A method, system, and apparatus for precise cell selection via reversible light-induced polymerization

Abstract

A method, system, and apparatus provides a precise cell selection via reversable light-induced polymerization of target cells in a heterogenous cell population in a. hydrogel system via reverse photopolymerization. A selection of cells suspended in a photopolymerizable carrier fluid in a reservoir are s elected and are exposed to a selective first wavelength illumination causing direct cell-selective light-induced polymerization and immobilization without disrupting cell viability. Non-selected cells remain unpolymerized and mobile for collection and separation. An integrated high-resolution imaging system enables a selective initial cell-selection, control of selected polymerization at the first wavelength using LCD panel masks, and verifying results and accuracy to single-cell resolution. A selected second wavelength illumination exposure enables release of immobilized cells when needed in a non-destructive and data-rich verifiable manner.

Description

PATENT 1 CORLA.P001PCT A METHOD, SYSTEM, AND APPARATUS FOR PRECISE CELL SELECTION VIA REVERSIBLE LIGHT-INDUCED POLYMERIZATION GOVERNMENT FUNDING RELATED TO APPLICATION(s)

[0001] None CROSS REFERENCES TO RELATED APPLICATIONS

[0002] This application relates to and claims priority from US Prov. Ser. No.: 63 / 655,540 filed June 3, 2024, the entire contents of which are incorporated herein fully by reference. FIGURE SELECTED FOR PUBLICATION

[0003] Fig. 9 BACKGROUND OF THE INVENTION Field of the Invention

[0004] The present invention relates to the field of biotechnology, specifically to methods, apparatus, and systems for precise cell selection via reversible polymerization and depolymerization using photopolymerization for precise cellular selection with high throughput. The invention also relates to and is applicable in various domains such as synthetic DNA production, single cell manipulation, pharmacological discovery, and cell-free systems. Description of the Related Art

[0005] In the field of biotechnology, precise control over biochemical reactions and molecular interactions is crucial. Traditional methods for partitioning reactions involve water-oil emulsions or separating reactions into micro-wells on a plate. These volumes are considered protected from other volumes by nature of their mechanical separation. There are substantial limitations to these methods, such as burdensome address retargeting and costs attendant to address per reaction within a micro-well. Existing technologies lack either address retargeting or cost-effective scalability needed for advanced applications like synthetic DNA production and single cell manipulation.PATENT 2 CORLA.P001PCT

[0006] Moreover, current cell selection methods and systems can be time-consuming and inefficient, particularly in high-throughput settings such as pharmacological discovery and cell- free systems. There is a need for a more efficient, reversible, and mechanically controlled method to protect and deprotect biological samples without compromising their integrity or functionality.

[0007] Current cell selection technologies have notable limitations. Fluorescence-Activated Cell Sorting (FACS) processes cells sequentially, reducing efficiency, particularly with rare subsets, and often compromising cell viability through mechanical stress. Bead-based selection methods, while gentler and faster, lack integrated inline quality control and detailed per-cell data, posing risks of off-target selection. Live Cell Analysis (LCA) provides extensive data but lacks direct selection capability. Similarly, techniques to build specific locational cage(s) around cells during live cell analysis by controlling cell adhesion (but failing to polymerize an entire target volume, including the desired isolated cell) mandates the creation of verification imaging errors, loss, and prevents verification of quality results.

[0008] Accordingly, there are a number of detriments in the present art. ASPECTS AND OBJECTS OF THE INVENTION

[0009] With knowledge of the aforementioned circumstances, the invention is based on the objects of addressing one or more of the concerns noted herein and above.

[0010] One adaptive aspect or object of the present invention is to provide an improvement to one or more of the concerns noted herein and above.

[0011] One adaptive aspect or object of the present invention is to provide a gentile, efficient, data-rich cell selection method that includes built-in, convenient verification to ensure therapeutic precision and safety.

