JP2011024423A - Particulate cell carrier and cell-introducing system containing the same and cell culture system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cell carrier which has a proper size not clogging an injector or a catheter, even when injected through the injector or the catheter, and can stably hold various cells in a living state. <P>SOLUTION: There is provided a particulate cell carrier comprising fine particles comprising low molecular heparin and protamine. The particulate cell carrier easily forms cell aggregates having suitable sizes together with various adhesive cells, and the cell aggregates can be injected into a body with an injector or the like. The particulate cell carrier can be used as a carrier for cell transplantation in regenerative medical treatments. Various cells, especially CD-34 positive hematopoietic system stem cells or mesenchymal stem cells which have been hardly obtained in a sufficient yield with a conventional technique, can be cultured in a serum-free state or in a low concentration serum state (about 1 to 2%) on a substrate coated with the fine particles. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to the use of microparticles composed of low molecular weight heparin and protamine as microparticle cell carriers (cell microcarriers) and coating materials. Furthermore, the present invention relates to an effective cell introduction method using the fine particle cell carrier and an effective cell growth method in a low serum medium using a culture substrate coated with the fine particles.

  In recent years, cell transfer therapy as regenerative medicine, which has been actively studied, aims to induce tissue regeneration by engrafting and proliferating the implanted cells themselves and the factors generated by the introduced cells into the target site. Is the method. Angiogenesis therapy uses vascular endothelial growth factor (VEGF) gene and endothelial progenitor cells (EPC), but research results are available as effective methods. There are concerns about safety and cost-effectiveness, and it has not been put into practical use. In addition, bone marrow transplantation has made great progress since the establishment of the bone marrow bank, but bone marrow transplant cases have been slow to grow, and research and development of efficient and effective bone marrow transplantation technology has been demanded through the proliferation of hematopoietic stem cells and the advancement of engraftment. ing.

  By the way, it is known that many tissues including bone marrow and adipose tissue contain precursor cells that retain pluripotency for tissue maintenance and repair. In particular, bone marrow cells include hematopoietic stem cells and mesenchymal stem cells and differentiate into not only various blood cells but also skin, bone, cartilage, blood vessel, and muscle cells to promote the regeneration of each tissue. Similarly, adipose tissue-derived mesenchymal stem cells are also known to differentiate into skin, bone, cartilage, blood vessel, and muscle cells, and a great deal of attention has been focused on fat, which is an abundant stem cell resource in humans.

  However, the yield of various stem cells is low, and there is a problem that a high concentration of animal serum is required for growth, and a culture method for efficiently growing in a medium containing a low concentration of serum is required. Furthermore, in cell transplantation, it is necessary to use a cell carrier that enhances engraftment and proliferation.

The present inventors have proposed that photocurable chitosan hydrogel (Patent Document 1) or 6-O-position desulfated heparin composite hydrogel (Patent Document 2) is a basic fibroblast growth factor (FGF-2) or the like. It has been reported that it protects the activity of various growth factors and is effective as a carrier for sustained release. It has been demonstrated that FGF-2, which is protected in the hydrogel and retains its activity, is gradually released to the surrounding sites as the hydrogel biodegrades, contributing to the promotion of angiogenesis and granulation in vivo. .
WO03 / 090765 pamphlet WO2005 / 025538 pamphlet

  However, these hydrogels are intended to be applied to an injury site such as a skin ulcer, and are designed to have high viscosity to prevent dripping. Therefore, when administered through a syringe or catheter, clogging may occur frequently. Moreover, if the viscosity is lowered by simply lowering the concentration of chitosan or the like, the amount of drug that can be carried is limited, and the desired effect may not be obtained. In particular, these hydrogels could not hold and survive various adhesive cells in the gel.

By using a combination of low molecular weight heparin and protamine, the present inventors can easily create microparticles having an average particle size of less than 10 μm and eventually 1 μm or less. It has been found that aggregates can be formed and used as injectable cell carriers. Further, the present inventors have found that the fine particles can easily coat (coat) the glass or plastic surface and promote the growth of various cells on the obtained substrate, and have made the present invention.
That is, the present invention provides a fine particle cell carrier comprising fine particles having a particle size of less than 5 μm composed of low molecular weight heparin and protamine, and a coating material comprising the fine particles.

