Intra-culture perfusion methods and applications thereof

• US 9,701,938 B2
• Filed: 10/10/2013
• Issued: 07/11/2017
• Est. Priority Date: 10/12/2012
• Status: Active Grant

• Current Assignee: Lena Biosciences, Inc.
• Original Assignee: Lena Biosciences, Inc.
• Inventors: Vukasinovic, Jelena
• Primary Examiner(s): DAVIS, RUTH A
• Application Number:
  US14/050,619
• Publication Number:
  US 20140106452A1
• Time in Patent Office: 1,370 Days
• CPC Class Codes: C12N 2533/30 Synthetic polymers thermoreactive polymers, e.g. PNIPAm, C12N2539/10
• CPC Class Codes: C12N 5/0068 General culture methods using substrates for specific animal cell type C12N5/06
• CPC Class Codes: Y10T 29/49826 Assembling or joining

Abstract

Disclosed are intra-culture perfusion methods applied to standard cell culture disposables using three-dimensional cell culture scaffold and synthetic vasculature compositions in high-throughput screening and high-content screening applications, and assay development.

Claims

1. A method of culturing cells, comprising providing the cells, and culturing each of the cells in a plurality of liquids, wherein the liquids are of a different density and two or more of the plurality of liquids form separate air-liquid interfaces.

2. The method of claim 1, wherein the plurality of liquids are immiscible.

3. The method of claim 1, wherein the plurality of liquids include a cell culture medium and perfluorodecalin.

4. The method of claim 1, wherein the cells are cell aggregates or spheroids.

5. The method of claim 1, wherein the cells are in a sol-state suspension and are placed in a hydraulically conductive scaffold.

6. The method of claim 5, wherein the hydraulically conductive scaffold has a void volume of between approximately 60% and 95%, and comprises between approximately 70% and 95% w/w insoluble borosilicate glass fibers having an absorbent polymer coating.

7. The method of claim 6, wherein the hydraulically conductive scaffold further comprises an absorbent material which extends to all surfaces of the scaffold.

8. The method of claim 6, wherein the absorbent polymer coating is polyvinyl alcohol.

9. The method of claim 5, wherein the hydraulically conductive scaffold is coated with a cell-adhesive coating or a non-cell adhesive coating.

10. The method of claim 1, wherein the cells are cultured in a housing for use in cell culture experiments, wherein the housing comprises a plate, a well insert, and a lid, and wherein

a. the plate comprises sides and a bottom which form a plate reservoir,

b. the well insert comprises one or more air openings and a plurality of insert wells configured to contain a cell culture, wherein each insert well has an open top portion, an open bottom portion, and a hydraulically conductive three dimensional scaffold disposed in between,

c. the well insert is disposed within the plate reservoir,

d. a first liquid is added to the insert wells containing cells, and

e. a second liquid is added to the plate reservoir.

11. The method of claim 10, wherein the plate reservoir comprises

a. a plurality of large wells configured to contain a cell culture, each large well having a height lower than the plate sides, and

b. wherein each of the plurality of insert wells is smaller in width and depth and greater in height than the corresponding large well that it fits within.

12. The method of claim 11, wherein the first liquid has a higher density than the second liquid.

13. The method of claim 10, wherein the first liquid has a lower density than the second liquid.

14. The method of claim 13, wherein the first liquid is a cell culture medium and the second liquid is perfluorodecalin.

15. The method of claim 10, further comprising adding the cells in a sol-state suspension to the each hydraulically conductive scaffold.

16. The method of claim 15, wherein the sol-state suspension is a sol-state composition comprising Laminin, Collagen IV, Entactin, and heparin sulfate proteoglycan.

17. The method of claim 10, wherein the plate further comprises at least one perfusion port and the second liquid is injected and then withdrawn via the at least one perfusion port.

18. The method of claim 17, wherein the plate comprises an inlet port and an outlet port, each disposed on opposite sides of the plate, and the second liquid is injected via the inlet and withdrawn via the outlet.

19. The method of claim 18, wherein the process of the second liquid injection and withdrawal is repeated.

20. The method of claim 19, wherein a continuous flow of the second liquid is further superimposed by way of an additional inlet and outlet perfusion port disposed in the opposing sides of the plate.

21. The method of claim 19, wherein the injected liquid volume is the volume of the injected liquid which was not in the plate prior to injection.