5° C. to 10° C. Under this condition, LASCol can be stored in a liquid state.
Referring to
This indicated that the storage elastic modulus G′ of the solution containing LASCol increased to a certain value that depended on the LASCol concentration when the pH and concentration of the LASCol solution were adjusted and the temperature thereof was raised. Furthermore, it was found that the storage elastic modulus reached an almost stable value 30 minutes after the LASCol solution was prepared so as to have a predefined concentration and the temperature thereof was raised to 37° C. For this reason, the storage elastic modulus at this time point is referred to as “practical elastic modulus” of LASCol.
It was shown that, when LASCol was exposed to an appropriate condition, the property thereof changed from sol having an unmeasurable elastic modulus to gel having a quantifiable elastic modulus, and thus LASCol could be used as an injectable gel particularly for injection into a living body.
When the LASCol concentration was further increased (
When LASCol is used for the therapeutic agent for nerve damage, the lower limit of the storage elastic modulus thereof in a gel form is believed to be 20 Pa. LASCol also functions as a scaffold for cells, and thus needs to stay in one place to some extent. The reason why the lower limit is 20 Pa is that LASCol with an elastic modulus of less than 20 Pa does not behave as gel and thus is believed to have difficulty in staying in the affected part.
The LASCol solution prepared as described above was used to confirm an ability thereof to maintain survival of the nerve cell. LASCol, atelocollagen, and poly-L-lysine (also referred to as “PLL” hereinafter) were coated onto a 24-well microplate. A non-coated well having no coating thereon was prepared as a control. Nerve cells derived from a neonatal rat hippocampus (a nerve cell that is not a mesenchymal stem cell and has completed differentiation; hereinafter simply referred to as “nerve cell”) were suspended in Neurobasal medium with B-27 supplement (manufactured by Thermo Fisher Scientific Inc., hereinafter referred to as “NB/B27”) and seeded in the above-mentioned wells.
PLL promotes adhesion mediated by an electric charge between a cell membrane surface and the culture dish. Therefore, use of PLL can make nerve cells adhere to a commercially available plastic plate, although a hydrophilicity-enhancing treatment generally applied to such a plate does not provide, by itself, sufficient adhesiveness. PLL is commonly used in culturing nerve cells.
The state of the nerve cells was observed by microscopy after 48 hours of culture and the result is shown in
In
In
Next, the LASCol solution, the atelocollagen solution, and the PLL solution were coated onto 20 mm×20 mm slide glasses and the nerve cells were seeded thereon. Then, the nerve cells were observed by scanning electron microscopy (SEM) after 24 hours. Specifically, the culture sample was fixed with 4% paraformaldehyde, dehydrated in alcohol, immersed in isoamyl acetate, and dried by critical point drying using liquefied carbon dioxide. Subsequently, the sample was coated with platinum palladium and observed by Hitachi S5000 SEM.
A plurality of long thread-like pseudopodia (filopodia, indicated by arrowheads) have extended from the growth cone that formed on the LASCol, which indicated that this growth cone was very active. Additionally, a new growth cone (arrow) had formed on the axon that further extended from the filopodia. Furthermore, the projection had a regular surface and had formed a shape typical of the projection. The nerve cell whose neurite has extended in such a state may be considered to be a form of a successfully cultured nerve cell.
Furthermore, LASCol fibers had formed densely in a layer underlying the growth cone and each filopodium had adhered to the LASCol fibers distinctly. This indicates that signal from the LASCol is involved in vigorous activity of the nerve cell.
It was found from the above that LASCol was effective not only for successful survival of the nerve cell but also for extension of the projection from the nerve cell.
The result of culturing of an astrocyte on LASCol is shown below. LASCol and atelocollagen were coated onto a 96-well microplate. Non-coated one was also prepared as a control group. Then, astrocytes derived from a rat cerebrum were seeded at 3×104 and 1×105 cells/mL, and the number of cells were measured by the WST-1 method after 48 hours. The WST-1 method is one of colorimetric MMT methods. The MMT method is colorimetry by measuring the activity of an enzyme that reduces MTT or a similar dye to a formazan dye (purple).
The WST-1 method is based on conversion of a tetrazolium salt (WST-1) into a formazan dye by mitochondrial dehydrogenase in a living cell and there is a linear relationship between the absorbance of the formazan dye solution and the number of living cells. Therefore, the number of cells can be quantitatively measured by measuring the absorbance. The results are shown in
Referring to
Since the astrocyte is a cell (glial cell) other than the nerve cell in the central system, the result shown in
A glial cell is known to increase in a lesion in nerve tissue. When glial cells increase in a damaged area in a part with aggregated nerves, such as a spine, a nerve fiber cannot extend beyond the glial cells and thus the nerve remains severed. LASCol is believed to be able to enhance extension of the nerve fiber because LASCol suppresses growth of the glial cells.