[0012] According to alternative and adaptive aspects of the present invention there is provided a method, system, and apparatus for reversible mechanical volumetric polymerization (e.g. protection) and depolymerization (e.g., deprotection) of a precise cell selection using reversible light-induced polymerization. This system enables precise control over the protection and deprotection process (also noted as polymerization and depolymerization) through the photo- catalyzation of polymer monomers into polymer blocks as directed by precise light exposure, and dissolving polymerized blocks via degradable polymer crosslinkers.PATENT 3 CORLA.P001PCT

[0013] A method, system, and apparatus provides for a precise cell selection via reversable light-induced polymerization of target cells in a heterogenous cell population in a hydrogel system via reverse photopolymerization. A selection of cells suspended in a photopolymerizable carrier fluid in a reservoir are selected and are exposed to a selective first wavelength illumination causing direct cell-selective light-induced polymerization and immobilization without disrupting cell viability. Non-selected cells remain unpolymerized and mobile for collection and separation. An integrated high-resolution imaging system enables a selective initial cell-selection, control of selected polymerization at the first wavelength using LCD panel masks, and verifying results and accuracy to single-cell resolution. A selected second wavelength illumination exposure enables release of immobilized cells when needed in a non-destructive and data-rich verifiable manner.

[0014] According to additional alternative and adaptive aspects of the present invention the invention additionally includes a device or apparatus or system, including operatable programmable components, LCD masks, controllers, inputs, and flow cell arrangements so that the proposed system, method, and apparatus can selectively expose an aqueous volume to the appropriate wavelength of light with sufficient intensity to photo-catalyze polymer monomers in the aqueous reagent. The polymerization results in inclusions of materials that are then protected from the unpolymerized volume. A subsequent solvent exchange is excluded from the polymerized volume. In this way, unpolymerized areas (zones) that contain multiple cells can be exposed to reagents while polymerized areas (zones) that contain multiple cells are protected. The polymer can be degraded and removed by the addition of chemicals that break polymer cross-linkers. This results in resetting the features, exposing the entire surface area to the aqueous volume. This process can be done cyclically, allowing any address in the volume to be protected and unprotected as needed. This device can be finely controlled to ensure accurate and reversible protection, making it ideal for applications in synthetic DNA production, single cell manipulation, pharmacological discovery, and even in cell-free systems. The system enhances efficiency, reduces processing time, and maintains the integrity of the biological samples.

[0015] According to an alternative aspect of the present invention, there is provided a device, system, and apparatus for reversible mechanical volumetric polymerization (protection) and depolymerization (deprotection) of biological samples, comprising: a chamber, a microfluidic chip, a control unit, an aqueous polymerization mechanism, and a sensor and feedback system, along with user input and controls (such as programming controller, LED panel masks, anPATENT 4 CORLA.P001PCT autosampler, program processors that store memory programs of required operative steps as noted herein).

[0016] According to another alternative aspect of the present invention, there is provided a device, system, and apparatus for reversible mechanical volumetric light-induced polymerization or immobilization (protection of biological target cells) and reversable light-induced de- polymerization or mobilization (deprotection of biological target cells) (e.g., prevent flow that removes unbound cells so that, after depolymerization, the unbound cells can be collected from the flow cells output well for further characterization), and a region wherein: the aqueous polymerization mechanism includes a degradable moiety for applying and releasing mechanical protection of a volume in a hydrogel system using dual-wavelength light.

[0017] According to another alternative aspect of the present invention, there is provided a device, system, and apparatus for reversible mechanical volumetric protection and deprotection (polymerization and depolymerization) of biological samples further comprising an electronic control unit for regulating the selection and properties of applied protective features within the chamber.

[0018] According to another alternative aspect of the present invention, there is presented a method for reversible mechanical volumetric protection and deprotection (polymerization and depolymerization encapsulation) of biological samples using the device, system, and apparatus discussed herein, further comprising the steps of: placing the sample in the protective chamber, applying a photopolymerizable carrier fluid (polymer) to protect the sample (e.g., polymerization at a first wavelength), and degrading the polymer to deprotect (e.g., depolymerization at a second wavelength) the sample so that it may be further collected and analyzed.

[0019] According to another alternative aspect of the present invention, there is presented a method for reversible mechanical volumetric protection and deprotection of biological samples wherein: the protective mechanism is applied and then degraded in a controlled manner based on feedback from integrated sensors, or programmed patterns with electronic program control units.