  The particulate cell carrier of the present invention is bonded to the surface of cells such as bone marrow or adipose tissue-derived mesenchymal stem cells, capillary endothelial cells, fibroblasts, etc., and promotes the formation of cell aggregates (spheroids). Viability and engraftment can be improved. Since the fine particle cell carrier of the present invention has an average particle size of less than 10 μm or 1 μm or less, the size of the formed cell aggregate can be easily controlled, and a cell aggregate that can be injected even with a syringe equipped with a fine needle is obtained. It is excellent in handling and operability. Low molecular weight heparins such as dalteparin and protamine used in the present invention are commercially available materials, and their safety is ensured. In addition, since the particulate cell carrier of the present invention protects cells in vivo and enhances their viability and biodegrades, when spheroids are injected into the body, spheroids disappear due to their biodegradation and cell migration. It is particularly excellent as a carrier for cell transplantation.

  On the other hand, glass or plastic plates coated with the microparticles of the present invention are efficiently adsorbed with heparin-binding growth factors such as FGF-2 and cytokines at high concentrations, and various cells are expressed by their efficient activity expression. Adhesiveness to the coated plate and proliferation are improved.

  As described above, the fine particle cell carrier and the coating material of the present invention use fine particles composed of low molecular weight heparin and protamine, and the average particle size is less than 10 μm, preferably 5 μm or less, more preferably 1 μm or less. About 0.5 μm. The lower limit of the particle size is not particularly limited, but is generally about 0.01 μm or more.

  The low molecular weight heparin constituting the particulate cell carrier and coating material of the present invention is generally a low molecular weight heparin obtained by depolymerizing natural heparin (molecular weight of about 15,000 to 20,000). The molecular weight range is not limited so long as it can form a particulate cell carrier (average particle size: 1 to 0.5 μm) by mixing with protamine, generally less than about 10,000, preferably less than about 8,000. More preferably, those having an average molecular weight of less than about 6,000 are used.

  For example, depolymerized heparin (dalteparin) obtained by nitrous acid decomposition of heparin derived from porcine small intestinal mucosa is commercially available as Fragmin (trade name) and has an average molecular weight of about 3,000 to 5,000. Similarly, depolymerized heparin (reviparin) obtained by nitrous acid decomposition of heparin derived from porcine small intestinal mucosa is expressed as lomorin (trade name), heparin derived from bovine or porcine small intestinal mucosa by hydrogen peroxide and cupric acetate. Depolymerized heparin (parnaparin) obtained by decomposition is commercially available as Rhohepa (trade name), and any of these can be used as the low molecular weight heparin in the present invention.

  Heparin alone does not have an anticoagulant effect and exerts its effect by binding to plasma ATIII, and coagulation enzymes such as factor IIa, factor XIIa, factor XIa, factor Xa, factor IXa Inhibits and inactivates. On the other hand, although low molecular weight heparin used in the present invention has anti-factor XIIa and anti-factor Xa activities, it has been clarified as a pharmaceutical that its inhibitory activity against factor IIa, XIa and factor IXa is slight. Even if it is injected into a wound site, it can be used without promoting the bleeding tendency at the site.

  The commercially available low molecular weight heparin of the present invention may be used. For example, heparin reduced in molecular weight by periodate oxidation / reduction or specific desulfated heparin can also be used. The low molecular weight heparin of the present invention generally has a molecular weight of less than about 10,000, preferably less than 10,000 to 7,000, more preferably about 3,000 to 5,000. By using such low molecular weight heparin, a particulate cell carrier can be obtained when mixed with protamine.

  On the other hand, protamine that constitutes the particulate cell carrier and coating material of the present invention is known as a highly basic protein that exists by binding to DNA in the nucleus of animal sperm. Generally, it is a low molecular weight protein consisting of 27 to 65 residues, and arginine accounts for 40 to 70% of amino acids. Protamine is also commercially available as a pharmaceutical. In the present invention, commercially available protamine can be used as it is. The weight ratio between the low molecular weight heparin and protamine is not particularly limited, but the yield of fine particles obtained tends to be improved when the weight of the low molecular weight heparin with respect to protamine is equal or slightly excessive.