Bone marrow stromal cells were seeded at a concentration of 3×103 cells/ml onto a dish that was coated with the LASCol solution, the atelocollagen solution, or the PLL solution and Mesenchymal Stem Cell Basal Medium with MSCGM SingleQuots (manufactured by Lonza) was added to, and observed after seven days. The results are shown in
For the atelocollagen-coated group of
Based on the above, one can conclude that the bone marrow stromal cells in the LASCol-coated group (
Furthermore, the tendency for the bone marrow stromal cells not to proliferate on LASCol was examined again. The LASCol solution was coated onto a 48-well microplate. The bone marrow stromal cells were seeded at 1×105, 3×104, 1×104, and 3×103 cells/well. After 24 hours, the number of cells was counted by using a Luna automated cell counter (manufactured by Logos Biosystems, Inc.). Non-coated one was also prepared as a control group.
Furthermore, the LASCol solution was coated onto a 96-well microplate and the bone marrow stromal cells were seeded at 1×105, 5×104, 2×104, and 1×104 cells/well. An assay using the WST-1 method was performed after two days.
The results are shown in
The above results demonstrated the tendency for the bone marrow stromal cells to proliferate less on LASCol.
The result of culturing of macrophages on LASCol is shown below. LASCol or atelocollagen was coated onto an eight-well chamber. Then, rat peritoneal macrophages were seeded at 2×105 cells/mL and observed after 48 hours. The results are shown in
As described above, the astrocytes, the bone marrow stromal cells, and the macrophages were hardly able to proliferate on LASCol. These findings demonstrated that LASCol exerted the effect of maintaining cell survival and the effect of promoting neurite extension on the nerve cell. Therefore, it can be concluded that LASCol could be used favorably as a nerve cell culture material. Particularly, it is almost impossible to culture a non-nerve cell on LASCol, and thus the nerve cell culture material using LASCol may allow for culturing the nerve cell in a state close to an actual state even when other cells coexist.
The above indicates that the nerve cells are successfully cultured in vitro in the LASCol-coated group. If LASCol exerted this effect in vivo, LASCol could become a useful pharmaceutical agent for nerve cell regeneration. Thus, the ability of LASCol to enable nerve cell culture in vivo was examined.
9-week-old male Sprague-Dawley (SD) rats were used. Each group described below consisted of seven rats. Crush injury was induced by using a standard New York University weight-drop device. The settings of the device were 10 g and 7.5 cm for a drop height. Impact was applied once.
One week after injury, 10 μL of the LASCol solution or phosphate buffered saline (hereinafter simply referred to as “PBS”) was administered to the injured part of the spinal cord. At this time, the temperature of the LASCol solution was at room temperature. Administration was performed by placing the rat on a stereotaxic apparatus and then using a screw-type injector to slowly push a fixed insulin syringe, thereby administering the samples. After allowing the syringe to stand for about two minutes, the needle was withdrawn. This method is the same as a method for performing a standard cell implantation.
For the above-mentioned administration, the practical elastic modulus of the LASCol solution to be injected into the injured part of the spinal cord was measured in advance (rheometer, HAAKE MARS III, Thermo Fisher Scientific Inc.) and was adjusted to 500 Pa to 600 Pa (37° C., pH 7.4). As already described, this value is a value measured on the rheometer 30 minutes after the temperature became 37° C. The practical elastic modulus of 500 Pa is roughly equivalent to the stickiness of honey.
The rat recovered rapidly for the first three weeks, and thereafter showed a tendency to recover slowly. The BBB score after five weeks was 11 for the LASCol-receiving group and 9 for the PBS-receiving group. In other words, the LASCol-receiving group recovered better than the PBS-receiving group, with difference in the BBB score being about two. In this context, the score of 9 represents a level at which the rat shows plantar paw placement with weight support when the rat is stationary but not during stepping. The score of 11 represents a level at which the rat shows highly frequent or consistent stepping with weight support. Particularly, significant recovery of the LASCol-receiving group compared with the PBS-receiving group was observed two weeks and five weeks after administration.
Separately from the experiment for BBB measurement, LASCol was administrated to a six-week-old SD rat immediately after crush injury was induced in the rat, and the tissue of the injured part of the spinal cord was observed eight days after administration.
The above-mentioned staining was performed by staining a non-labeled primary antibody with a fluorescently labeled secondary antibody. More specifically, a goat anti-rabbit IgG antibody conjugated to a fluorescent dye (green) excited at a wavelength of 488 nm (CF 488A goat anti-rabbit IgG) was used as the secondary antibody for the anti-GFAP antibody that had been used for staining astrocytes.