[0020] According to another alternative aspect of the present invention, there is presented a method for reversible mechanical volumetric protection and deprotection of biological samples, wherein: the process of protecting and exposing material in the chamber is done in a cyclical manner using photopolymerizable carrier fluid and a light-induced polymerization of selectedPATENT 5 CORLA.P001PCT masked regions and a depolymerization step at different wavelengths allowing for one or many rounds of the process to occur in series to result in a purified sample.

[0021] According to another alternative aspect of the present invention, there is provided a reversible mechanical volumetric protection and deprotection, system, method, device and apparatus as shown and described herein.

[0022] According to another alternative aspect of the present invention, there is provided a method, system, and apparatus for reversible mechanical volumetric protection and deprotection. This system enables precise control over the protection and deprotection process (photo- polymerization / depolymerization) through the photo-catalyzation of polymer monomers into polymer regions (e.g., blocks) as directed by precise light exposure, and dissolving polymerized blocks via degradable polymer crosslinkers, as noted herein.

[0023] According to another alternative aspect of the present invention, the photopolymerizable carrier fluid may additionally include an effective amount of photocatalysts such as titanium dioxide (TiO2) or a photo initiator such as 2,2-dimethoxy-2-phenylacetaphenone (DPMA) may be used to generate free radicals, promote uniform polymerization (acrylamide polymerization) and to provide additional reliability during an exposure polymerization process.

[0024] According to another and alternative process the photopolymerizable carrier fluid (when polymerized such as with a Bis(acryloyl)cystamine crosslinker) can be quickly dissolved (unpolymerized / depolyermized) by dithiothreitol (DTT).

[0025] According to another alternative and adaptive aspect of the present invention, there is provided a method for precise, rapid, and gentle selection of target cells from heterogeneous populations. A device has been developed that achieves selection of cells using standard microscope slides, sandwiched around a gasket, as a reservoir for cells. Cells are suspended in a photopolymerizable carrier fluid, that may include additional photocatalysts, and are exposed to selective illumination of a first wavelength using a programmable controlled LCD panel mask or other system for selective illumination, that is linked with an image system which causes specific cells to be exposed (polymerized) and become immobilized within the photopolymerizable carrier fluid without disrupting cell viability. Cells remaining in the unpolymerized portions remain mobile for collection. An integrated high-resolution imaging system and controller guides selection and verifies outcomes, ensuring accuracy at single-cell resolution, and records image and count data in a memory. A second wavelength, which is different from a first wavelength, enablesPATENT 6 CORLA.P001PCT the release of immobilized cells when needed and may be combined with a compound to enhance dissolution and depolymerization (e.g., unprotection of the targeted cells in a flow cell). This approach combines the strengths of Fluorescence-Activated Cell Sorting (FACS), bead-based methods, and live-cell imaging, offering a scalable, non-destructive, and data-rich solution for cell selection.

[0026] According to another alternative and adaptive aspect of the present invention, the system integrates high-resolution imaging to guide and verify selection of portions for polymerization / depolymerization (e.g., protection and deprotection). Initial imaging using the proposed system identifies cells based on morphological or fluorescent features, or both, and directs the illumination pattern to an LCD panel masks, or uses other illumination-precise means, to control an exposure to UV-A light in a selected wavelength. After photo-polymerization, a follow-up imaging of the flow cell is used to confirm that the desired cells were successfully retained (or excluded), and imagery may be electronically recorded, enabling quality control at single-cell resolution.

[0027] According to another alternative aspect of the present invention, there is provided a method for selective immobilization and recovery of viable cells via reverse photopolymerization.

[0028] According to another alternative aspect of the present invention, there is provided a hydrogel system responsive to dual-wavelength light enabling programmable (designate-able) cell encapsulation and reversable release.

[0029] According to another alternative aspect of the present invention, there is provided a hydrogel system integrated with high-resolution imaging enabling light-induced cell selection for inline verification.

