JP2006246770A - Method of inducing cell differentiation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of inducing cell differentiation capable of differentiating embryonic stem cells to vascular endothelial cells near the surface of histoengineering scaffold material on which the embryonic stem cells are engrafted and to smooth muscle cells in the histoengineering scaffold material, thus capable of producing hybrid-type artificial blood vessel having three-dimensional hierarchical structure. <P>SOLUTION: The method of inducing the differentiation of cells by engrafting the cells on a histoengineering scaffold material of thermoplastic resin having porous three-dimensional network structure is provided, involving culturing the cells under applying pulsatile flow thereto. Thus, endothelial cells are differentiated by shearing force caused by the pulsatile flow burden, while smooth muscle cells are differentiated by periodical stretch/contraction and pressure pulse. By this method, as multiple vascular wall-constructive cells can be differentiated simultaneously, the hybrid-type artificial blood vessel having three-dimensional hierarchical structure can be constructed. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a cell differentiation induction method, and more particularly to a cell differentiation induction method suitable for producing an artificial blood vessel.

  Unlike cell division, cell differentiation may change into several cells. In particular, cells having pluripotency such as stem cells can differentiate into a wide variety of cells. In tissue engineering, it is necessary to induce differentiation of a cultured cell or a cultured cell into a target cell. Therefore, selective differentiation induction of a cell is an important technique.

Regarding the induction of differentiation of undifferentiated embryonic stem (ES) cells into vascular wall constituent cells in vitro, vascular endothelial cells and pericytes are induced by cytokines such as VEGF and PDGF using biochemical techniques, Furthermore, it has been reported that capillary-like tubular tissues are formed by three-dimensional culture.
Yamashita J, Itoh H, et al. Flk1 positive cells derived from embryonic stem cells serveas vascular progenitors.Nature 408: 92-96, 2000. Yurugi-Kobayashi T, Itoh H, Yamashita J, et al. Effective contribution of transplanted vascular progenitor cells derived from embryonic stem cells to about neovascularization in proper differentiation stage.Blood 101: 2675-2678, 2003.

  Embryonic stem cells are cells that have a high possibility of being used in regenerative medicine and regenerative medicine that have attracted attention in recent years, but it is known that there is a risk of becoming tumor cells when transplanted into a living body in an undifferentiated state. It is a fact. Therefore, differentiation of embryonic stem cells in regenerative medicine without leaving undifferentiated cells, in particular, establishment of a technique for inducing differentiation of cells engrafted in a scaffold into a target cell is a major issue.

  The present invention can differentiate embryonic stem cells into vascular endothelial cells near the surface of the tissue engineering scaffold material engrafted with embryonic stem cells, and differentiate into smooth muscle cells inside the tissue engineering scaffold material. An object of the present invention is to provide a cell differentiation induction method capable of producing a hybrid type artificial blood vessel having a three-dimensional hierarchical structure.

  The method of inducing cell differentiation according to claim 1 is a method of engrafting cells in a scaffold material for tissue engineering made of a thermoplastic resin having a porous three-dimensional network structure to induce differentiation of the cells. In contrast, the culture is performed while applying a pulsatile flow.

  The cell differentiation induction method according to claim 2 is the method according to claim 1, wherein the thermoplastic resin is made of a polyurethane resin, a polyamide resin, a polylactic acid resin, a polyolefin resin, a polyester resin, a fluororesin, an acrylic resin, a methacrylic resin, and derivatives thereof. It is 1 type or 2 types or more selected from a group, It is characterized by the above-mentioned.

  The cell differentiation induction method according to claim 3 is characterized in that, in claim 2, the thermoplastic resin is a polyurethane resin.

  The cell differentiation induction method according to claim 4 is characterized in that, in claim 3, the polyurethane resin is coated with a biocompatible substance.

  The cell differentiation inducing method according to claim 5 is characterized in that, in any one of claims 1 to 4, the pulsation period is 0.1 Hz to 10000 Hz.

  The cell differentiation induction method according to claim 6 is characterized in that, in any one of claims 1 to 5, the difference between the highest and lowest pulsation pressures is 1 mmHg to 500 mmHg.

  The cell differentiation induction method according to claim 7 is characterized in that, in any one of claims 1 to 6, the cell is a stem cell.

  The cell differentiation induction method according to claim 8 is characterized in that, in claim 7, the stem cell is an embryonic stem cell.

  The cell differentiation inducing method according to claim 9 is characterized in that, in any one of claims 1 to 8, the tissue engineering scaffold is tubular, and an artificial blood vessel is produced by cell differentiation. is there.

  According to the present invention, it is considered that endothelial cells are induced to differentiate by shear stress generated by a pulsatile flow load, and smooth muscle cells are induced to differentiate by periodic stretching and pressure pulsation.

