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

• US 20140178992A1
• Filed: 03/30/2012
• Published: 06/26/2014
• Est. Priority Date: 03/30/2011
• Status: Abandoned Application

• Current Assignee: National Center For Geriatrics And Gerontology, Nepa Gene Co., Ltd.
• Original Assignee: National Center For Geriatrics And Gerontology
• Inventors: Osabe, Masahiro, Nakashima, Misako, Iohara, Koichiro, Yamada, Kazumasa, Shimagaki, Masaaki
• Application Number:
  US14/008,760
• Publication Number:
  US 20140178992A1
• Time in Patent Office: Days
• US Class Current: 435/375
• CPC Class Codes: B01D 2315/06 Submerged-type; Immersion type
• CPC Class Codes: B01D 63/087 Single membrane modules
• CPC Class Codes: B01D 67/0093 Chemical modification
• CPC Class Codes: B01D 67/00933 by addition of a layer chemically bonded to the membrane
• CPC Class Codes: B01D 71/50 Polycarbonates
• CPC Class Codes: B05D 1/18 performed by dipping
• CPC Class Codes: B05D 3/068 using ionising radiations (gamma, X, electrons)
• CPC Class Codes: C12M 23/12 Well or multiwell plates C12M25/04 takes precedence
• CPC Class Codes: C12M 25/04 in combination with well or multiwell plates, i.e. culture inserts
• CPC Class Codes: C12M 29/04 Filters; Permeable or porous membranes or plates, e.g. dialysis
• CPC Class Codes: C12M 47/04 Cell isolation or sorting purging biological preparations of unwanted cells C12N5/0081, determining the presence or kind of microorganism C12Q1/04
• CPC Class Codes: C12N 5/0663 Bone marrow mesenchymal stem cells (BM-MSC)
• CPC Class Codes: C12N 5/0664 Dental pulp stem cells, Dental follicle stem cells
• CPC Class Codes: C12N 5/0667 Adipose-derived stem cells [ADSC]; Adipose stromal stem cells

Abstract

A membrane separation culture device includes an upper structure including 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 including a vessel that retains a fluid in which the membrane of the upper structure is immersed.

Claims

1. A membrane separation culture device comprising:

an upper structure comprising 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 comprising a vessel that retains a fluid in which the membrane of the upper structure is immersed.

2. The membrane separation culture device according to claim 1, 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

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.

3. The membrane separation culture device according to claim 1, wherein the size of the pore is 3 μ

m to 10 μ

m and the density of the pore is 1×

10⁵ to 4×

10⁶ pores/cm².

4. The membrane separation culture device according to claim 1, comprising a plurality of the upper structures, and

further comprising a frame body accommodated in the lower structure and comprises a plate-like member having a plurality of holes each established to lock the plurality of the upper structures.

5. The membrane separation culture device according to claim 1,

comprising a plurality of the upper structures, and further comprising a frame body accommodated in the lower structure and comprises a plate-like member having a plurality of holes each established to lock the plurality of the upper structures, wherein

the lower structure comprises a plurality of vessels each corresponding to the plurality of the upper structures.

6. The membrane separation culture device according to claim 4, wherein the plurality of the upper structures have membranes each having a different pore size and/or a different pore density.

7. The membrane separation culture device according to claim 1, further comprising a lid structure that covers or hermetically seals the upper structure and the lower structure.

8. The membrane separation culture device according to claim 7, wherein the lid structure further comprises a gas exchanger comprising a gas inlet port and a gas discharge port.

9. The membrane separation culture device according to claim 7, wherein at least a portion of the lid structure is formed with a membrane having pores whose pore size is 1 to 100 nm.

10. The membrane separation culture device according claim 7, wherein a hermetic sealing elastic body is established between the lid structure and the lower structure.

11. The membrane separation culture device according to claim 7, further comprising a temperature control system containing a temperature-measuring device and a temperature-controlling device.

12. The membrane separation culture device according to claim 1, wherein the lower structure further comprises a medium replacement system comprising a medium inlet port and a medium outlet port.

13. A membrane separation culture kit comprising the membrane separation culture device according to claim 1 and cell migration factor(s) to be poured into the lower structure.

14. The kit according to claim 13, 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, SCF, MMP3, Slit, GM-CSF, LIF, HGF, S1P, protocatechuic acid, and serum.

15. The kit according to claim 13, wherein the concentration of the cell migration factor(s) is 1 ng/ml to 500 ng/ml.

16. The kit according to claim 13, further comprising serum to be poured into the lower structure and wherein the cell migration factor is G-CSF or bFGF.

17. A method of separating stem cells with the membrane separation culture device according to claim 1, comprising:

dispersing test cells or test tissues on the membrane of the upper structure;

filling the vessel as a lower structure with a medium containing cell migration factor(s); and

causing the membrane of the upper structure to contact the medium in the lower structure.