[0030] According to another alternative aspect of the present invention, there is provided a method, for cell selection via reversible light-induced polymerization, comprising the steps of: preparing an aqueous cell slurry containing a heterogenous group of cells in a photopolymerizable carrier fluid; the heterogenous group of cells having identifiable cellular features including morphological features or fluorescent features; transferring the cell slurry to a flowcell; visualizing the flow cell with a visualization system; designating a targeted region and a nontargeted region of the flow cell based upon the identifiable cellular features of the heterogenous group of cells in the cell slurry visualized by the visualization system; masking the targeted region of the flow cell with a masking device and preventing a light-induced photopolymerization of the masked targetedPATENT 7 CORLA.P001PCT region thereby maintaining a mobility of cells in the cell slurry of the targeted region of the flowcell; illuminating the unmasked nontargeted regions of the flow cell with a first wavelength of light causing a light-induced polymerization and immobilization of the cell slurry in the unmasked nontargeted regions; washing the flowcell and collecting mobile cells remaining in the cell slurry from the masked targeted region that are not light-induced polymerized; illuminating the unmasked nontargeted regions of the flow cell with a second wavelength of light causing a light-induced depolymerization and remobilization of the cell slurry in the unmasked nontarget region; and washing the flowcell and collecting remobilized cells from the unmasked nontargeted region of the flow cell.

[0031] According to another alternative aspect of the present invention, there is provided a method, for cell selection via reversible light-induced polymerization, wherein: the second wavelength of light is different than the first wavelength of light.

[0032] According to another alternative aspect of the present invention, there is provided a method, for cell selection via reversible light-induced polymerization, wherein: the first wavelength of light includes illumination with a 405 nm light; and the second wavelength of light includes illumination with a 505nm light.

[0033] According to another alternative aspect of the present invention, there is provided a method, for cell selection via reversible light-induced polymerization, wherein: the carrier fluid includes a multiarmed PEG derivative with malemide groups functionalized with a protein prepared with Donpra145N.

[0034] According to another alternative aspect of the present invention, there is provided a method, for cell selection via reversible light-induced polymerization, wherein: the carrier fluid includes a photocatalyst.

[0035] According to another alternative aspect of the present invention, there is provided a method, for cell selection via reversible light-induced polymerization, wherein: the masking device is selected from one of a Liquid Crystal Display (LCD), a spatially controlled digital micromirror device (DMDs), and a precision laser scanning system.

[0036] According to another alternative aspect of the present invention, there is provided a method, for cell selection via reversible light-induced polymerization, wherein: the step of transferring the cell slurry to a flowcell and further comprises: placing the flowcell in a chamber of an autosampler; and supplying the autosampler with access to a reagent supply.PATENT 8 CORLA.P001PCT

[0037] According to another alternative aspect of the present invention, there is provided a method, for cell selection via reversible light-induced polymerization, wherein: the step of visualizing the flow cell with the visualization system, further comprises: designating a plurality of targeted regions and a plurality of nontargeted regions of the flow cell of the flow cell.

[0038] According to another alternative aspect of the present invention, there is provided a method, for cell selection via reversible light-induced polymerization, further comprising repeating the steps of: visualizing the cell slurry in the flowcell, designating one or more targeted regions and nontargeted regions; masking the one or more targeted regions; illuminating the one or more nontargeted regions with the first wavelength; washing the flowcell and collecting mobile cells that are not light polymerized; illuminating the one or more nontargeted regions with the second wavelength; and washing the flowcell and collecting remobilized cells, whereby multiple cycles of the method allow for multiple cell selections from said heterogenous group of cells.

[0039] According to another alternative aspect of the present invention, there is provided a method, for cell selection via reversible light-induced polymerization, wherein: the first wavelength of light includes illumination with a wavelength selected from the wavelengths from 400 nm to 409 nm.

[0040] According to another alternative aspect of the present invention, there is provided a method, for cell selection via reversible light-induced polymerization, wherein: the second wavelength of light includes illumination with a wavelength selected from the wavelengths from 500 nm to 509 nm.

[0041] According to another alternative aspect of the present invention, there is provided a method, for cell selection via reversible light-induced polymerization, wherein: the visualization system, further comprises: a controllable light source; a microscopic objective system; and at least one of a compound columinating lens, a plano-convex lens, and a dichroic mirror.

[0042] According to another alternative aspect of the present invention, there is provided a method, for cell selection via reversible light-induced polymerization, wherein: the visualization system, further comprises: a camera system in electronic communication with an electronic control unit receiving a visual image from said visualization system.

