JP2005040060A - Highly efficient seeding of cells on porous biocompatible materials - Google Patents

Abstract

An object of the present invention is to provide a method for efficiently seeding and holding cultured cells in the pores of a porous biomaterial within the scope of regenerative medical techniques for treating injured organs using cells. And
In the present invention, cells are seeded in the pores of a porous biocompatible material, and the cells are damaged by a method of introducing cells into the pores of the porous biomaterial using an external force. The cells were efficiently retained in the pores of the porous material. By transplanting this cell / porous biomaterial complex into a living body, long-term function maintenance of cells due to the construction of new blood vessels in the pores was realized.
[Selection] Figure 1

Description

  The present invention belongs to a technique for efficiently seeding cells in pores of a porous biocompatible material that cannot be realized by a normal cell seeding method and retaining the cells in the pores.

  The cell-embedded device obtained by the method of the present invention can be successfully transplanted into a human or mammalian body as an artificial organ after transplantation, and can retain the function of cells for a long time even after transplantation.

  Regenerative medicine for treating various organ injuries using cells has been developed. Although organ transplantation is considered as a fundamental treatment, donor shortages are serious. Artificial organs are also an important option, but their therapeutic effects are short-term because they have no biological function.

As a solution to this, cell therapy in which cells themselves are transplanted has been attempted, but there is a problem that a large number of cells must be secured due to the low engraftment rate of the recipient. Therefore, there is a demand for the development of hybrid artificial organs that incorporate cells into materials and utilize their metabolic functions. Until now, cell-embedded devices typified by bioreactors that function in vitro have been used, but there is a drawback that their functions only last for a short period of time while requiring a large number of cells.
Many porous biomaterials for seeding and holding cells are known (Patent Documents 1 to 5). However, in the method of seeding cells on a porous biomaterial by ordinary free fall, cells are placed in the pores. It was difficult to effectively sow and maintain.

Further, Patent Document 6 discloses a method of seeding cells using centrifugal force, reduced pressure, pressurization, etc., but there is no disclosure of specific conditions, and it is compared with a seeding method under normal pressure. In addition, it does not describe whether or not the cell function was maintained.
JP2003-010309 JP2002-146086 JP2002-146084 JP2002-143291 JP2002-143290 JP2002-282285

  An object of the present invention is to provide a cell-integrated device typified by a bioreactor whose function can be sustained for a long period of time by seeding and holding a large number of cells.

  As a result of repeated examinations in view of the above problems, the present inventor can densely fill a large number of cultured cells into the pores of the porous material by applying external force to the cells by centrifugation, decompression, pressurization, vibration, etc. They found that cell function can be sustained for a long time.