A goat anti-mouse IgG antibody conjugated to a fluorescent dye (red) excited at a wavelength of 546 nm (Alexa Fluor 546 goat anti-mouse IgG) was used as the secondary antibody for the anti-phosphorylated GAP-43 antibody. Axio Imager M1 microscope was used as a fluorescent microscope and AxioVision software was used for image acquisition (both were manufactured by Carl Zeiss AG, Tokyo, Japan).
Reference is made to
In
On the other hand,
Reference is made to
On the other hand,
Referring to
The part where the astrocyte disappeared and became negative for GFAP (white part in
Therefore, it can be concluded that administration of the LASCol solution to the rat with spinal cord injury led to regeneration of the nerve cell in the injured part and recovery.
Next, an effect of LASCol that had been dried and made into a spongy object of a certain shape was examined. Sponge samples that were used were those made by drying different concentrations of LASCol and atelocollagen. The concentrations of LASCol before drying was 30 mg/ml and 50 mg/ml, and the concentration of atelocollagen before drying was 20 mg/ml. The sponge samples are referred to as LA30, LA50, and AC20, respectively. The concentration of each sponge sample before drying is shown in Table 3. Each sponge sample was formed into a shape with a diameter of 2 to 3 mm and a length of 5 mm.
A part of the spinal cord (about 5 mm in the vertical direction and about 1 mm in the horizontal direction from the center of the spinal cord) at the level of the eighth to ninth thoracic vertebrae (the same part as the part for crush injury) was excised from a 9-week-old female SD rat. The sponge sample was immediately implanted into the resulting space with tweezers. Each sponge sample was implanted into three rats (the experiment was performed with n=3).
Two weeks after implantation, all the rats were exsanguinated by perfusion with PBS (phosphate buffered saline), and then fixed by perfusion with 4% PFA (perfluoroalkoxy alkane). The spinal cord including the injured part (the part where the sponge sample was implanted) was removed and fixed by immersion in 4% PFA for one day, and after replacing PFA with 30% sucrose (saccharose), was embedded in Surgipath (registered trademark) FSC22 embedding compound (manufactured by Leica Biosystems Inc.). The specimen was horizontally sectioned at a thickness of 10 μm using a cryostat.
After washing the section with PBS, the section was subjected to permeabilization treatment and blocking treatment at room temperature for five minutes using 3% Triton X-100-containing Blocking One Histo (Nacalai Tesque Inc.). A rabbit anti-βIII-tubulin polyclonal antibody (marker of a nerve cell, Abcam plc., ab18207) and a mouse anti-type I collagen monoclonal antibody (for detecting implanted LASCol and AteloCol, Sigma-Aldrich Co., C2456) were used at a 1:200 dilution as primary antibodies, and were reacted with the section at room temperature overnight.
CF488A goat anti-Rabbit IgG (Biotium, Inc.) and Alexa Fluor 555 goat anti-mouse IgG (Thermo Fisher Scientific Inc.) were used at a 1:200 dilution as secondary antibodies and were reacted with the section at room temperature for 30 minutes. The nuclei of the cells were stained with 0.3 μM DAPI. After putting a cover glass on the section by using Fluoromount/Plus (Diagnostic BioSystems Inc.), a fluorescent microscope, specifically Axio Imager M1 microscope with AxioVision software (Carl Zeiss AG) was used to observe the section and acquire image data thereof.
The part in the center of
The dark part in the center of
In particular, in
These findings demonstrated that the neural axon had extended into the implanted LASCol sponge sample.
The horizontal axis represents the type of the sponge samples. In the case of AC20, the atelocollagen sponge sample, little β-Tubulin was present in Col1. In contrast, in the case of the LASCol sponge sample, the axon was found in the sponge sample. Furthermore, when the LASCol concentrations of 30 mg/ml and 50 mg/ml were compared, the axon area was larger for the concentration of 30 mg/ml (LA30).
A higher LASCol concentration before drying results in a denser sponge sample. Therefore, the result of
It was found from the above that LASCol (gel and a dry product) could be favorably used as the therapeutic agent for nerve damage. Because a nerve cell has a similar property regardless of the place where the nerve cell exists, LASCol can be used favorably as a therapeutic agent for injury of spinal cord, which is a central nerve, but also the therapeutic agent for damage of nerve including a peripheral nerve.
The nerve cell culture material according to the present invention can be favorably used as a scaffold material or an additive for culturing nerve cells. The therapeutic agent for nerve damage according to the present invention can also be used favorably for regenerative treatment of a damaged part of a nerve severed due to spinal cord injury. Furthermore, the inventive therapeutic agent can be used as a therapeutic agent for regeneration of a nerve cell in other places.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2018-015496 | Jan 2018 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2019/003502 | 1/31/2019 | WO | 00 |
