PHENOTYPIC CHARACTERIZATION OF CELLS

• US 20200131557A1
• Filed: 03/19/2018
• Published: 04/30/2020
• Est. Priority Date: 03/22/2017
• Status: Active Grant

• Current Assignee: Astrego Diagnostics AB (Sysmex Corporation)
• Original Assignee: Astrego Diagnostics AB (Sysmex Corporation)
• Inventors: ELF, Johan, READ, Michael, BALTEKIN, Ozden, LOVMAR, Martin, HAMMAR, Petter, AMSELEM, Elias, OLSSON, Mikael, OHMAN, Ove
• Granted Patent: US 11,976,316 B2
• Time in Patent Office: Days
• CPC Class Codes: C12Q 1/18 Testing for antimicrobial activity of a material
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/5011 for testing antineoplastic activity

Abstract

Phenotyping of cells involves loading a biological sample into a microfluidic device (1, 100) comprising cell compartments (20, 120) to capture biological material, including target cells, in the sample in the cell compartments (20, 120). A subset (20A) of the cell compartments (20, 120) is identified as comprising target cells exhibiting target phenotype characteristic(s) as determined based on monitoring biological material in the cell compartments (20, 120) prior to addition of a test agent. The biological material is exposed to a test agent and a phenotypic response of the target cells to the test agent is determined based on monitoring target cells in the identified subset (20A) of the cell compartments (20, 120). The phenotyping of the target cells is thereby not overshadowed by the response of other cells and non-cell material present in the biological sample.

Claims

1-16. -16. (canceled)

17. A method for phenotyping cells, said method comprising:

a) loading a biological sample comprising biological material including target cells into a microfluidic device comprising spatially defined and separated cell compartments to capture biological material in said spatially defined and separated cell compartments;

b) monitoring biological material in said spatially defined and separated cell compartments prior to exposing biological material in said spatially defined and separated cell compartments to a test agent;

c) identifying a subset of said spatially defined and separated cell compartments as comprising target cells exhibiting at least one target phenotype characteristic as determined based on said monitoring of biological material in said spatially defined and separated cell compartments in b);

d) exposing biological material in said spatially defined and separated cell compartments to said test agent;

e) monitoring target cells in said identified subset of said spatially defined and separated cell compartments; and

f) determining a phenotypic response of said target cells to said test agent based on said monitoring of target cells in said identified subset of said spatially defined and separated cell compartments in e).

18. The method according to claim 17, further comprising:

exposing biological material in said spatially defined and separated cell compartments to a culture medium, wherein

monitoring biological material comprises monitoring biological material exposed to said culture medium in said spatially defined and separated cell compartments.

19. The method according to claim 18, wherein exposing biological material to said test agent comprises exposing biological material in said spatially defined and separated cell compartments to said culture medium comprising said test agent or to another culture medium comprising said test agent.

20. The method according to claim 17, wherein identifying said subset of said spatially defined and separated cell compartments comprises:

identifying target cells exhibiting said at least one target phenotype characteristic as determined based on said monitoring of biological material in said spatially defined and separated cell compartments in b); and

selecting said subset of said spatially defined and separated cell compartments as a subset of said spatially defined and separated cell compartments comprising said identified target cells.

21. The method according to claim 17, wherein monitoring biological material comprises monitoring, at multiple time instances, biological material in said spatially defined and separated cell compartments.

22. The method according to claim 17, wherein monitoring target cells comprises monitoring, at multiple time instances, target cells in said identified subset of said spatially defined and separated cell compartments.

23. The method according to claim 17 wherein monitoring biological material comprises taking at least one image of said spatially defined and separated cell compartments.

24. The method according to claim 23, wherein identifying said subset of said spatially defined and separated cell compartments comprises:

processing said at least one image for detection of said spatially defined and separated cell compartments and biological material in said spatially defined and separated cell compartments;

determining at least one respective phenotype characteristic of biological material in said spatially defined and separated cell compartments based on said processing;

identifying target cells as biological material in said spatially defined and separated cell compartments and having at least one respective determined phenotype characteristic corresponding to said at least one target phenotype characteristic; and

selecting said subset of said spatially defined and separated cell compartments as a subset of said spatially defined and separated cell compartments comprising said identified target cells.