  When high molecular weight heparin, hyaluronic acid, or chondroitin sulfate is used instead of low molecular weight heparin, it is not possible to obtain fine particles as obtained in the present invention. By employing a unique combination of low molecular weight heparin and protamine, it is possible to obtain fine particles having an average particle size of less than 10 μm and further 1 μm or less.

The first aspect of the present invention is a fine particle cell carrier comprising the fine particles.
The particulate cell carrier of the present invention has a property of easily binding to various adherent cells. Therefore, by incubating with adherent cells, various adherent cell aggregates (spheroids) composed of several to several tens of cells are easily formed in a floating state. Moreover, the average particle size of cell aggregates formed by incubation for about 1 to 5 hours is about 50 to 100 μm, and does not clog syringes and catheters. Suitable for use as a composition. Furthermore, additional cell maintenance, proliferation, differentiation induction, and engraftment effects can be expected by simultaneously adding to the composition a heparin-binding growth factor or cytokine involved in the maintenance, proliferation, and differentiation of various cells.

  Therefore, the present invention provides a cell aggregate comprising the fine particle cell carrier and adherent cells. In this cell aggregate, cells forming the aggregate can stably survive. And since the cell aggregate of the present invention can be sized to be injected into the body, it can be transplanted into a target part of the body and allowed to engraft, which enables cell transplantation that has been difficult in the past. To.

  The cell aggregate has a micro size that can be injected and can be dispersed in a suspended state in a medium, and thus can be applied as various pharmaceuticals. For example, cells are engrafted at the target site by injecting into the target site a dispersion of cell aggregates comprising the particulate cell carrier of the present invention and adipose tissue-derived mesenchymal stem cells. As a result, angiogenesis at the target site can be promoted. Furthermore, by allowing various physiologically active molecules suitable for cell growth to coexist, cell growth at the target site is further promoted, and the function of the cell can be exhibited.

Therefore, the present invention provides a pharmaceutical composition containing the cell aggregate of the present invention and, if necessary, an appropriate physiologically active substance.
The cell constituting the cell aggregate used in the pharmaceutical composition of the present invention is not particularly limited. For example, adipose tissue or bone marrow-derived mesenchymal stem cells used in regenerative medicine involving cell introduction, CD34 positive Hematopoietic stem cells, vascular endothelial cells and their precursor cells, hepatocytes and their precursor cells, dermal fibroblasts, chondrocytes and their precursor cells, bone cells and their precursor cells, and other universal cells used in regenerative medicine (IPS cells), embryonic stem cells (ES cells), cell lines and the like.

  The physiologically active substance optionally added to the pharmaceutical composition of the present invention is preferably a substance that has a high affinity with low molecular weight heparin or protamine and is easily carried on fine particles. Especially growth factors such as fibroblast growth factor (FGF), liver growth factor (HGF), vascular endothelial growth factor (VEGF), stem cell factor (SCF), thrombopoietin (Tpo), interleukin-3 (IL-3), A heparin-binding protein such as granulocyte-macrophage colony-stimulating factor (GM-CSF) is preferred.

The second aspect of the present invention is a coating material comprising the fine particles.
The fine particles are well dispersed in an aqueous solution, and a fine particle-coated substrate can be obtained by simply adding the fine particles onto a glass or plastic substrate plate and allowing to stand for several hours. Proteins necessary for cell growth such as various growth factors and cytokines are easily adsorbed on the surface of the substrate. When this substrate is used, various cells can be cultured in low-concentration serum (about 1 to 2%) simply by adding a low-concentration heparin-binding growth factor or cytokine. In particular, hematopoietic cytokines (SCF: stem cell factor, Tpo: thrombopoietin, Flt-3: Flt-3 ligand) are well adsorbed on the substrate coated with the microparticles of the present invention. CD34-positive hematopoietic stem cells and mesenchymal stem cells whose yield was not obtained can be grown in a low (no) serum medium.

