Cell culture

• US 9,828,576 B2
• Filed: 07/29/2013
• Issued: 11/28/2017
• Est. Priority Date: 08/01/2012
• Status: Expired due to Fees

• Current Assignee: Centre National De La Recherche Scientifique, National University of Singapore
• Original Assignee: Centre National De La Recherche Scientifique, National University of Singapore
• Inventors: Viasnoff, Virgile Nicolas Robert, Li, Qiushi
• Primary Examiner(s): DINES, KARLA A
• Application Number:
  US14/416,330
• Publication Number:
  US 20150203809A1
• Time in Patent Office: 1,583 Days
• Field of Search: 506 9
• CPC Class Codes: B01L 3/5085 for multiple samples, e.g. microtitration plates
• CPC Class Codes: C12M 1/18 Multiple fields or compartments
• CPC Class Codes: C12M 23/12 Well or multiwell plates C12M25/04 takes precedence
• CPC Class Codes: C12M 23/20 Material Coatings immunocoatings C12M25/00
• CPC Class Codes: C12M 25/02 Membranes; Filters filters or filtration in general B01D24/00-B01D41/00
• CPC Class Codes: C12M 25/04 in combination with well or multiwell plates, i.e. culture inserts
• CPC Class Codes: C12N 2529/10 Stimulation by light
• CPC Class Codes: C12N 2533/30 Synthetic polymers thermoreactive polymers, e.g. PNIPAm, C12N2539/10
• CPC Class Codes: C12N 2533/52 Fibronectin; Laminin
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 2537/10 Cross-linking
• CPC Class Codes: C12N 2539/00 Supports and/or coatings for cell culture characterised by properties
• CPC Class Codes: C12N 5/0062 General methods for three-dimensional culture
• CPC Class Codes: C12N 5/0068 General culture methods using substrates for specific animal cell type C12N5/06
• CPC Class Codes: G01N 33/5014 for testing toxicity

Abstract

The disclosure relates to the fabrication of a three dimensional [3-D] cell culture membrane comprising one or more functionalized surfaces adapted to provide cell culture conditions suitable for the analysis of proliferation, differentiation or function of cells, typically eukaryotic or prokaryotic cells.

Claims

1. A cell culture vessel comprising a three dimensional cell culture substrate comprising:

a perforated membrane with through holes,

the perforated membrane having three membrane surfaces, namely an upper surface, a lower surface opposite to the upper surface, and a plurality of inner surfaces inside the through holes,

the perforated membrane comprising a cured polymer adapted for cell culture having at least two modified cell culture surfaces among the three surfaces of the perforated membrane,

wherein the first cell culture surface comprises at least one first cell culture agent for enhancing the proliferation and/or differentiation or function of the cells and the second cell culture surface does not comprise the first cell culture agent but a second different cell culture agent for enhancing the proliferation and/or differentiation or function of the cells,

the first and the second cell culture surfaces being two surfaces out of the three membrane surfaces.

2. The cell culture vessel of claim 1, further comprising at least one cell, and cell culture medium.

3. The cell culture vessel according to claim 2, wherein said cell culture substrate is suspended and supported in said cell culture medium.

4. The cell culture vessel according to claim 3, wherein said cell culture substrate comprises one or more cell culture surfaces, wherein said cell culture surfaces do not contact a cell culture vessel surface.

5. The cell culture vessel according to claim 4, wherein said cell is genetically modified by transfection with an isolated nucleic acid or expression vector to recombinantly express a selected nucleic acid in said cell.

6. The cell culture vessel according to claim 1, wherein said cell is a mammalian cell or a prokaryotic cell.

7. The cell culture vessel according to claim 6, wherein said mammalian cell is an epidermal keratinocyte, a fibroblast cell, an epithelial cell, a neuronal glial cell, a neural cell, a hepatocyte, a hepatocyte stellate cell, a mesenchymal cell, a muscle cell, a kidney cell, a blood cell, a pancreatic β

cell, cancer cell, or an endothelial cell.

8. The cell culture vessel according to claim 1, wherein said membrane comprises a plurality of perforations wherein said perforations are at least 5-1000 μ

m in diameter.

9. The cell culture vessel according to claim 8, wherein the perforations have an aspect ratio not greater than 2.

10. The cell culture vessel according to claim 1, wherein said curable polymer is UV curable.

11. The cell culture vessel according to claim 10, wherein said UV curable polymer is an acrylate based polymer.

12. The cell culture vessel according to claim 1, wherein said cell culture agent and/or said membrane is further modified by inclusion of a cross-linking agent that facilitates the cross-linking of the cell culture agent to said membrane to provide a modified cell culture surface.

13. The cell culture vessel according to claim 1, wherein said membrane has a refractive index of between about 1.30 to about 1.50.

14. The cell culture vessel according to claim 1, wherein said cell substrate comprises a network of interconnected cell culture microwells.

15. The cell culture vessel according to claim 14, wherein the network comprises a plurality of elongate cell culture microwells adapted to provide at least said first and second modified cell culture surfaces.

16. A method for the culture of cells, comprising:

i) providing a cell culture vessel according to claim 1, comprising;

a) cells; and

b) cell culture medium sufficient to support the growth of said cells; and

ii) providing cell culture conditions which promote the proliferation and/or differentiation and/or function of said cells.

17. The method according to claim 16, wherein said cell culture substrate is suspended and supported in said cell culture medium.

18. The method according to claim 17 wherein said cell culture substrate comprises one or more cell culture surfaces wherein said cell culture surface does not contact a cell culture vessel surface.

19. A method to screen for an agent that affects the proliferation, differentiation or function of a cell, comprising:

i) providing a cell culture comprising at least one cell and a cell culture vessel according to claim 1;

ii) adding at least one agent to be tested; and

iii) monitoring the activity of the agent with respect to the proliferation, differentiation or function of said cells.

20. A method to test the liver toxicity of an agent, comprising:

i) providing a cell culture comprising at least one hepatocyte cell and a cell culture vessel according to claim 1;

ii) adding at least one agent to be tested; and

iii) monitoring the activity of the agent with respect to the proliferation, differentiation or function of said hepatocyte cells as a measure of toxicity of the agent.