25. The method according to claim 17, wherein

loading said biological sample comprises loading a biological sample comprising biological material including said target cells and non-target cells into said microfluidic device to capture biological material in said spatially defined and separated cell compartments;

identifying said subset of said spatially defined and separated cell compartments comprises;

identifying a subset of said spatially defined and separated cell compartments as comprising target cells exhibiting said at least one target phenotype characteristic as determined based on said monitoring of biological material in said spatially defined and separated cell compartments in b); and

identifying remaining spatially defined and separated cell compartments as comprising non-target cells and/or non-cell material not exhibiting said at least one target phenotype characteristic as determined based on said monitoring of biological material in said spatially defined and separated cell compartments in b); and

monitoring target cells comprises;

taking at least one image of said spatially defined and separated cell compartments; and

processing said at least one image for detection of said identified subset of said spatially defined and separated cell compartments and target cells in said identified subset of said spatially defined and separated cell compartments while disregarding said remaining spatially defined and separated cell compartments and non-target cells and/or non-cell material in said remaining spatially defined and separated cell compartments.

26. The method according to claim 17, wherein said target phenotype characteristic is selected from a group consisting of growth rate, shape, size, form of growth rate curve defining growth rate over time, form of length curve defining cell length over time, form of area curve defining cell area over time, color, optical density, electrical conductivity, heat production, surface antigen composition, absorption spectra, and a mixture thereof.

27. The method according to claim 17, further comprising:

adding a labeled probe to said spatially defined and separated cell compartments, wherein

monitoring biological material comprises monitoring said labeled probe in said spatially defined and separated cell compartments; and

identifying said subset of said spatially defined and separated cell compartments comprises identifying said subset of said spatially defined and separated cell compartments as comprising target cells exhibiting at least one target phenotype characteristic as determined based on said monitoring of said labeled probe in said spatially defined and separated cell compartments.

28. The method according to claim 17, wherein

identifying said subset of said spatially defined and separated cell compartments comprises identifying a first subset of said spatially defined and separated cell compartments as comprising a first population of target cells exhibiting at least one first target phenotype characteristic and a second subset of said spatially defined and separated cell compartments as comprising a second population of target cells exhibiting at least one second target phenotype characteristic as determined based on said monitoring of biological material in said spatially defined and separated cell compartments;

monitoring target cells comprises monitoring said first population of target cells in said identified first subset of said spatially defined and separated cell compartments and said second population of target cells in said identified second subset of said spatially defined and separated cell compartments; and

determining said phenotypic response comprises determining a respective phenotypic response of said first population of target cells and said second population of target cells to said test agent based on said monitoring of said first population of target cells in said identified first subset of said spatially defined and separated cell compartments and of said second population of target cells in said identified second subset of said spatially defined and separated cell compartments.

29. The method according to claim 17, wherein

said test agent is an antibiotic;

and

determining said phenotypic response comprises determining a susceptibility of said target cells to said antibiotic based on said monitoring of target cells in said identified subset of said spatially defined and separated cell compartments.

30. The method according to claim 17, wherein

said test agent is a cytostatic;

and

determining said phenotypic response comprises determining a susceptibility of said target cells to said cytostatic based on said monitoring of target cells in said identified subset of said spatially defined and separated cell compartments.

31. The method according to claim 17, wherein

said microfluidic device comprises:

a substrate having spatially defined and separated cell channels having a dimension to accommodate cells;

a respective first end of said spatially defined and separated cell channels is in fluid connection with a flow input channel having a first end in fluid connection with a first fluid port and a second end in fluid connection with a second fluid port;

a respective second end of said spatially defined and separated cell channels is in fluid connection with a flow output channel in fluid connection with a third fluid port; and

said spatially defined and separated cell channels comprise a respective channel obstruction designed to prevent selected cells from passing said respective channel obstruction and into said flow output channel; and

loading said biological sample comprises loading said biological sample into said first fluid port allowing excessive biological sample to flow out through said third fluid port to capture biological material in said spatially defined and separated cell channels by said channel obstructions.

32. The method according to claim 31, wherein loading said biological sample comprises loading said biological sample into said first fluid port allowing excessive biological sample to flow out through said third fluid port and said second fluid port to capture biological material in said spatially defined and separated cell channels by said channel obstructions.

33. The method according to claim 31, wherein exposing biological material to said test agent comprises loading said test agent into said second fluid port allowing said test agent to flow through said flow input channel, said spatially defined and separated cell channels, said flow output channel and out from said third fluid port.

34. The method according to claim 33, wherein exposing biological material to said test agent comprises loading said test agent into said second fluid port allowing said test agent to flow through said flow input channel, said spatially defined and separated cell channels, said flow output channel and out from said third fluid port and said first fluid port.