Therefore, the present invention provides a cell culture substrate comprising a substrate whose surface is coated with the fine particle coating material of the present invention. Prior to cell culture, physiologically active substances such as various growth factors and cytokines may be adsorbed on the cell culture substrate of the present invention. Further, these physiologically active substances may coexist in a medium for cell culture.
For example, when using the cell culture substrate of the present invention, the present inventors have found that the proliferation rate of CD34 positive-hematopoietic stem cells has a low concentration of hematopoietic cytokines (SCF; 5 ng / ml, Tpo; 10 ng / It was confirmed that the serum-free medium containing ml, Flt-3; 10 ng / ml) was significantly improved.

  Cells to be cultured on the cell culture substrate of the present invention are not particularly limited, but are used in regenerative medicine, adipose tissue or bone marrow-derived mesenchymal stem cells, CD34 positive hematopoietic stem cells, vascular endothelial cells and precursors thereof Cells, hepatocytes and their precursor cells, dermal fibroblasts, chondrocytes and their precursor cells, bone cells and their precursor cells, other universal cells (iPS cells) used in regenerative medicine, embryonic stem cells (ES cells) ), Cell lines and the like. The physiologically active substance to be administered simultaneously is preferably a substance that has a high affinity with low molecular weight heparin or protamine and is easily carried on fine particles. Especially growth factors such as fibroblast growth factor (FGF), liver growth factor (HGF), vascular endothelial growth factor (VEGF), stem cell factor (SCF), thrombopoietin (Tpo), interleukin-3 (IL-3), Heparin-binding proteins such as granulocyte-macrophage colony stimulating factor (GM-CSF) are preferred.

EXAMPLES Hereinafter, although this invention is demonstrated further in detail with reference to an Example, these Examples do not limit this invention.
Example 1 Promotion of Aggregation of Suspended Cells and Improvement of Cell Viability by the Fine Particle Cell Carrier of the Present Invention As low molecular weight heparin, commercially available dalteparin, Fragmin (trade name) (6.4 mg / ml, 1,000 IU / ml, Similarly, commercially available protamine (10 mg / ml, Mochida Pharmaceutical Co., Ltd.) was mixed at a volume ratio of 7: 3 to obtain a fine particle dispersion. The dry fine particle yield per 1 ml of the fine particle dispersion at this time was about 7 mg.

  The fine particle dispersion was incubated with human capillary endothelial cells, human dermal fibroblasts, and mouse adipose tissue-derived mesenchymal stem cells for about 1 hour. As a result, as shown in FIG. 1, the microparticles are bonded to the surface of human capillary endothelial cells (A), human skin fibroblasts (B), and mouse adipose tissue-derived mesenchymal stem cells (C). Cell aggregates (spheroids) were formed by culturing within the time.

  This cell aggregate was suspended in culture using a medium containing 10% fetal bovine serum and 1.4 mg / ml microparticles, and viable cells were quantified using a cell counting kit (Dojindo Laboratories). As a result, human skin fibroblasts and mouse adipose tissue-derived mesenchymal stem cells were present in the presence of 1.4 mg / ml microparticles, and capillary endothelial cells were present in the presence of 1.4 mg / ml microparticles and 5 ng / ml FGF-2. Prolonged cell viability was observed for at least 3 days (FIG. 2).

(Example 2) Adsorption of various growth factors and cytokines on the surface of the fine particle-coated substrate of the present invention Various growth factors and cytokines (FGF-1, FGF-2, HGF, IL-3, GM-CSF, SCF, Tpo, Flt 3) Protamine / Fragmin fine particle coating-Adsorbability to plastic plate was measured using ELISA method. The cell culture plate was allowed to stand at 4 ° C. for 24 hours with a phosphate buffer (PBS) containing 1.4 mg / ml microparticles (FIG. 3A), and then the PBS solution was removed and the coating was performed by drying (FIG. 3B). Further, microparticles were similarly prepared using fluorescently labeled protamine (Texas Red-X Protein Labeling Kit; Invitrogen Japan KK, Tokyo, Japan) and coated (FIG. 3C). It was found that the fine particles changed to a paste form upon drying and were stably coated on the surface of plastic or the like.

