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US 2024/0254431 A1Published application
Systems and methods for high-throughput cell line development
AssigneeOnecyte Biotechnologies, Inc.
InventorsKonstantinos Tsioris, Ayca Yalcin Ozkumur
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Abstract
Provided herein are methods and systems for high-throughput cell line development.
Claims186
A method for selecting a target cell;
- comprising:
- a) placing a plurality of cells into a plurality of chambers, wherein each individual chamber of a subset of the plurality of chambers contains one or no more than 2, 3, 5, 10, 15 or 20 individual cells of the plurality of cells;
- b) exposing at least the subset of the plurality of chambers from a) to a condition, wherein the condition is exposing the individual chamber with one or more regents, or treating the individual chamber with a plurality of secondary cells, or applying a membrane to the individual chamber to form an individual membrane-modified chamber, or contacting the individual chamber with a capture substrate, or contacting the individual chamber with a secondary cell-immobilized capture substrate, or a combination thereof;
- c) detecting a signal or a change thereof from a particular chamber of the subset of the plurality of chambers during or after the exposing in b), wherein the signal or the change thereof is indicative of (i) the presence of a target cell in the particular chamber, or (ii) the presence of a product produced by the target cell in the particular chamber;
- and
- d) selecting the target cell in the particular chamber from the plurality of cells at least based on a pre-determined value of the signal or the change thereof in c).
3 more elements
A method for facilitating clone selection of a cell line, from among a plurality of candidate single cells, comprising:
- a) generating, by an imaging unit, a first plurality of images of each of the plurality of candidate single cells individually, wherein each of the plurality of candidate single cells resides in an individual chamber of a plurality of chambers;
- b) detecting, by one or more processors analyzing the first plurality of images for each of the plurality of candidate single cells, one or more cell features of each of the plurality of candidate single cells depicted in the first plurality of images;
- and
- c) based on the one or more cell features, determining, by the one or more processors and according to a finalized single cell-to-colony machine learning model, one or more predicted attributes for a colony expanded from each of the plurality of candidate single cells;
- d) ranking the plurality of candidate single cells according to the one or more predicted attributes for each of the plurality of candidate single cells,
- wherein the finalized single cell-to-colony model predicts attributes of a hypothetical colony based on at least the one or more cell features of a single cell.
3 more elements
A method for high-throughput cell line development, comprising:
- a) providing a plurality of target cells, an array of nano-wells, one or more reagents and instructions;
- b) loading the plurality of target cells into said array of nano-wells such that an individual well of the array of nano-wells contains an individual target cell;
- c) exposing said plurality of target cells to one or more reagents;
- d) obtaining measurements of individual target cells and quantitative measurements of individual articles associated with said individual target cells;
- e) selecting a target cell from said individual target cells to be recovered based on predetermined values of said measurements;
- wherein a time to reach a decision for selecting said target cell for recovery does not exceed 3 hours from the initialization of said method;
- and
- wherein said method yields clones with a mean productivity of at least 5 grams per liter.
5 more elements
A method for isolated co-culture utilizing a secondary cell suspension, comprising:
- a) providing a plurality of target cells, an array of nano-wells, one or more reagents and instructions;
- b) loading individual target cells of said plurality of target cells into said array of nano-wells;
- c) applying a membrane to said array of nano-wells to form a membrane-modified array of nano-wells;
- d) providing a suspension of a plurality of secondary cells, one or more reagents, or a combination thereof, near or in contact with said membrane-modified array of nano-wells;
- e) obtaining measurements of individual target cells and measurements of individual articles associated with said individual target cells;
- f) selecting a target cell from said individual target cells to be recovered based on predetermined values of said measurements;
- and
- wherein a time to reach a decision for selecting said target cell for recovery does not exceed 3 hours from the initialization of said method.
5 more elements
A method for isolated co-culture utilizing a secondary cell immobilized-capture substrate, comprising:
- a) providing a plurality of target cells, an array of nano-wells, one or more reagents, instructions;
- and a plurality of secondary cells immobilized to a capture substrate;
- b) loading individual target cells of said plurality of target cells into said array of nano-wells;
- c) applying a membrane to said array of nano-wells to form a membrane-modified array of nano-wells;
- d) simultaneously contacting said membrane-modified array of nano-wells and the secondary cell-immobilized capture substrate with one or more reagents;
- e) obtaining measurements of individual target cells and measurements of individual articles associated with said individual target cells;
- f) selecting a target cell from said individual target cells to be recovered based on predetermined values of said measurements;
- wherein a time to reach a decision for selecting said target cell for recovery does not exceed 3 hours from the initialization of said method;
- and
- wherein said method yields clones with a mean productivity of 5 grams per liter.
7 more elements
A system for high-throughput cell line development, comprising:
- a) an array of nano-wells comprising individual nano-wells, wherein said individual nano-wells contain zero or more target cells;
- b) an apparatus for reversibly sealing a capture substrate with said array of nano-wells;
- c) a reagent module configured for supplying one or more reagents to said array of nano-wells;
- d) a detection module configured for performing measurements of biomolecules secreted by said target cell onto said capture substrate at discrete positions indexed to said individual wells;
- e) a cell recovery apparatus configured for recovery of said individual cells, wherein values extracted from said measurements of biomolecules and cells are compared to predetermined criteria and used for the selection of said individual cells to be recovered;
- wherein said system is configured to reach a decision for selecting said target cell for recovery within 3 hours from initialization;
- and
- wherein said system is configured to yield clones with a mean productivity of 5 grams per liter.
