Multi-well micropatterning by ablation

Patent metadata

Field	Value
Publication number	US-11366103-B2
Application number	US-202016741423-A
Country	US
Kind code	B2
Filing date	Jan 13, 2020
Priority date	Oct 12, 2006
Publication date	Jun 21, 2022
Grant date	Jun 21, 2022

Abstract

The present invention is drawn to the generation of micropatterns of biomolecules and cells on standard laboratory materials through selective ablation of a physisorbed biomolecule with oxygen plasma. In certain embodiments, oxygen plasma is able to ablate selectively physisorbed layers of biomolecules (e.g., type-I collagen, fibronectin, laminin, and Matrigel) along complex non-linear paths which are difficult or impossible to pattern using alternative methods. In addition, certain embodiments of the present invention relate to the micropatterning of multiple cell types on curved surfaces, multiwell plates, and flat bottom flasks. The invention also features kits for use with the subject methods.

First claim

The invention claimed is:

1. A monolithic multi-well cell plate comprising a plurality of wells, each well comprising a micropatterned substrate prepared by a process comprising the steps of:
adsorbing biomolecules onto a surface of a substrate within each well of the multi-well plate, thereby forming a coated surface of the substrate;
compressing a micropatterned etch mask onto the coated surface of the substrate, wherein said compression step seals the etch mask to the coated surface of the substrate such that the biomolecules are protected from ablation;
exposing the compressed micropatterned etch mask and coated surface of the substrate to a gas plasma for a period of time sufficient to ablate the exposed surfaces of the substrate, thereby ablating the exposed surfaces of the substrate,
removing the micropatterned etch mask, whereby the biomolecules remain adsorbed, such that the micropatterned substrate is formed,
thereby providing a multi-well plate comprising a plurality of wells, each well comprising a micropatterned substrate.

2. The multi-well plate of claim 1 , wherein said multi-well cell plate is a 24-well or a 96-well or a 384-well cell plate.

3. The multi-well plate of claim 1 , wherein said biomolecule is selected from the group consisting of peptides, polypeptides, nucleic acids, nucleic acid binding partners, proteins, receptors, antibodies, enzymes, carbohydrates, oligosaccharides, polysaccharides, cells, cell aggregates, cell components, lipids, arrays of ligands, non-protein ligands, liposomes, and microorganisms.

4. The multi-well plate of claim 1 , wherein the process further comprises rinsing and drying said coated surface after the adsorbing step.

5. The multi-well plate of claim 1 , wherein said exposing step is carried out in a plasma asher.

6. The multi-well plate of claim 1 , wherein the micropatterned etch mask is one solid elastomeric piece.

7. The multi-well plate of claim 1 , wherein the micropatterned etch mask comprises a plurality of pillars.

8. The multi-well plate of claim 1 , wherein the micropatterned etch mask comprises chrome or elastomeric poly(dimethylsiloxane) or rubber or plastic.

9. The multi-well plate of claim 1 , wherein the adsorbed molecules are different.

10. The multi-well plate of claim 9 , wherein the different molecules each have a different pattern.

11. The multi-well plate of claim 1 , wherein the micropatterned etch mask comprises elastomeric poly(dimethylsiloxane).

12. The multi-well plate of claim 1 , wherein the micropatterned etch mask comprises an about 50 μm to about 1 mm thick piece of elastomeric poly(dimethylsiloxane).

13. The multi-well plate of claim 1 , wherein said substrate surface is ceramic, metal, glass, or plastic.

14. The multi-well plate of claim 1 , wherein said substrate comprises fluoropolymers, fluorinatedethylene propylene, polyvinylidene, polydimethylsiloxane, polystyrene, polycarbonate, and polyvinyl chloride, fused silica, polysilicon, or single silicon crystals.

15. The multi-well plate of claim 1 , wherein said biomolecules are hyaluronic acid, collagen, fibronectin, lamanin, or matrigel.

16. The multi-well plate of claim 1 , wherein the process further comprises:
contacting said micropatterned substrate with cells.

17. The multi-well plate of claim 16 , wherein said cells are hepatocytes, endothelial cells, kidney, muscle, pancreas, epithelium cells, tissue/skin cells, intestinal cells or stem-cell derived cells.

18. The multi-well plate of claim 17 , wherein said cells are rat cells, human cells, mouse cells, monkey cells, or guinea pig cells.

19. The multi-well plate of claim 1 , wherein the compression step is accomplished by use of a clamp.

20. The multi-well plate of claim 1 , wherein the gas plasma penetrates more than 10 cm along non-linear path in forming the micropatterned substrate.

21. The multi-well plate of claim 1 , wherein the substrate to be micropatterned is not directly exposed to the oxygen plasma.

22. The multi-well plate of claim 1 , wherein the period of time sufficient to ablate the exposed surfaces of the substrate while preserving activity of the biomolecules is from about 5 to about 1000 seconds.

23. The multi-well plate of claim 1 , wherein the period of time sufficient to ablate the exposed surfaces of the substrate while preserving activity of the biomolecules is from about 5 to about 120 seconds.

Assignees

• Massachusetts Inst Technology

Inventors

• Eddington David T
• Bhatia Sangeeta N

Classifications

• B01L3/5025 Primary

  for parallel transport of multiple samples · CPC title

• C12M23/12

  Well or multiwell plates (C12M25/04 takes precedence) · CPC title

• C12N2502/14

  hepatocytes · CPC title

• B01J2219/00587

  High throughput processes · CPC title

• G01N33/5436

  with ligand physically entrapped within the solid phase (liposomes G01N33/5432; immunological test elements G01N33/54386) · CPC title