This 48-well-coated-plastic plate contains 2% FBS containing 8, 4, 2 ng / ml growth factors (FGF-1, FGF-2, HGF, IL-3, GM-CSF, SCF). Add 200 μl of medium or 200 μl of hematopoietic progenitor growth medium (HPGM) containing 20, 10, 5 ng / ml of cytokine (Tpo, Flt-3) and let stand for 2 hours at room temperature. Cytokines were adsorbed. Next, each used solution (200 μl) was placed in another coating-well, and the second adsorption amount was quantified (FIG. 4). Each growth factor and cytokine adsorbed per 48-well plate (well area: 0.78 cm ² ) by incubation at room temperature for 2 hours by reducing the amount of adsorption from the first adsorption to the second (used solution) Estimated amount.

  In the ELISA method, each primary antibody was reacted with a plate on which each growth factor and cytokine were adsorbed, and after washing, a secondary antibody was reacted. Finally, it was quantified using a peroxidase substrate (Bio-Rad). When 200 μl of various growth factors and cytokines (4 ng / ml) solution was added to the coating-well and incubated at room temperature for 2 hours, the adsorbed amounts were (FGF-1: 0.42 ng, FGF-2: 0.36 ng, HGF: 0.32) ng / ml, IL-3: 0.36 ng / ml, GM-CSF: 0.36 ng / ml, SCF: 0.34 ng / ml, Tpo: 0.27 ng / ml, Flt-3: 0.32 ng / ml) .

(Example 3) Growth promotion of human bone marrow and mouse adipose tissue-derived mesenchymal stem cells using the microparticle-coated substrate of the present invention and 2% serum medium containing 5 ng / ml FGF-2. Bone marrow and mouse adipose tissue-derived mesenchymal stem cells were seeded, cultured in 2% serum DMEM medium containing 5 ng / ml FGF-2 for 5 days, and compared with the control of a 48-well tissue culture plate without coating the growth rate (Fig. 5).

  Apparently, the microparticle coating greatly promoted the proliferation of both bone marrow and adipose tissue-derived mesenchymal stem cells with 1% low serum in the presence of FGF-2. Although not shown, it was confirmed that both mesenchymal stem cells cultured and expanded under these conditions were induced to differentiate into adipocytes, osteoblasts, chondrocytes and the like. Therefore, the method for growing mesenchymal stem cells using the present fine particle-coated plate is an effective culture method that enables serum concentration reduction and growth promotion of the medium to be used.

(Example 4) Growth promotion of TF-1 cell line using microparticle-coated substrate of the present invention and 1% serum medium containing IL-3 and GM-CSF Since human TF-1 cell line normally grows in suspension culture Require more than 5% fetal bovine serum and hematopoietic cytokines such as IL-3 and GM-CSF. TF-1 cell line is seeded in a 48-well tissue culture plate coated with fine particles, suspended in 1% serum DMEM medium containing 5 ng / ml IL-3 and 5 ng / ml GM-CSF for 5 days, and its growth rate is not coated Compared to the control of a 48-well tissue culture plate (FIG. 6).

  Apparently, the microparticle coating greatly promoted the proliferation of TF-1 cells with 1% low serum in the presence of IL-3 and GM-CSF. Also, TF-1 cells are good on microparticle-coated 48-well tissue culture plates in which cytokines are pre-adsorbed at 4 ° C for 18 h in 2% serum DMEM medium containing 5 ng / ml IL-3 and 5 ng / ml GM-CSF It was revealed that the cytokine adsorbed on the fine particle coating plate maintained the active form (FIG. 7).

(Example 5) Human using the microparticle-coated substrate of the present invention and a serum-free medium containing low concentration cytokines (SCF (5 ng / ml), Tpo (10 ng / ml), Flt-3 (10 ng / ml)) Promotion of proliferation of CD 34-positive hematopoietic stem cells Human CD 34-positive hematopoietic stem cells are usually grown in suspension culture, so high concentrations of cytokines (20 ng / ml SCF, 40 ng / ml IL-3, 40 ng / It is necessary to culture in hematopoietic stem cell medium (HPGM) containing ml Flt-3). Seed CD 34-positive hematopoietic stem cells in a 24-well tissue culture plate coated with microparticles and contain no low concentrations (SCF (5 ng / ml), Tpo (10 ng / ml), Flt-3 (10 ng / ml)) The suspension was cultured in serum HPGM medium for 6 days, and the growth rate was compared with a control of a 24-well tissue culture plate without coating (FIG. 8).