5 more elements
A mechanism sealing a capture substrate to an array of nano-wells in a sterile fashion, comprising:
- a) a top piece configured to immobilize capture substrate;
- b) a base configured to immobilize an array of nano-wells;
- wherein said base comprises one or more alignment rods to align said top piece to said base such that said capture substrate and said array of nano-wells are fixed in coplanar and rotationally aligned orientation;
- and
- wherein the distance between said capture substrate and said array of nano-wells can be controllably varied along an axis perpendicular to the coplanar planes of said capture substrate and said array of nano-wells, thus placing said capture substrate and said array of nano-wells in alignment and forming a sterile, fluid tight seal.
2 more elements
A method for cell line development utilizing a terminal assay for cell selection, comprising:
- a) providing a plurality of target cells, an array of nano-wells, one or more reagents and instructions;
- b) loading individual target cells of said plurality of target cells into individual nano-wells of said array of nano-wells and exposing said individual target cells to one or more reagents;
- c) contacting a capture substrate to said array of nano-wells, thereby sealing said individual target cells into said individual nano-wells;
- d) growing a colony of cells from said individual target cell, such that one or more colony cells of the colony of cells are transferred to said capture substrate onto positions registered to corresponding individual wells from which said one or more colony cells originated;
- e) separating said capture substrate from said array of nano-wells and performing an assay that may result in the death of said colony cells that transferred to said indexed positions;
- and
- f) selecting colony cells for recovery from said corresponding individual wells based on predetermined values of said measurements.
4 more elements
A method for high-throughput identification of a B cell or antibody secreting cells (ASCs), comprising:
- (a) obtaining a plurality of B cells or ASCs from a subject;
- (b) loading the individual B cells or ASCs into individual wells of an array of nano-wells;
- (c) detecting a secreted product of the individual B-cell or ASCs;
- (d) selecting the individual B-cell or ASCs;
- wherein a time to reach a decision for selecting said target cell does not exceed 5.5 hours from the initialization of said method.
2 more elements
A method cell line development utilizing identification of glycosylation patterns on a biomolecule, comprising:
- a) providing a plurality of target cells, an array of nano-wells, one or more reagents and instructions;
- b) loading the plurality of target cells into an array of nano-wells such that an individual well of the array of nano-wells contains an individual target cell;
- c) exposing the plurality of target cells to one or more reagents, wherein each individual target cell can produce a biomolecule;
- d) capturing said biomolecule produced by each individual target cell on a capture substrate;
- wherein said capture substrate is configured to keep the biomolecule produced by each individual target cell in the array of nano-wells spatially distinct and registered to said individual nano-well of origin;
- wherein the capture substrate comprises a glycan binding reagent;
- e) comparing said captured biomolecule produced by each individual target cell in the array of nano-wells on the capture substrate to a reference;
- f) ascertaining a glycosylation profile for each individual biomolecule;
- and
- g) selecting a target cell from the plurality of target cells to be recovered based on the glycosylation profile of the biomolecule produced by the individual target cell.
7 more elements
A method for cell line development utilizing mass spectrometry for identification of a biomolecule, comprising:
- a) providing a plurality of target cells, an array of nano-wells, one or more reagents and instructions;
- b) loading the plurality of target cells into array of nano-wells such that an individual well of the array of nano-wells contains an individual target cell;
- c) exposing the plurality of target cells to one or more reagents, wherein each individual target cell can produce a biomolecule;
- d) capturing each biomolecule produced by each individual target cell on a capture substrate;
- wherein the capture substrate is configured to keep each biomolecule produced by each individual target cell in the array of nano-wells spatially distinct and registered to said individual well from which said individual target cell originated;
- e) analyzing the biomolecule with mass spectrometry (MS);
- f) comparing and the captured biomolecule on the capture substrate to a mass spectrometry-based reference;
- g) identifying the captured biomolecule;
- and
- h) selecting a target cell from the individual target cells to be recovered based on the identify of biomolecule produced by the individual target cell.
7 more elements
A method of selecting a target cell based on an aggregation property of a secreted biomolecule, comprising:
- a) providing a plurality of cells, an array of nano-wells, and one or more reagents, wherein the plurality of cells comprises a target cell;
- b) loading the plurality of cells into said array of nano-wells such that an individual well of the array of nano-wells contains an individual cell;
- c) exposing the plurality of cells to one or more reagents, wherein each of the plurality of cells can produce a biomolecule;
- wherein the target cell can produce a target biomolecule;
- d) capturing said biomolecule produced by each individual target cell on a capture substrate;
- wherein said capture substrate is configured to keep said biomolecule produced by each individual cell in the array of nano-wells spatially distinct and registered to said individual nano-well from which said biomolecule originated;
- e) exposing the biomolecule or cell to a reagent or other perturbant that induces aggregation of the biomolecule;
- determining an aggregation property of the biomolecule to identify the target biomolecule;
- and
- f) selecting the target cell from the plurality of cells to be recovered based on the aggregation property of the target biomolecule.
7 more elements
Dependencies are inferred from the claim text.