  When a tubular tissue engineering scaffold is used, a plurality of blood vessel wall-constituting cells can be induced to differentiate simultaneously, so that a hybrid artificial blood vessel having a three-dimensional hierarchical structure can be constructed.

  Hereinafter, preferred embodiments of the present invention will be described in detail.

  First, the scaffold material for tissue engineering used in the cell differentiation induction method of the present invention will be described.

  The three-dimensional network structure portion made of a thermoplastic resin constituting the tissue engineering scaffold is preferably tubular, and may be a continuous, ie, porous, three-dimensional network structure having a continuous porosity. Even if the whole from the outer wall to the outer wall may have a similar structure, it may be different between the vicinity of the inner wall and the vicinity of the outer wall. Further, the average pore diameter and the apparent density may partially change, and for example, it may have so-called anisotropy in which the average pore diameter gradually changes from the inner wall toward the outer wall. A skin layer may be provided on the outer peripheral surface of the tubular body.

The average pore diameter of the porous three-dimensional network structure made of this thermoplastic resin is preferably 20 to 650 μm, and the apparent density is 0.01 to 0.5 g / cm ³ , more preferably 30 to 100 μm. If the apparent density is in the range of 0.01 to 0.5 g / cm ³ , cell engraftment is good, excellent physical strength is maintained, and elastic properties approximate to a living body are obtained. 0.01 to 0.2 g / cm ³ , more preferably 0.01 to 0.1 g / cm ³ .

  The distribution of the pore size is preferably monodispersed, and it is desirable that the contribution ratio of pores having a pore size of 30 to 100 μm, which is a pore size important for cell invasion, is high. When the contribution ratio of pores having a pore diameter of 30 to 100 μm is 10% or more, preferably 20% or more, more preferably 30% or more, still more preferably 40% or more, and particularly preferably 50% or more, cells easily invade, Since the invading cells are likely to adhere and grow, it is effective for use as a scaffold material and an artificial blood vessel.

  With such a porous three-dimensional network structure having an average pore size, apparent density, and pore size distribution, cells, culture solution, and the like can easily penetrate into the pores, and cells can easily adhere to and grow in the porous structure layer. Therefore, when this is formed into a tubular shape, cells can be engrafted from the inner wall to the outer periphery, so that an artificial blood vessel with a low risk of occlusion and a high patency rate can be realized.

  Examples of the thermoplastic resin constituting the scaffold for tissue engineering include polyurethane resin, polyamide resin, polylactic acid resin, polyolefin resin, polyester resin, fluororesin, acrylic resin, methacrylic resin and derivatives thereof. These may be used alone or in combination of two or more, but are preferably polyurethane resins, and segmented polyurethane resins are particularly excellent in antithrombogenicity and physical properties. An artificial blood vessel can be obtained, which is preferable.

  Also, if the thermoplastic resin is hydrolyzable or biodegradable, it is gradually decomposed and absorbed after the transplantation of the artificial blood vessel, and finally the resin base material leaving the engrafted cells. It is also possible to exclude itself from the living body.

  By coating the surface of this scaffold material with a biocompatible compound such as fibronectin, the adherence of cells can be improved.

  As cells to be engrafted in this scaffold material, embryonic stem cells (ES cells) and ES cell-derived Flk-1 positive cells are suitable.

  In order to engraft the cells on the scaffold material, at least a part of the cell suspension may be purified by MACS (magnetic cell separation device) as necessary, and then seeded on the scaffold material. Examples of this purification treatment method include a method in which FLK-1 positive cells are magnetically labeled with magnetic beads to which an FLK-1 antibody is immobilized, and then separated on a column. The purification rate is preferably 90% or more. In addition, the magnetically labeled cells are preferably seeded on the scaffold as they are (without removing the magnetic beads on which the antibody is immobilized).

  When an artificial blood vessel is manufactured by the method of the present invention, the scaffold material is a hollow tube, and the cell suspension is seeded on the inner surface of the lumen. Specifically, by inserting a nozzle into the lumen and rotating the tubular scaffold material around the axis while discharging the cell suspension from the nozzle, the cell suspension can be uniformly attached to the lumen surface, Either one end of the tubular scaffold material is sealed, the cell suspension is injected from the other end, and then seeded by pressurization, or the cell suspension is injected into the hollow tubular scaffold material and continuously Or a method of stationary culture while rotating intermittently.

The cell adhesion amount per unit area of the lumen surface is preferably about 1 to 500,000 cells / cm ^2, but is not limited thereto.

  About 0.5 to 4 days after sowing, VEGF, platelet-derived growth factor, epidermal growth factor, transforming growth factor α, insulin-like growth factor, insulin-like growth factor binding protein, hepatocyte growth factor, angiopoietin , Nerve growth factor, brain-derived neurotrophic factor, ciliary neurotrophic factor, transforming growth factor β, latent transforming growth factor β, activin, bone trait protein, fibroblast growth factor, tumor growth factor β, two About 0.1-10 ug / mL of cytokines such as ploidy fibroblast growth factor, heparin-binding epidermal growth factor-like growth factor, schwanoma-derived growth factor, amphiregulin, betacellulin, epigrelin, lymphotoxin, erythroepoetin After stationary culture in the middle of the culture, the culture solution containing no cytokines such as VEGF and PDGF is distributed in the lumen. That. During this distribution, the culture solution is beaten.