  Clearly, the microparticle coating greatly promoted the proliferation of CD 34-positive hematopoietic stem cells in the presence of low concentrations of cytokines. In addition, including the results of measurement of the CD 34-positive cell rate by flow cytometry, this microparticle coating-plastic plate and low concentrations (SCF (5 ng / ml), Tpo (10 ng / ml), Flt-3 ( Human CD 34-positive hematopoietic stem cells using a serum-free medium containing 10 ng / ml)) were able to grow more than 10 times in 7 days.

(Example 6) Cell engraftment and angiogenesis promotion by administration of aggregates of particulate cell carrier of the present invention and adipose tissue-derived mesenchymal stem cells
After proliferating adipose tissue-derived mesenchymal stem cells collected from GFP (Green Fluorescent Protein) mice, suspend 5 million cells and microparticles (3.5 mg) in 1 ml of medium, and then float suspension culture at room temperature for 1 h 200 μl of the nude mouse was injected subcutaneously into the back of the nude mouse. The fluorescence at the injection site was observed with a fluorescence microscope after 3, 7 and 10 days. From the 10th day onward, the fluorescence was observed only in the group to which the aggregate solution with fine particles was administered, and the survival and engraftment of the administered cells were observed. Was confirmed (FIG. 9).

  Moreover, when the number of new capillaries per microscopic field was measured based on the pathological photograph of the tissue at the injection site, the number of new capillaries per microscopic field was significantly greater than the other groups over the 14th day from the seventh day. It became clear that it was high (FIG. 10). Thus, cell engraftment and angiogenesis promotion by administration of aggregates of this microparticle cell carrier and adipose tissue-derived mesenchymal stem cells are significant only in the group administered with an aggregate solution with microparticles compared to other control groups. The effectiveness of the particulate cell carrier of the present invention in angiogenesis therapy by introducing adipose tissue-derived mesenchymal stem cells was confirmed.

(Example 7) Blood regeneration promotion by femur transplantation of aggregates of microparticle cell carrier and bone marrow tissue-derived cells of the present invention 10G radiation that is lethal to mice (C57BL / 6J; Nippon SLC Co., Ltd., Shizuoka) (Radiation irradiation device MBR-1505R2 Hitachi Medical Co., Ltd.), 2 hours later, only 500,000 bone marrow cells (control group), equal amount of bone marrow cells + 350 μg of microparticles (particle group), and equal amount 25 μl of a medium solution containing bone marrow cells + 350 μg microparticles + cytokine (SCF: 3.125 ng, Tpo: 6.25 ng, Flt-3: 6.25 ng) (microparticle + cytokine group) was administered by intrafemoral injection. Under this condition, all three groups of mice could be saved from bone marrow death. Then, 1, 2, 3 and 4 weeks after administration, the blood regeneration was evaluated by examining changes in white blood cells, hemoglobin, and platelets in peripheral blood.

  As shown in FIG. 11, in the fine particle group and the fine particle + cytokine group, as in the control group, leukocytes and the like greatly decreased after 1 week, but regeneration of leukocytes and the like was observed after 2 weeks. However, no additional promoting activity by cytokines was observed. This result is thought to be due to promotion of aggregation of floating cells, improvement of cell viability, and promotion of cell engraftment by the particulate cell carrier of the present invention rather than direct effects of cytokines.

