CELL CULTURE

• US 20150203809A1
• Filed: 07/29/2013
• Published: 07/23/2015
• Est. Priority Date: 08/01/2012
• Status: Active Grant

• 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
• Granted Patent: US 9,828,576 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• 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 three dimensional cell culture substrate comprising:

a perforated membrane comprising a cured polymer adapted for cell culture characterized in that said membrane has at least two modified cell culture surfaces wherein the first surface comprises at least one cell culture agent and the second surface comprises a second different cell culture agent, wherein said membrane enhances the proliferation and/or differentiation or function of cells associated with said membrane.

2. The substrate according to claim 1 wherein said membrane comprises a plurality of perforations wherein said perforations are at least 5-1000 μ

m in diameter.

3. The substrate according to claim 2, wherein the perforations have an aspect ratio not greater than 2.

4. The substrate according to claim 1, wherein said curable polymer is UV curable.

5. The substrate according to claim 4 wherein said UV curable polymer is an acrylate based polymer.

6. The substrate 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.

7. The substrate according to claim 1, wherein said membrane has a refractive index of between about 1.30 to about 1.50.

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

9. The substrate according to claim 8 wherein the network comprises a plurality of elongate cell culture microwells adapted to provide at least said first and second modified cell culture surfaces.

22. A cell culture vessel comprising the cell culture substrate of claim 1.

23. A cell culture vessel comprising the cell culture substrate according to claim 1, at least one cell, and cell culture medium.

24. The cell culture vessel according to claim 23, wherein said cell culture substrate is suspended and supported in said cell culture medium.

25. The cell culture vessel according to claim 24, 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.

26. The cell culture vessel according to claim 22, wherein said cell is a mammalian cell or a prokaryotic cell.

29. The cell culture vessel according to claim 26, 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.

35. The cell culture vessel according to claim 25, 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.

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

i) providing a cell culture vessel comprising;

a) cells;

b) the cell culture substrate according to claim 1;

c) 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.

42. The method according to claim 37, wherein said cell culture substrate is suspended and supported in said cell culture medium.

43. The method according to claim 42 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.

44. 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 substrate 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.

46. 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 substrate 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.

10-11. -11. (canceled)

12. A process for the micro-fabrication of a cell culture membrane comprising at least one cell culture micro well wherein said micro well is adapted with at least two functionalized cell culture surface, comprising:

i) contacting the surface of a support with a substrate adapted by the provision of one or more raised projections wherein one or more of said projections contact the surface of said support;

ii) applying a cell culture compatible polymer solution to said substrate wherein said polymer is curable;

iii) curing the applied polymer to form a cell culture membrane;

iv) removing said membrane from said substrate and contacting a first membrane surface with one or more cell culture agents;

v) re-orienting said membrane to expose a second membrane surface; and

contacting said second membrane surface with a second, different, cell culture agent; and

optionally

vi) repeating step iv) and/or step v) to further functionalize said cell culture membrane.

13. The method according to claim 12 wherein steps ii)-vi) are further modified by the inclusion of a chemical cross-linking agent which facilitates the cross-linking of said cell culture agent to said cell culture membrane.

14. The method according to claim 13, wherein said chemical cross-linking agent is a hetero-bifunctional cross-linker.

16. The method according to claim 12, wherein the one or more cell culture agents is a protein, carbohydrate, lipid, or anti-fouling agent, or comprises a poly-amino coating.

21. A cell culture membrane obtained by the method of claim 12.

15. (canceled)

17-20. -20. (canceled)

27-28. -28. (canceled)

30-34. -34. (canceled)

36. (canceled)

38-41. -41. (canceled)

45. (canceled)

47-48. -48. (canceled)