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EP-2692853-B1 - MEMBRANE-SEPARATION-TYPE CULTURE DEVICE, MEMBRANE-SEPARATION-TYPE CULTURE KIT AND STEM CELL SEPARATION METHOD USING SAME

EP2692853B1EP 2692853 B1EP2692853 B1EP 2692853B1EP-2692853-B1

Inventors

  • NAKASHIMA, MISAKO
  • IOHARA, KOICHIRO
  • YAMADA, KAZUMASA
  • SHIMAGAKI, MASAAKI
  • OSABE, MASAHIRO

Dates

Publication Date
20260506
Application Date
20120330

Claims (7)

  1. A membrane separation culture device comprising: an upper structure constituted with a vessel in which at least a portion of the bottom thereof is formed with a separation membrane having pores that allow stem cells to permeate therethrough; and a lower structure constituted with a vessel that retains a fluid in which the separation membrane of the upper structure is immersed, wherein the separation membrane comprises: a base material membrane consisting of a hydrophobic polymer; and a functional layer formed by allowing one or more hydrophilic polymers selected from a vinyl pyrrolidone polymer, a polyethylene glycol polymer and a vinyl alcohol polymer to bind to the surface of the base material membrane via a covalent bond; wherein the weight percentage of the hydrophilic polymer(s) constituting the functional layer is 1.5% to 35% based on the total weight of the separation membrane, and wherein the size of the pore is 3 µm to 10 µm and the pore density is 1 × 10 5 to 4 × 10 6 pores/cm 2 .
  2. The membrane separation culture device according to claim 1, wherein the plurality of the upper structures have separation membranes each having a different pore size and/or a different pore density.
  3. A membrane separation culture kit comprising a membrane separation culture device according to any one of claims 1 and 2 and cell migration factor(s) to be poured into the lower structure, wherein the cell migration factor(s) are one or more selected from SDF-1, G-CSF, bFGF, TGF-β, NGF, PDGF, BDNF, GDNF, EGF, VEGF, MMP3, Slit, GM-CSF, LIF, HGF, and serum.
  4. The kit according to claim 3, wherein the concentration of the cell migration factor(s) is 1 ng/ml to 500 ng/ml.
  5. The kit according to claim 3, which further comprises serum to be poured into the lower structure and wherein the cell migration factor is G-CSF or bFGF.
  6. A method for separating stem cells using the membrane separation culture device according to any one of claims 1 and 2, wherein the method comprises: a step of dispersing test cells or test tissues on the separation membrane of the upper structure; a step of filling the vessel as a lower structure with a medium containing cell migration factor(s); and a step of allowing the separation membrane of the upper structure to come into contact with the medium in the lower structure, wherein the cell migration factor(s) are one or more selected from SDF-1, G-CSF, bFGF, TGF-β, NGF, PDGF, BDNF, GDNF, EGF, VEGF, MMP3, Slit, GM-CSF, LIF, HGF, and serum.
  7. The method according to claim 6, wherein the concentration of the cell migration factor(s) is 1 ng/ml to 500 ng/ml.

Description

Technical Field The present invention relates to a membrane separation culture device and to a membrane separation culture kit, which may be used to separate the stem cells and dental pulp stem cells of an organism of any species, and a method for separating stem cells using the same. In particular, the present invention relates to a membrane separation culture device and a membrane separation culture kit, which are used to separate dental pulp stem cells or mesenchymal stem cells with which the root canal is to be filled for regeneration of the dental pulp, and a method for separating stem cells using the same. A separation membrane useful for the membrane separation culture device of the invention, and production of the separation membrane using a surface modification method are also disclosed. The present invention uses a separation membrane, in which adhesion of cells to the membrane upon separation of the cells by permeation is suppressed by hydrophilizing a membrane consisting of a hydrophobic polymer without impairing separation performance. Accordingly, the present invention is preferably used in the fields of separation and purification of cells including blood purification field or regenerative medicine as typical examples. Moreover, by such a polymer surface modification method, only the surface of a polymer can be simply modified, and sterilization can be simultaneously carried out. Hence, when compared with conventional methods, the polymer surface modification method can contribute to production efficiency. Background Art At present, dental pulp stem cells used in biological root canal fillers for treatments such as treatment by extirpation of the pulp or the treatment of the infected root canal are fractions that are excellent in terms of angiogenic ability, nerve regeneration ability and dental pulp regeneration ability. Dental pulp-derived CD31-SP (side population) cells, CD105+ cells, or CXCR4+ cells, have been mainly used. SP cells are labeled with Hoechst33342, and a fraction that highly emits this pigment is then separated by flow cytometer using Hoechst Blue and Hoechst Red. Large quantities of stem cells are contained in this fraction. However, since Hoechst33342 is a DNA-binding pigment and it essentially requires the use of flow cytometer, it is said that Hoechst33342 is problematic in terms of the safety of cells. On the other hand, in the case of using an antibody against a stem cell-specific membrane surface antigen, a method of using magnetic beads without using a flow cytometer has been developed. For example, as bone marrow stem cells, CD34 or CD133 antibody beads have been known. This method requires the use of considerable quantities of tissues or cells. Thus, if such tissues or cells are separated from dental pulp tissues, this method is inappropriate. In addition, in the case of human dental pulp, CD34-positive cells, and CD133-positive cells are hardly present in dental pulp test cells (0.01% and 0.5%, respectively), and thus, the existing (commercially available) magnetic beads methods are not appropriate. Since CD105 or CXCR4 antibody beads must be prepared to order, they may be extremely expensive. Moreover, as a device for separating stem cells from adipose tissues, Celution 800/CRS system that is based on cell separation according to enzymatic digestion or centrifugation has already been used in clinical sites. However, this device requires large quantities of tissues, cells are obtained as a heterogeneous cell group containing large quantities of precursor cells, and it is also expensive. Furthermore, as a device for separating stem cells from bone marrow tissues, Bone Marrow MSC Separation Device has been commercialized. It is considered that this device is able to collect stem cells in a short time (20 minutes) by trapping bone marrow mesenchymal cells with fibers consisting of rayon and polyethylene. This device is relatively inexpensive, but it requires relatively large quantities of bone marrow tissues (spinal fluid) and the obtained cells are of a heterogeneous cell group containing large quantities of precursor cells. Hence, a device for separating stem cells from solid tissues without using enzymatic digestion and amplifying them has not yet been developed. Cellculture Insert (Polycarbonate Membrane Transwell (registered trademark) Inserts; 2 x 105 pores/cm2, pore size: 8 µm, diameter of bottom surface: 6.4 mm, diameter of opening portion: 11.0 mm, height: 17.5 mm) (Corning) used as an upper structure, can be inserted into a 24-well plate (diameter: 15.0 mm, diameter of opening portion: 15.0 mm, height: 22.0 mm) (Falcon) used as a lower structure, and the thus prepared device can be used as a membrane separation device. However, since large quantities of cells adhere to a PET membrane or a polycarbonate membrane, migration of the cells to a lower layer cannot be carried out efficiently. Further, since this device has an open shape, it has a high risk of bein