FIG. 2 is a photomicrograph of aggregates of floating human capillary endothelial cells (A), aggregates of human skin fibroblasts (B), and aggregates of adipose tissue-derived mesenchymal stem cells (C) using the particulate cell carrier of the present invention. . It is a graph which shows the lifetime of the cell aggregate of the microparticle cell carrier of this invention, and a human capillary endothelial cell (A), a human skin fibroblast (B), or an adipose tissue origin mesenchymal stem cell (C). Aggregates of human capillary endothelial cells were FGF-2-containing 10% FBS-containing DMEM and were able to survive for 3 days in suspension culture (A). Aggregates of microparticles and human skin fibroblasts (B) and adipose tissue-derived mesenchymal stem cells are aggregated in 10% FBS-containing DMEM (in the absence of FGF-2) and survived in suspension culture for 3 days. I was able to. It is the microscope picture of the external appearance (C) which coat | covered the microparticles | fine-particles (A) of this invention, the external appearance (B) coated on the plastic plate, and the fluorescent-labeled (Texas Red-X Protein Labeling Kit) microparticles | fine-particles. Each cytokine adsorbed per 48-well plate (well area: 0.78 cm ² ) by incubation at room temperature for 2 hours by subtracting the second adsorption amount (□) from the first adsorption amount (O). It is the graph which estimated quantity. The amount adsorbed on the control plate not coated with fine particles is indicated by (Δ). Human bone marrow mesenchymal stem cells (A) and mouse adipose tissue-derived mesenchymal stem cells (B) on a fine particle coated plate (●) and an uncoated control plate (◯) with 2% FBS and 5 ng / ml FGF-2 It is a graph which shows the growth rate when it culture | cultivates with the included DMEM. Growth rate when the human TF-1 cell line was cultured in DMEM containing 1% FBS, 5 ng / ml IL-3 and GM-CSF on the fine particle coated plate (●) and uncoated control plate (○) It is a graph to show. Fine particle coating plate (●) and uncoated control plate (○) were allowed to adsorb IL-3 and GM-CSF at 4 ° C for 18 hours, and the plate was washed 3 times with medium. It is a graph which shows the growth rate when the human TF-1 cell line is cultured on DMEM containing only 1% FBS for 5 days. Culture human CD34-positive hematopoietic stem cells on serum-free HPGM containing 5 ng / ml SCF, 10 ng / ml Tpo, and 10 ng / ml Flt-3 on microparticle-coated plates (●) and uncoated control plates (○) It is a graph which shows the proliferation rate when doing. Adipose tissue-derived mesenchymal GFP luminescent cell alone administration group (upper) and adipose tissue-derived mesenchymal GFP luminescent cell spheroid administration group (lower) containing fine particles were injected subcutaneously into the back of nude mice. It is a fluorescent stereomicrograph with time. It is a graph which shows the time-dependent change of the number of capillaries of the injection site of the spheroid injection group of microparticles and adipose tissue-derived mesenchymal GFP luminescent cells, the adipose tissue-derived mesenchymal GFP luminescent cell alone injection group, and the microparticle-only injection group . This is data obtained by taking a histopathological image (H & E staining) at the injection site and counting the number of capillaries per field of view from the photograph. Mice were exposed to a lethal dose of 10G radiation, and after 2 hours, only 500,000 bone marrow cells (control group; white bar), equal amount of bone marrow cells + 350 μg microparticles (microparticle group; black bar) (B ), And equal amounts of bone marrow cells + 350 μg of microparticles + cytokines (SCF: 3.125 ng, Tpo: 6.25 ng, Flt-3: 6.25 ng) (microparticles + cytokine group; black bars), equivalent amounts of bone marrow cells + cytokines (SCF : 3.125 ng, Tpo: 6.25 ng, Flt-3: 6.25 ng) (cytokine (+) control group; white bar) White blood cells (WBC) when 25 μl of medium solution containing (A) was administered by intrafemoral injection, It is a graph which shows the change of hemoglobin (Hb) and platelets (PLT). 1, 2, 3, and 4 weeks after administration, the results of blood regeneration were evaluated by examining changes in white blood cells, hemoglobin, and platelets in peripheral blood.

Claims (5)

A fine particle cell carrier comprising fine particles having a particle size of less than 10 μm composed of low molecular weight heparin and protamine. A cell aggregate comprising the particulate cell carrier according to claim 1 and adherent cells. A pharmaceutical composition comprising the cell aggregate according to claim 2 and optionally a physiologically active substance. A coating material comprising fine particles having a particle size of less than 10 μm composed of low molecular weight heparin and protamine. A cell culture substrate obtained by coating a substrate with the coating material according to claim 4.