# US 10,370,630 B2 · Method and apparatus for cell isolation, growth, replication, manipulation, and analysis

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- Number: US 10,370,630 B2
- Document ID: US10370630B2
- Status: Granted
- Kind: B2
- Application number: 15/117,933
- Filed: 2015-02-10
- Published: 2016-12-01
- Granted: 2019-08-06
- Earliest claimed: 2014-02-10
- Assignees: Technion Research & Development Foundation Limited
- Applicants: Technion Research & Development Foundation Limited
- Inventors: Uri Sivan; Elad Brod
- Web page: https://patentgrep.com/patent/US10370630B2

## CPC

- C12M 23/12: {Well or multiwell plates (C12M25/04 takes precedence)}
- B01L 3/5027: {by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip}
- B01L 3/50273: {characterised by the means or forces applied to move the fluids}
- B01L 3/502753: {characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation}
- B01L 3/50857: {using arrays or bundles of open capillaries for holding samples}
- C12M 21/08: {for producing artificial tissue or for ex-vivo cultivation of tissue (prostheses A61F2/00, grafts A61L27/00)}
- C12M 23/16: {Microfluidic devices; Capillary tubes (integrated microfluidic structures B01L3/5027; microreactors B01J19/0093)}
- C12M 29/00: {Means for introduction, extraction or recirculation of materials, e.g. pumps (pumps per se F04B)}
- C12M 41/48: {Automatic or computerized control (automatic analysis G01N35/00)}
- C12M 47/02: {Separating microorganisms from the culture medium; Concentration of biomass (separating microorganisms from their culture media C12N1/02)}
- G01N 33/56966: {Animal cells}
- B01L 2300/0645: Electrodes
- B01L 2300/0681: Filter
- B01L 2300/069: Absorbents; Gels to retain a fluid
- B01L 2300/0819: Microarrays; Biochips
- B01L 2300/0829: Multi-well plates; Microtitration plates
- B01L 2400/0487: fluid pressure, pneumatics
- B01L 2400/049: vacuum
- C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS}
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

According to an aspect of some embodiments, an apparatus for isolation and cytometric analysis of cells from a liquid medium is provided. The apparatus comprises a cell reservoir, for receiving a liquid medium containing a suspension of cells. The apparatus further comprises a cell cage array between the cell reservoir and an enclosed reservoir, each cell cage comprising a large opening adjacent to the first cell reservoir, a cell cage, and one or more small opening adjacent to the enclosed reservoir. A connected liquid medium pump may move the suspended cells into cell cages by flowing the liquid medium from the cell reservoir to the enclosed reservoir. The apparatus further comprises a mechanical element which when actuated, pushes the cell cage array against a gel layer, forming a contiguous barrier to isolate each cell in a cell cage, and allowing cytometric analysis of isolated cells.

## Claims

### Claim 1

An apparatus for isolation of cells from a liquid medium for cytometric analysis, comprising:
a cell cage array that comprises a plurality of cell cages, each cell cage comprising a large opening that opens to a cell reservoir, a cell enclosure and at least one small opening adjacent and linking to an enclosed reservoir;
a liquid medium pump configured to draw liquid medium, comprising a cell suspension, from said cell reservoir to thereby cause liquid to flow from said cell reservoir through cell cages of said cell cage array and to said enclosed reservoir;
a mechanical element configured to push said cell cage array against a gel layer, thereby said gel layer forming a contiguous barrier to said large openings, to thereby isolate a cell contained in said cell enclosure; and
wherein said large opening has dimensions large enough for a cell to enter said cell cage, and said at least one small opening has dimensions smaller than said cell, prohibiting said cell from exiting said cell cage into said enclosed reservoir.

### Claim 2 (depends on 1)

The apparatus of claim 1, wherein said cell cage array is detachable from said apparatus and wherein said cell cage array is a single-use cell cage array.

### Claim 3 (depends on 1)

The apparatus of claim 1, wherein said apparatus further comprises:
at least one first electrode, in electric contact with said liquid medium in said cell reservoir;
a semipermeable membrane between said enclosed reservoir and an ion reservoir;
said ion reservoir containing an ionic fluid; and
at least one second electrode, in electric contact with said ionic fluid, such that a current flowing between said at least one first and second electrodes inject ions into said cell cages.