  As the culture medium, DMEM, BME, glutamax and the like can be used.

  The average flow rate (average flow rate in the tube axis direction) of the culture solution is preferably about 5 to 250 ml / min.

  A pulsating pump may be used for pulsation. The period of pulsation is preferably about 0.7 to 1.2 seconds, and in one pulsation, the systole is about 0.2 to 0.4 seconds, and the diastolic period is 0.4 to 0.6 seconds. The degree is preferred. The average pulsation pressure is preferably about 10 to 100 mmHg, and the difference between the maximum pulsation pressure and the minimum pressure is preferably about 10 to 150 mmHg.

Example 1
After preparing a cell suspension with a concentration of 5 × 10 ⁹ cells / mL mainly composed of Flk-1 positive cells derived from undifferentiated mouse ES cells, and magnetically labeling them with magnetic beads fixed with FLK-1 antibody, Then, the cells were purified by magnetic cell separation and then mixed with unpurified cells to obtain a cell suspension.

Polyurethane sponge tubes having a skin layer on the outer peripheral surface were prepared with an inner diameter of 5 mm, an outer diameter of 6 mm, and a length of 30 mm, and an inner diameter of 1.8 mm, an outer diameter of 30 mm, and a length of 30 mm. The thickness of the skin layer is about 0.05 to 0.2 mm. This sponge tube has an average pore diameter of 40 μm and an apparent density of 0.23 g / cm ³ . Fibronectin was thinly coated by immersing it in a PBS solution containing 0.1% fibronectin on the inner peripheral surface of each tube for 30 minutes.

While holding these tubes with a holder and rotating them around the axis at 2000 rpm, a nozzle was inserted into the lumen to discharge the cell suspension and seeded from the lumen surface. The seeding amount per unit area of the lumen surface is 3 million cells / mm ² .

Subsequently, pre-stationary culture was performed for 2 days in a culture medium composed of α-MEM medium (containing 10% FBS and 5 × 10 ⁻⁵ M 2-mercaptoethanol) containing 0.1 μg / mL of VEGF. Thereafter, the same culture solution as above except that VEGF was not contained was passed through the lumen of each tube using a pulsating pump.

The average linear velocity of liquid flow was 50 mL / min. The pulsation is as follows: systole 0.3 seconds, diastolic 0.5 seconds, pulsation maximum pressure 30 mmHg, pulsation minimum pressure 10 mmHg, shear stress −0.98 to 2.2 dyn / cm ² , peripheral stress 4.6 to The measurement was performed under the condition of 9.6 × 10 ⁴ dyn / cm ² .

  After passing this solution for 2 days, the cell type was examined immunohistologically, and the cell morphology of the luminal surface was observed with SEM. As a result, vascular endothelial cell marker (CD31, CD144) positive cells and smooth muscle cell marker (αSMA) positive cells were observed.

  This is thought to be because endothelial cells were induced to differentiate by shear stress generated by pulsatile flow load, and smooth muscle cells were induced to differentiate by periodic stretching and pressure pulsation.

Claims (9)

  In a method for engrafting cells in a tissue engineering scaffold made of thermoplastic resin having a porous three-dimensional network structure and inducing differentiation of the cells, culturing the cells while applying a pulsatile flow A cell differentiation inducing method characterized by the above.   2. The thermoplastic resin according to claim 1, wherein the thermoplastic resin is selected from the group consisting of polyurethane resin, polyamide resin, polylactic acid resin, polyolefin resin, polyester resin, fluororesin, acrylic resin, methacrylic resin, and derivatives thereof. A method for inducing cell differentiation characterized by the above.   The cell differentiation induction method according to claim 2, wherein the thermoplastic resin is a polyurethane resin.   The cell differentiation induction method according to claim 3, wherein the polyurethane resin is coated with a biocompatible substance.   The method for inducing cell differentiation according to any one of claims 1 to 4, wherein a cycle of pulsation is 0.1 Hz to 10000 Hz.   6. The method for inducing cell differentiation according to any one of claims 1 to 5, wherein the difference between the highest pressure and the lowest pressure of pulsation is 1 mmHg to 500 mmHg.   The cell differentiation induction method according to any one of claims 1 to 6, wherein the cell is a stem cell.   8. The method for inducing cell differentiation according to claim 7, wherein the stem cell is an embryonic stem cell.   9. The method for inducing cell differentiation according to claim 1, wherein the tissue engineering scaffold is tubular and an artificial blood vessel is produced by cell differentiation.