[0043] According to another alternative aspect of the present invention, there is provided a method, for cell selection via reversible light-induced polymerization, wherein: the flow cell is aPATENT 9 CORLA.P001PCT plurality of flowcells in the chamber; and the step of transferring the cell slurry to the flowcell further includes a step of transferring the cell slurry to the plurality of flowcells.

[0044] According to another alternative aspect of the present invention, there is provided a system, for conducting a method of cell selection via reversible light-induced polymerization, according to the methods disclosed herein.

[0045] According to another alternative aspect of the present invention, there is provided a system, for conducting a method of cell selection via reversible light-induced polymerization, comprising: a flowcell containing an aqueous cell slurry with a heterogenous group of cells in a photopolymerizezable carrier fluid; the heterogenous group of cells having identifiable cellular features including morphological features or fluorescent features; a visualization system for visualizing the aqueous cell slurry; the visualization system designating a targeted region and a nontargeted region of the flow cell based upon the identifiable cellular features of the heterogenous group of cells in the cell slurry visualized by the visualization system; a masking device that masks that masks the targeted region of the flow cell and prevents a light-induced photopolymerization of the masked targeted region thereby maintaining a mobility of cells in the cell slurry of the targeted region of the flowcell; a wavelength emitter that illuminates the unmasked nontargeted regions of the flow cell with a first wavelength of light causing a light-induced polymerization and immobilization of the cell slurry in the unmasked nontargeted regions; whereby a washing of the flowcell collects mobile cells remaining in the cell slurry from the masked targeted region that are not light-induced polymerized; the wavelength emitter that illuminates the unmasked nontargeted regions of the flow cell with a second wavelength of light causing a light-induced depolymerization and remobilization of the cell slurry in the unmasked nontarget region; and whereby a washing the flowcell collects remobilized cells from the unmasked nontargeted region of the flow cell.

[0046] According to other alternative aspects or objects of the present invention, there are provided the technical advantage of high-throughput parallel processing for target cell collection (instead of sequential sorting), the gentle handling of target cells to preserve cell viability, the operability of real-time, image-guided, selection of target cells and quality control verification, the ability to fully recover fractions for both retained (and excluded) cells; and a method, system, and device that is scalable and compatible with good manufacturing practice (GPM) compliant workflows.PATENT 10 CORLA.P001PCT

[0047] According to further alternative aspects or objects of the present invention, there are provided a method, system, and device that a enables operable selection of genetically edited hematopoietic stem cells (HSCs), of enrichment of desired CAR-T or TCR-T cell populations, of a non-destructive isolation of selected rare immune or progenitor cell subtypes, provides for in- process and inline quality control (QC) analysis of quality samples during cell therapy manufacturing, and enables collection of designated live / dead / optimal targeted cells.

[0048] Further explanations of the invention, advantageous details and features will become apparent from the following description of the exemplary embodiments of the invention schematically illustrated in the figures or respective parts thereof.

[0049] The above presents a simplified aspect and summary in order to provide a basic understanding of some aspects of what is claimed below. This summary is not an exhaustive overview of the claimed subject matter. It is not intended to identify key or critical elements of the disclosure or to delineate the scope of the claims. Its sole purpose is to present some concepts in a simplified form as a prelude to the more detailed description that is discussed below.

[0050] Additionally, the above and other aspects, features and advantages of the present invention will become apparent from the following description read in conjunction with the accompanying drawings, in which like reference numerals designate the same elements. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Fig. 1 is a process illustration representing selective photopolymerization and depolymerization of a carrier fluid within a flow cell, using the present invention.

[0052] Fig. 2 is a further graphical process illustration designating the arrangement of the monomer units bonding to each other and photopolymerizing (and de-photopolymerizing) when induced with specific wavelengths of light according to the present invention.

[0053] Fig. 3 is a graphic representation of the gene, provided inserted into the Donpra145N.

[0054] Fig. 4 is an illustrative view of synthetic DNA production regarding nucleotide (NTP) additions as discussed herein.

[0055] Fig. 5 is an illustrative partial view of cell sorting in a flow cell and system.

[0056] Fig. 6 is an illustrative partial view of the proposed system and method.

[0057] Fig. 7 is an illustrative partial view of the proposed system and method.PATENT 11 CORLA.P001PCT

[0058] Fig. 8 is an illustrative step depiction of selective steps of the proposed method.