18. The method according to claim 17, wherein the cell migration factor(s) are one or more selected from SDF-1, G-CSF, bFGF, TGF-13, NGF, PDGF, BDNF, GDNF, EGF, VEGF, SCF, MMP3, Slit, GM-CSF, LIF, HGF, SIP, protocatechuic acid, and serum.

19. The method according to claim 17, wherein concentration of the cell migration factor(s) is 1 ng/ml to 500 ng/ml.

20. The method according to claim 17, wherein the test cells are dispersed at a density of 3×

10² cells to 3×

10⁴ cells per mm² of the separation membrane.

21. The method according to claim 17, wherein the stem cells are dental pulp stem cells, the cell migration factor is G-CSF or bFGF, the concentration of the G-CSF or bFGF is 50 to 150 ng/ml, the test cells are dispersed at a density of 3×

10² to 1.5×

10³ cells per mm² of the separation membrane, or the test tissues are left at rest at a density of 0.1 mg to 1 mg per mm² of the separation membrane, and serum is added to a medium containing the cell migration factor at a volume percentage of 5% to 20% based on the volume of the medium.

22. The method according to claim 17, wherein the stem cells are bone marrow stem cells or adipose stem cells, the cell migration factor is G-CSF or bFGF, the concentration of the G-CSF or bFGF is 50 to 150 ng/ml, the test cells are dispersed at a density of 3×

10² to 1.5×

10³ cells per mm² of the separation membrane, or the test tissues are left at rest at a density of 0.1 mg to 1 mg per mm² of the separation membrane, and serum is added to a medium containing the cell migration factor at a volume percentage of 5% to 20% based on the volume of the medium.

23. A separation membrane comprising:

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

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.

24. The separation membrane according to claim 23, wherein the base material membrane has pores with a pore size of 1 to 10 μ

m and the base material membrane is used for cell separation.

25. The separation membrane according to claim 23, wherein the hydrophobic polymer is selected from the group consisting of a sulfone polymer, an amide polymer, a carbonate polymer, an ester polymer, a urethane polymer, an olefin polymer, and an imide polymer.

26. The separation membrane according to claim 23, which separates cells by permeation.

27. A method of producing the separation membrane according to claim 23, comprising:

immersing a base material membrane consisting of a hydrophobic polymer having a water absorption percentage of 2% or less in a treating aqueous solution containing one or more hydrophilic polymers selected from a vinyl pyrrolidone polymer, a polyethylene glycol polymer and a vinyl alcohol polymer at a concentration of 10 to 2000 ppm, and also containing a 0.01% to 0.2% alcohol; and

irradiating the base material membrane with a high-energy beam to modify the surface of the membrane to have a protein adhesion-suppressing property and a cell adhesion-suppressing property.

28. A method of producing the separation membrane according to claim 23, comprising:

immersing a base material membrane consisting of a hydrophobic polymer having a water absorption percentage of more than 2% in an aqueous solution containing one or more hydrophilic polymers selected from a vinyl pyrrolidone polymer, a polyethylene glycol polymer and a vinyl alcohol polymer at a concentration of 10 to 2000 ppm; and

irradiating the base material membrane with a high-energy beam to modify the surface of the membrane to have a protein adhesion-suppressing property and a cell adhesion-suppressing property.

29. The method according to claim 27, wherein the hydrophobic polymer is selected from the group consisting of a sulfone polymer, an amide polymer, a carbonate polymer, an ester polymer, a urethane polymer, an olefin polymer, and an imide polymer.

32. A method of producing the separation membrane according to claim 23 by a method of modifying a surface of a molded body comprising:

immersing a molded body having a water absorption percentage of 2% or less in a treating aqueous solution containing one or more hydrophilic polymers selected from a vinyl pyrrolidone polymer, a polyethylene glycol polymer and a vinyl alcohol polymer at a concentration of 10 to 2000 ppm, and also containing a 0.01% to 0.2% alcohol; and

irradiating the molded body with a high-energy beam to modify the surface of the molded body to have a protein adhesion-suppressing property and a cell adhesion-suppressing property.

30. A method of modifying a surface of a molded body, comprising:

immersing a molded body having a water absorption percentage of 2% or less in a treating aqueous solution containing one or more hydrophilic polymers selected from a vinyl pyrrolidone polymer, a polyethylene glycol polymer and a vinyl alcohol polymer at a concentration of 10 to 2000 ppm, and also containing a 0.01% to 0.2% alcohol; and

irradiating the molded body with a high-energy beam to modify the surface of the molded body to have a protein adhesion-suppressing property and a cell adhesion-suppressing property.

31. A method of modifying a surface of a molded body comprising:

of immersing a molded body having a water absorption percentage of more than 2% in an aqueous solution containing one or more hydrophilic polymers selected from a vinyl pyrrolidone polymer, a polyethylene glycol polymer and a vinyl alcohol polymer at a concentration of 10 to 2000 ppm; and

irradiating the molded body with a high-energy beam to modify the surface of the molded body to have a protein adhesion-suppressing property and a cell adhesion-suppressing property.