Method for isolation and morphological analysis of cells
Inventors
Jung, Yong Gyun • Kim, Joo Ho • Park, Wook • Yoon, Jin Sik • Song, Suk Heung
Assignees
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Publication Number
US-12059682-B2
Publication Date
2024-08-13
Expiration Date
Abstract
Provided is a method for cell isolation and morphological analysis. The method includes: providing a substrate with one or more wells; inserting cell isolation devices, each of which has one side patterned with one or more microwells, into the wells; introducing a liquid medium including cells into the wells; allowing the microwells of the cell isolation devices to come into contact with the inner surfaces of the wells such that some of the cells are isolated and trapped at the level of individual cells in the microwells; and observing the morphological changes of the isolated cells.
Core Innovation
The invention relates to a cell isolation device for cell analysis in which a body is made of a swellable material. The body includes a top portion and a bottom portion, where bubble holes are formed on a flank of the top portion and one or more microwells are patterned at least on the bottom surface. The top portion and the bottom portion have a cylindrical shape, with the bottom portion having a smaller diameter than the top portion.
The device enables cell trapping at an individual-cell level using the microwells that contact inner well surfaces upon swelling of the swellable body. This supports time-lapse optical observation of morphological changes of isolated cells, including division/no-change, filament formation, and swelling formation, as well as associated outcomes such as cytotoxicity and cytoproliferation/survival/apoptosis as assessed by time-lapse morphology.
The disclosed system is applied to cell and microbial analysis, including antibiotic susceptibility testing. In this use case, cytotoxicity and minimum inhibitory concentration (MIC) are determined based on time-lapse morphology after exposure to antibiotics such as ampicillin, chloramphenicol, and methicillin, for organisms including Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and methicillin-resistant Staphylococcus aureus (MRSA). Experimental examples are described as agreeing with CLSI MIC ranges while reducing assay runtime (e.g., about 4 hours versus 18–24 hours).
Claims Coverage
The partial content provides one independent claim (clm-00001). It is supported by dependent claims (clm-00002 through clm-00005) that refine the swellable material behavior and add geometric and packaging features.
Swellable cylindrical body with bubble holes and microwells
A cell isolation device for cell analysis comprising a body made of a swellable material, having a top portion and a bottom portion with bubble holes formed on a flank of the top portion and one or more microwells patterned at least on the bottom surface, wherein the top portion and the bottom portion have a cylindrical shape.
Bottom portion smaller diameter than top portion
The device wherein the bottom portion has a smaller diameter than the top portion.
Stimulus-responsive swelling
The device wherein the swellable material is swelled in response to an externally applied stimulus selected from electricity, light, sound, heat, magnetism, specific substances, or combinations thereof.
Hydrogel swellable material
The device wherein the swellable material is a hydrogel.
Concave top surface
The device wherein the top surface is concave.
Kit with substrate wells and inserted cell isolation devices
A kit for isolating cells and performing morphological analysis comprising a substrate with one or more wells and a plurality of the cell-isolation devices inserted and arranged in each well.
Overall, the claims cover a hydrogel-based, cylindrical, swellable cell isolation device with bubble holes on a top portion and microwells on a bottom surface, including optional concave top geometry and bottom/top diameter difference, and extending the device concept to a kit configuration using a substrate with wells for morphological analysis.
Stated Advantages
Enables time-lapse optical observation of isolated single cells based on morphological changes.
Provides antibiotic susceptibility testing with cytotoxicity and minimum inhibitory concentration (MIC) determination based on time-lapse morphology.
Provides agreement with CLSI MIC ranges in experimental examples.
Reduces assay runtime (e.g., about 4 hours versus 18–24 hours).
Documented Applications
Cell and microbial analysis using time-lapse optical observation of isolated single cells.
Antibiotic susceptibility testing to determine cytotoxicity and minimum inhibitory concentration (MIC) for antibiotics including ampicillin, chloramphenicol, and methicillin, for organisms including E. coli, S. aureus, and MRSA.
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