The present invention provides the following methods.
Item 1. A method of seeding cells in pores of a porous biocompatible material, wherein the cells are introduced into the pores of the porous biomaterial using an external force.
Item 2. The biocompatible material is
(1) Hydroxyapatite, tricalcium phosphate, calcium deficient apatite, amorphous calcium phosphate, tetracalcium phosphate, octacalcium phosphate, fluorinated apatite, carbonate apatite, calcium carbonate, calcium pyrophosphate, monetite, calcium phosphate in vivo Ceramic-based materials including inorganic compounds capable of adsorbing species;
(2) Collagen, gelatin, chitin, chitosan, glycosaminoglycan, hyaluronic acid,
Natural polymers including chondroitin sulfate, fibrin, alginic acid, fibroin, starch, pectin, pectic acid, agarose and their partial degradation products, oxides, alkylene oxide adducts, carboxymethylates and cross-linked products; and
(3) A homopolymer or copolymer of a bioabsorbable polymer such as polyvinyl alcohol, polyethylene glycol, polymethyl methacrylate, methacrylic acid ester polymer, silicone resin, polylactic acid, polyglycolic acid, polyε-caprolactone, etc. Item 2. The method according to Item 1, which is at least one selected from the group consisting of:
Item 3. The biocompatible material is hydroxyapatite, tricalcium phosphate, calcium deficient apatite, amorphous calcium phosphate, tetracalcium phosphate, octacalcium phosphate, fluorinated apatite, carbonate apatite, calcium carbonate, calcium pyrophosphate, monetite, Including at least one ceramic material selected from the group consisting of inorganic compounds capable of adsorbing calcium phosphates in vivo, collagen, gelatin, chitin, chitosan, glycosaminoglycan, hyaluronic acid, chondroitin sulfate as optional components , Fibrin, alginic acid, fibroin, starch, pectin, pectic acid, agarose and their partial degradation products, oxides, alkylene oxide adducts, carboxymethylated products and cross-linked natural polymers Includes homopolymers or copolymers of bioabsorbable polymers such as polyvinyl alcohol, polyethylene glycol, polymethyl methacrylate, methacrylate polymers, silicone resins, polylactic acid, polyglycolic acid, and poly-ε-caprolactone. Item 3. The method according to Item 1 or 2, further comprising at least one selected from the group consisting of synthetic polymers.
Item 4. Item 4. The method according to any one of Items 1 to 3, wherein an external force for introducing cells into the pores of the porous biomaterial is greater than 1G and not greater than 200G.
Item 5. Item 5. The method according to any one of Items 1 to 4, wherein the external force is selected from the group consisting of centrifugal force, pressurization, decompression, and vibration.
Item 6. Item 6. The method according to any one of Items 1 to 5, wherein the porous biocompatible material has a porosity of 20% to 95% and a pore diameter range of 50 μm to 600 μm.
Item 7. The porosity and external force conditions when cells are effectively seeded on the porous biocompatible material are external force of over 1G and porosity of 30G or less when the porosity is 95% to 65%, and external force of 30G when the porosity is 65% to 40%. Item 7. The method according to any one of Items 1 to 6, wherein the external force is 120G to 200G when the porosity is 40% to 20%.
Item 8. The pore diameter and external force conditions when cells are effectively seeded on the porous biocompatible material are as follows: external force is more than 1G and 30G or less when the pore diameter is 600 μm to 500 μm, and external force is 30G when the pore diameter is 500 μm to 200 μm Item 7. The method according to any one of Items 1 to 6, wherein the external force is 120 G to 200 G when the pore diameter is 200 μm to 50 μm.
Item 9. Item 9. The method according to any one of Items 1 to 8, wherein the porous biocompatible material has a lump shape, a sheet shape, a bead shape, a disk shape, or a sponge shape.
Item 10. Item 10. A transplantation method comprising transplanting a porous biomaterial into which a cell obtained by the method according to any one of Items 1 to 9 is introduced into a pore, to a human or a mammal.

In the present invention, the porosity (porosity) of the porous biocompatible material is usually about 20 to 95%, preferably about 30 to 80%, more preferably about 40 to 65%. If the porosity is too high, sufficient strength cannot be obtained, and if the porosity is too low, a sufficient number of cells cannot be retained.

The pore size of the porous biocompatible material is not particularly limited as long as a sufficient number of cells can be retained, but is usually about 50 to 600 μm, preferably about 100 to 500 μm, more preferably 200 to 400 μm.
Degree. If the pore size is too small, cells are difficult to enter even when an external force is applied. If the pore size is too large, cells enter and exit easily, and the cells are difficult to settle inside.

  The porous biocompatible material may be either an inorganic material or an organic material, and may be used alone or in combination of two or more materials.

  Inorganic materials include hydroxyapatite, tricalcium phosphate, calcium deficient apatite, amorphous calcium phosphate, tetracalcium phosphate, octacalcium phosphate, fluorinated apatite, carbonate apatite, calcium carbonate, calcium pyrophosphate, monetite, raw Examples include inorganic compounds capable of adsorbing calcium phosphates in the body. Examples of the “inorganic compound capable of adsorbing calcium phosphates in vivo” include titanium, titanium compounds, glass ceramics, zeolite, and silica.