### Claim 4 (depends on 3)

The apparatus of claim 3, wherein said semipermeable membrane is selected from the list consisting of a cation exchange membrane (CEM), a charge mosaic membrane (CMM), a bipolar membrane (BPM), an anion exchange membrane (AEM), an alkali anion exchange membrane (AAEM), and a proton exchange membrane (PEM).

### Claim 5 (depends on 3)

The apparatus of claim 3, wherein said at least one first electrode is in electric contact with said liquid medium on one side of said enclosed reservoir, and said second electrode is in electric contact with said liquid medium on an opposite side of said enclosed reservoir, such that ion injection into said cell cages is by diffusion.

### Claim 6 (depends on 1)

The apparatus of claim 1, wherein each of said cell enclosures have internal dimensions so that at least one cell fits in each cell cage.

### Claim 7 (depends on 6)

The apparatus of claim 6, wherein said cell has a dimension between 1 to 30 micrometers, said cell enclosures have a diameter between 10% to 90% larger than said cell dimension, said large opening has dimensions between 10% to 90% larger than said cell dimension, and said at least one small opening has dimensions between 10% to 90% of said cell dimension.

### Claim 8 (depends on 1)

The apparatus of claim 1, wherein said cell enclosures have internal dimensions so that a plurality of cells fit in each said cell cage, wherein said at least one small opening is a plurality of small openings.

### Claim 9 (depends on 1)

The apparatus of claim 1, wherein said cell enclosures have an internal shape of a target cell to isolate.

### Claim 10 (depends on 1)

The apparatus of claim 1, wherein said cell enclosures have an internal cylindrical shape.

### Claim 11 (depends on 1)

The apparatus of claim 1, wherein said cell enclosures have an internal conical shape.

### Claim 12 (depends on 1)

The apparatus of claim 1, wherein said cell enclosures have an internal hemispherical shape.

### Claim 13 (depends on 1)

The apparatus of claim 1, wherein said cell enclosures have internal dimensions large enough to fit a plurality of individual cells.

### Claim 14 (depends on 13)

The apparatus of claim 13, wherein said individual cell has a dimension between 1 to 30 micrometers, said plurality of individual cells comprises N cells, said cell enclosures have a diameter between 10% to 90% larger than said individual cell dimension multiplied by said N cells, said large opening has dimensions between 10% to 90% larger than said individual cell dimension multiplied by said N cells, and said at least one small opening has dimensions between 10% to 90% of said individual cell dimension.

### Claim 15 (depends on 1)

The apparatus of claim 1, wherein said liquid medium pump is selected from the list consisting of an electrical pump, a micropump, a manual syringe with an attached caliper, an automatic programmable syringe, a computerized syringe, a syringe driver, a syringe pump, a programmable syringe pump, a media dispenser, an inductive pump, a pressure injection cell dispenser, a peristaltic pump, and an infusion pump.

### Claim 16 (depends on 1)

The apparatus of claim 1, wherein between said cell cage array and said enclosed reservoir there is an array support structure, wherein said array support structure maintains said cell cage array in a substantially flat plane.

### Claim 17 (depends on 1)

The apparatus of claim 1, wherein said cell cage array is visible through an opaque window in said apparatus.

### Claim 18 (depends on 1)

The apparatus of claim 1, wherein said cell cage array further comprises attached antibodies.

### Claim 19 (depends on 1)

The apparatus of claim 1, wherein said gel layer further comprises attached antibodies.

### Claim 20 (depends on 1)

The apparatus of claim 1, wherein said apparatus further comprises a diffusing gel layer between said cell cage array and said enclosed reservoir.

### Claim 21 (depends on 1)

The apparatus of claim 1, wherein said apparatus further comprises an adsorbing layer adjacent to said gel layer, and said adsorbing layer comprises a material selected from the list consisting of nitrocellulose, polyvinylidene difluoride (PVDF) and low fluorescence PVDF.

### Claim 22 (depends on 1)

The apparatus of claim 1, wherein said gel layer is composed of any from the list of agarose, acrylamide, and bisacrylamide mixtures.

### Claim 23 (depends on 1)

The apparatus of claim 1, wherein said mechanical element is selected from the list consisting of a screw, a level, a step motor, a computerized step motor, linear actuator, and computerized linear actuator.

### Claim 24 (depends on 1)

The apparatus of claim 1, wherein said apparatus is a computerized apparatus comprising at least one user interface, at least one component interface, and at least one processing unit capable of controlling at least one component.