[0059] Fig.9 is an illustrative graphic of elements and features of the proposed system herein.

[0060] Fig.10 is an illustrative flow chart for operating the proposed system and devices herein for the proposed invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0061] Reference will now be made in detail to embodiments of the invention. Wherever possible, same or similar reference numerals are used in the drawings and the description refer to the same or like parts or steps. The drawings are in simplified form and are not to precise scale. The word 'couple' and similar terms do not necessarily denote direct and immediate connections, but also include connections through intermediate elements or devices. For purposes of convenience and clarity only, directional (up / down, etc.) or motional (forward / back, etc.) or position (front / back, etc.) terms may be used with respect to the drawings. These and similar directional terms should not be construed to limit the scope in any manner. It will also be understood that other embodiments may be utilized without departing from the scope of the present invention, and that the detailed description is not to be taken in a limiting sense, and that elements may be differently positioned, or otherwise noted as in the appended claims without requirements of the written description being required thereto.

[0062] Various operations may be described as multiple discrete operations in turn, in a manner that may be helpful in understanding embodiments of the present invention; however, the order of description should not be construed to imply that these operations are order dependent.

[0063] As will be reviewed in further illustrative examples in the figures below, the present invention provides a method, system, and device provide for a sorting of one or more heterogeneous group of cells into desired subsets, for example subsets A and B, or other multiple subsets, based on identifiable cellular features. In the process, cells are first suspended in a photopolymerizable carrier fluid. Upon selective illumination of a first wavelength (for example 405 nm light), targeted regions of the carrier fluid polymerize, immobilizing specific cells within the targeted region of the hydrogel on a microscope slide (or series of slides or containers in a chamber). It will be understood that non-targeted cells that remain in non-targeted non- polymerized regions stay suspended in the aqueous carrier solution and can be easily collected,PATENT 12 CORLA.P001PCT achieving clear separation of the desired subsets (e.g., subsets A and B). Subsequently, immobilized cells can be released from the photopolymer gel through dissolution by the application of a second wavelength different from the first wavelength (for example 505 nm light) and return back into an aqueous solution enabling full recovery of selected cells. It will be understood that related wavelengths (e.g., for the first wavelength 400 nm, 401 nm, 402 nm, 403 nm, 404 nm, 406 nm, 407 nm, 408 nm, 409 nm and for the second wavelength 500 nm, 501 nm, 502 nm, 503 nm, 504 nm, 506 nm, 507 nm, 508 nm, and 509 nm) may be used without departing from the scope and spirit of the present invention. Additionally as noted herein, other wavelengths may be used such as 350 nm or 410 nm as noted.

[0064] Discussion of the process and preparation of the carrier fluid: Referring now to Figs. 1 and 2, wherein Fig. 1 is a graphical process illustration representing an exemplary selective photopolymerization (by encapsulation of target cells in a flow cell) and depolymerization of a carrier fluid within a flow cell, according to the present invention, and Fig. 2 is a further graphical process illustration designating the arrangement of the monomer units bonding to each other and photopolymerizing when induced with specific wavelengths of light according to the present invention.

[0065] [[introducing chem / com / poly]] As noted in the illustrated processes, and further informed herein and below, the proposed photopolymerizing carrier fluid is an aqueous suspension of monomer units that bond to each other when induced with specific wavelengths of light. The aqueous component is pH buffered between pH 7.4 and 8.0. The suspended monomer is a 135.2 kilodalton (kDa) construct composed of one 20 kDa 4-armed PEG reagent (Creative PEGWorks, Inc., PSB-455, provided by www.creativepegworks.com which is a 4-armed PEG-MAL (a multiarmed PEG derivative with maleimide groups) that has been functionalized with a protein derived from the 22G, each roughly 28.8 kDa. This protein dictates what wavelengths of light are used to polymerize or depolymerize (e.g., protect and unprotect) the carrier fluid. In the case of this 22G derived protein, 405 nm light is used to induce polymerization and 505 nm light is used to dissolve polymerized carrier fluid.