  Examples of organic materials include collagen, gelatin, chitin, chitosan, glycosaminoglycan, hyaluronic acid, chondroitin sulfate, fibrin, alginic acid, fibroin, starch, pectin, pectic acid, agarose and their partial decomposition products, oxides, alkylene Natural polymers including oxide adducts, carboxymethylates and cross-linked products, and polyvinyl alcohol, polyethylene glycol, polymethyl methacrylate, methacrylic ester polymers, silicone resins, polylactic acid, polyglycolic acid, poly-ε-caprolactone Examples thereof include synthetic polymers including a homopolymer or a copolymer of a bioabsorbable polymer.

  The porous biocompatible material is preferably composed of an inorganic material or a combination of an inorganic material and an organic material. In the case of using a combination of an inorganic material and an organic material, a configuration in which the organic material is coated on the surface of the porous inorganic material can be preferably exemplified.

  The porous body of the material can be produced by a known method. For example, the porous body of the organic material can be obtained by a well-known method such as a foam molding method using a foaming agent or a porous agent removing method. In the foam molding method of the porous body, a foaming agent is added to a polymer compound to foam the foaming agent, and then the polymer is cured. A water-soluble saccharide or salt may be added to the polymer solution, and after curing, the water-soluble substance may be washed away with water. Further, the porous body of the inorganic material can be manufactured by mixing, shaping and sintering inorganic material powder together with an organic binder.

  When the biocompatible material includes an inorganic material and an organic material, the surface of the inorganic material can be coated with the organic material. It is also possible to treat the surface of the organic material with an inorganic material by any method such as precipitating calcium phosphate by treating the organic polymer material with a calcium salt or solution thereof and a phosphate salt or solution thereof. .

  Examples of the shape of the porous biocompatible material include a lump shape, a sheet shape, a bead shape, a disk shape, and a sponge shape.

  Cells are seeded in the porous biocompatible material. Examples of cells to be seeded include cells derived from organs such as liver, kidney, pancreas, brain, spinal cord, gallbladder, adrenal gland, spleen, stomach, thyroid gland, preferably hepatocytes, tubule epithelial cells, pancreatic β cells And dopaminergic cells, nerve cells, glial cells, chromaffin cells, thyroid epithelial cells and the like. Only one type of cell may be seeded, or a plurality of cells may be seeded simultaneously or sequentially. When using a plurality of cells, cells from the same organ may be used, or cells from different organs may be used. In addition, fibroblasts, vascular endothelial cells and the like may be mixed with other cells in order to introduce blood vessels into the porous body.

  As the cells, cells derived from humans or animals, particularly mammals, can be used. Moreover, you may use the fused cell which fuse | melted 2 or more types of cells.

  When a porous biomaterial in which cells obtained by the method of the present invention are introduced into pores is transplanted into a human, a human or other animal-derived cell, particularly a human-derived cell can be preferably used.

  In the present invention, a porous material having a high cell density is obtained by seeding the porous material using external force and efficiently introducing the cells.

  Examples of the external force include centrifugal force, pressurization, decompression, and vibration.

In the present invention, seeding cells means forming a high-density cell group inside the porous body. The cells occupy 20% or more, preferably 50% or more, more preferably 70% or more, particularly 80% or more of the space of the porous body. As long as nutrients are supplied into the cell, almost 100% of the space of the porous body may be occupied by the cell.

  Within the porous body, cells can exist as a group of populations or clusters.

The method of seeding cells using external force is:
(1) Pressurization: For example, a cell suspension (liquid medium or buffer, etc.) and a porous biocompatible material are placed in a container with a porous bottom, and for example, using a syringe for injection closely attached to the container, A method in which the inside of the container is pressurized, the culture solution is pushed out of the container, and the cells are introduced into the porous material;
(2) Depressurization: Place the cell suspension and porous biocompatible material in a container with a porous bottom, for example, depressurize the bottom to extract the culture solution in the container from the bottom of the container and make the cell porous Introducing into the material;
(3) Centrifugal force: Place the cell suspension and porous biocompatible material in a container, and use normal centrifugal devices to damage cells depending on the cell type, porous material, pore size, etc. A method of introducing cells into the porous material by applying a centrifugal force within a range that does not give
(4) Vibration: A method in which cells are introduced into a porous material using gravity by placing a cell suspension in a container containing a porous material at the bottom and applying mechanical vibration or sound waves;
Is exemplified.

In a particularly preferred embodiment of the present invention, the porosity or the pore diameter and the external force conditions when cells are effectively seeded on the porous biocompatible material are:
・ When the porosity is 95% to 65%, the external force exceeds 1G and 30G or less.
・ When the porosity is 65% ~ 40%, external force 30G ~ 120G,
・ When the porosity is 40% ~ 20%, external force 120G ~ 200G,
・ When the pore size is 600 μm to 500 μm, the external force exceeds 1G and 30G or less.
・ External force 30G ~ 120G when the pore size is 500μm ~ 200μm,
-When the pore diameter is 200 μm to 50 μm, the external force is 120 G to 200 G.

  When such conditions are satisfied, it is particularly preferable because the activity is not reduced by cell seeding.

  When cells are seeded on one side of the porous material when an external force is applied once, it is possible to increase the cell density of the porous material by turning the porous material over and applying multiple external forces. It is.

The porous material containing cells at a high density obtained by the seeding method of the present invention has the same functions as cultured cells even though the cells are forcibly introduced into the porous material at a high density by external force. The above is maintained. Therefore, even when transplanted in a living body, some functions of the organ can be fully exhibited.

  Examples of the transplant site include subcutaneous, intradermal, intramuscular and intraperitoneal.

  For example, when hepatocytes are seeded on a porous material and transplanted to appropriate in vivo sites such as subcutaneous, intradermal, intramuscular, intraperitoneal, etc., functions such as production of proteins and enzymes such as albumin and metabolism of various substances Can be demonstrated. Moreover, if it is a porous material which seed | inoculated the insulin producing cell, it is possible to reduce a blood glucose level by production of insulin.

  Even if various cells such as hepatocytes are transplanted as dissociated cultured cells or cell clusters, these cells are absorbed into surrounding tissues and cannot function.

  On the other hand, when cells are transplanted after seeding in a porous biocompatible material at a high density as in the present invention, the cells can not only maintain their functions for a long time independently of the surrounding tissue, but also the number of cells ( Since the cell density is large, its function can be exhibited to a sufficient extent.

The technique of seeding cells in the pores of a porous biocompatible material at a high density has made it possible to reduce the number of cells and to maintain the long-term function of the cells in vivo.

Example 1
As Example 1 of the present invention, a method for preparing a rat hepatocyte / hydroxyapatite ceramics composite containing rat hepatocytes at high density by centrifugation and the results after transplanting the prepared composite to a model animal will be described.

1. Cell culture hepatocytes were isolated from male SD rat liver according to the method of Seglen. Isolated hepatocytes in Williams E medium with 10% fetal bovine serum (FBS), 100 ng / mL aprotinin, 1 nM
The suspension was suspended in insulin (1 nM dexamethasone, 50 U / mL penicillin G, 100 μg / mL streptomycin, 50 ng / mL amphotericin B) (IDSA medium). Cell suspension diameter 5

Three ³ × 10 ⁵ seeds were seeded on hydroxyapatite ceramics having a height of 2 mm and an average pore diameter of 200 μm. Hepatocyte / hydroxyapatite ceramics composite, 37? C, in the presence of 5% CO ₂ I
After culturing in DSA medium for 6 hours, it was cultured in Williams E medium supplemented with 10% FBS, 50 U / mL penicillin G, 100 μg / mL streptomycin, 50 ng / mL amphotericin B. The medium was changed every 24 hours.

2. Cell seeding using a centrifugal operation To fill the hepatocytes with high density, place 4 hydroxyapatite ceramics in a 50 mL centrifuge tube containing 5 mL of a hepatocyte suspension (2.5X10 ⁶ cells / mL) and perform the centrifugation. (50G, 1 minute) was performed three times. Is this hepatocyte / hydroxyapatite ceramic composite 37? C, 5% CO ₂
Cultivate for 6 hours in IDSA medium in the presence of 10% FBS, 50 U / mL penicillin G, 100 in Williams E medium.


The cells were cultured in a medium supplemented with μg / mL streptomycin and 50 ng / mL amphotericin B. The medium was changed every 24 hours.

When analyzed on the 6th day of culture, the number of cells retained in the hydroxyapatite ceramics by this centrifugation operation was about 4 times per ceramic compared to the control in which the centrifugation operation was not performed (FIG. 1A).

3. Measurement of urea synthesis activity The measurement of urea synthesis activity using cultured hepatocytes was carried out by adding 1 mM ammonium chloride to the culture system of hepatocyte / hydroxyapatite ceramics complex on the 6th day after the start of culture, and culturing by 4 hours later. This was done by quantifying the amount of urea produced in the liquid. In the evaluation, the amount of urea produced per hour was converted to one per hydroxyapatite ceramic.

Since the number of hepatocytes retained in hydroxyapatite ceramics by centrifugation increased by a factor of 4 compared to the control without centrifugation, urea synthesis activity also increased by a factor of 4 per hydroxyapatite (Fig. 1B).

4). Transplantation of hepatocyte / hydroxyapatite ceramics composites into albumin-free rats 37? After culturing in an IDSA medium in the presence of C and 5% CO ₂ for 6 hours, the complex was transplanted into the abdominal cavity of male albumin-free rats derived from SD rats. Albumin-free rats that were not transplanted were used as controls.

5. Serum albumin concentration measurement method Blood was collected from the tail vein without albumin at 1, 2, and 3 weeks after transplantation according to the previous section 4, and the serum albumin concentration was quantified by sandwich ELISA using anti-rat albumin antibody.

Serum albumin concentrations at 1, 2, and 3 weeks after transplantation were significantly higher than those in the control without transplantation (FIG. 2). This indicates that hepatocytes survived and functioned in vivo at least until 3 weeks after transplantation.

6). Histochemical analysis The hepatocyte / hydroxyapatite ceramic composite was taken out from the abdominal cavity of the albumin-free rat 3 weeks after transplantation according to the previous section 4. This complex is fixed with a 10% formalin solution,
Thin slices were prepared after decalcification. The sections were stained with a hematoxylin / eosin solution and then observed with an optical microscope.

  When the complex 3 weeks after transplantation was observed, hepatocytes were engrafted in the hydroxyapatite pores. Obvious angiogenesis was observed in the pores (FIG. 3). It was inferred that hepatocytes engrafted in hydroxyapatite pores by centrifugation were able to survive and develop functions due to blood flow through the new blood vessels.

FIG. 1 shows the maintenance of the number of viable hepatocytes per disc seeded by centrifugation in hydroxyapatite (HA). “A” indicates the number of viable cells counted by trypan blue exclusion test after 6 days of culture. “B” indicates the amount of urea synthesis expressed as a value per disc after 6 days of culture. Values are mean ± SD (standard deviation) of 3 experiments. An asterisk (**) indicates a significant difference ( ^** p <0.01) by a statistical method. FIG. 2 shows an increase in Nagase albumin-free rat (NAR) serum albumin concentration transplanted with HA discs seeded with hepatocytes. Serum albumin levels in transplanted rats (♦) and serum albumin levels in non-transplanted rats (■) were measured once a week by sandwich ELISA. Values are mean ± SD (standard deviation) of 3 experiments. An asterisk (*) indicates a significant difference ( ^* p <0.05) from the non-transplant control. Figure 5 shows angiogenesis of HA discs implanted in Nagase albumin free rats (NAR). HA discs were removed 3 weeks after transplantation. The arrowhead is a cross-sectional view of the formed blood vessel.

Claims (10)

A method of seeding cells in pores of a porous biocompatible material, wherein the cells are introduced into the pores of the porous biomaterial using an external force. The biocompatible material is
(1) Hydroxyapatite, tricalcium phosphate, calcium deficient apatite, amorphous calcium phosphate, tetracalcium phosphate, octacalcium phosphate, fluorinated apatite, carbonate apatite, calcium carbonate, calcium pyrophosphate, monetite, raw Ceramic materials including inorganic compounds that can adsorb calcium phosphates in the body;
(2) Collagen, gelatin, chitin, chitosan, glycosaminoglycan, hyaluronic acid, chondroitin sulfate, fibrin, alginic acid, fibroin, starch, pectin, pectic acid, agarose and their partial degradation products, oxide, alkylene oxide addition Natural polymers including products, carboxymethylates and cross-linked products; and
(3) Homopolymers or copolymers of bioabsorbable polymers such as polyvinyl alcohol, polyethylene glycol, polymethacrylic acid methyl, methacrylic acid ester polymer, silicone resin, polylactic acid, polyglycolic acid, polyε-caprolactone A synthetic polymer comprising:
The method according to claim 1, wherein the method is at least one selected from the group consisting of: The biocompatible material is hydroxyapatite, tricalcium phosphate, calcium deficient apatite, amorphous calcium phosphate, tetracalcium phosphate, octacalcium phosphate, fluorinated apatite, carbonate apatite, calcium carbonate, calcium pyrophosphate, monetite, Including at least one ceramic material selected from the group consisting of inorganic compounds capable of adsorbing calcium phosphates in vivo, collagen, gelatin, chitin, chitosan, glycosaminoglycan, hyaluronic acid, chondroitin sulfate as optional components , Fibrin, alginic acid, fibroin, starch, pectin, pectic acid, agarose and their partial degradation products, oxides, alkylene oxide adducts, carboxymethylated products and cross-linked natural polymers Includes homopolymers or copolymers of bioabsorbable polymers such as polyvinyl alcohol, polyethylene glycol, polymethyl methacrylate, methacrylate polymers, silicone resins, polylactic acid, polyglycolic acid, and poly-ε-caprolactone. The method according to claim 1 or 2, further comprising at least one selected from the group consisting of synthetic polymers. The external force that introduces cells into the pores of the porous biomaterial is greater than 1G (G is the acceleration of gravity), and 200G
The method according to claim 1, wherein: The method according to claim 1, wherein the external force is selected from the group consisting of centrifugal force, pressurization, decompression, and vibration. The porosity of the porous biocompatible material is 20% to 95%, and the pore diameter range is 50 μm to 600 μm.
The method according to any one of claims 1 to 5. The porosity and external force conditions when cells are effectively seeded on the porous biocompatible material are: porosity 95
When external force is more than 1G and 30G or less when% to 65%, external force is 30G to 120G when porosity is 65% to 40%, porosity is 40% to 2
The method according to claim 1, wherein the external force is 120 G to 200 G when 0%. The pore diameter and external force conditions when cells are effectively seeded on the porous biocompatible material are: pore diameter 60
External force over 1G and 30G or less when 0 μm to 500 μm, external force 30G to 12 when pore diameter is 500 μm to 200 μm
The method according to any one of claims 1 to 6, wherein the external force is 120G to 200G when the pore size is 0 G and the pore diameter is 200 µm to 50 µm. The method according to claim 1, wherein the porous biocompatible material has a lump shape, a sheet shape, a bead shape, a disk shape, or a sponge shape. A transplantation method comprising transplanting a porous biomaterial into which pores obtained by the method according to any one of claims 1 to 9 are introduced into a human or a mammal.

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