[0066] Additionally and optionally, the present method and system may adaptively include a photocatalyst (add a small molecule for light absorption) to increase the precision, depth, uniformity, and completeness of polymerization. As discussed herein, light entering a flowcell may scatter, and adding a broad-spectrum absorption nano-particles or molecules (photocatalystPATENT 13 CORLA.P001PCT such as TiO2or DPMA (Dimethoxy-2-phenylacetophenone) will reduce off-target polymerization in masked areas during exposure to UV / visible light. The photocatalyst absorption molecule has the additional benefit of reducing (and controlling) polymerization depth. It is advantageous for the photopolymer discussed herein to solidify from the bottom up to avoid clogging the upper region of a flow cell (e.g., solidifying or polymerizing from the bottom upward will allow a more uniform result). This will increase perfusion performance of the fluidic system.

[0067] Discussion of the preparation of the carrier fluid: Dronpa145N functionalized PEG- Maleidamide was initially tested as a proof of concept in combination with monomers forming a photo-responsive hydrogel. Applicant also tested the prepared carrier fluid showing that polymerized hydrogel does not dissolve in water unless irradiated with 505 nm light. Beneficially, the Dronpa145N does not generate free radicals and provides a gentle aqueous polymer allowing full-encapsulation of target cells in the photopolymer as regions without concern and without a need for maintaining an aqueous volume around a target cell to avoid damage. This benefit serves to reduce the exposure of cells to free radicals (e.g., full photopolymerization of target cells in a flow cell provides protection against free radicles without the burden of maintaining an aqueous surround) and thereby maintain cell viability.

[0068] Additionally referring now to Fig. 3, in the preparation of the carrier fluid based on Donpra145N, as provided by a GeneArt custom order from ThermoFisher Scientific Corp.(Del), the Donpra145N gene (see Fig.3 sequence box) was inserted into a bBAD / His vector between the BglII and HindIII (see Fig. 3, cleavage site) and was transfected into E.coli K12 DH10B™ T1R (cells provided by Life Technologies Corp., Del). This was provided as a live E.coli culture, and as lyophilized plasmid DNA (pBAD vector).

[0069] Dronpa was then expressed in the provided library strain by growing the E.coli in LB media, with 100 ng / Ml Ampicilin, up to an OD600 reading of ~0.5. The culture was then induced with 0.02% (w / v) L-(+)-Arabinose and incubated for 4 hours at 37°C prior to harvesting.

[0070] The protein laden E. coli was then lysed with B-PER II protein extraction reagent. The protein was purified using a Pierce His Protein Pull-Down kit . The 4 kDa 6xhis tag was cleaved off with Enterokinase, and removed from solution via solvent exchange over a 10K MWCO spin column, which also concentrated the purified protein. The protein concentration was normalized to 100 uM in phosphate buffered saline, pH 7.2, and mixed in a 10 to 1 ratio of protein to PEG- MAL. This reaction was allowed to proceed at room temperature for 2 hours, then transferred to aPATENT 14 CORLA.P001PCT Seedat concentration device (provided by ThermoFisher) and concentrated until a stable gel forms. The hydrogel is then washed several times with deionized (DI) water to remove any non-reactive material.

[0071] Table 1 : Selected supplies provided from sources as follows: Material Supplier Catalogue Number

[0072] Masking and systems used with the carrier fluid in the present proposed invention provide for illumination of targeted regions within a flow cell (or multiple flow cells in a designated illustrative chamber (elsewhere noted as chamber 100) uses a digital mask such as a Liquid Crystal Display (LCD), or spatially controlled digital micromirror devices (DMDs), or precision laser scanning systems. This method captures target cells by blocking specific LCD pixels, or directing light at a specific region, as informed by the optical analysis of cells in that space. The accurate immobilization of targeted cells with 405nm light and efficient collection of mobile cells effectively depletes the sample of cells with targeted features. The polymerization process is selectively reversable. Applying 505 nm light when selectively illuminating the flowcell causes targeted areas to de-polymerize and release any cells contained within the space back to the aqueous state. Cells that were previously immobilized become mobile due to the breakdown of polymer and may be collected. Therefore, a stepwise process can be performed to collect any fraction of cells from the total input, at any time point.

[0073] Exemplary components are noted as follows: Masking was achieved using a SUMAOPAI monochromatic LCD, model number PJ3D925V1, which has a resolution of 5760 by 3600 in a 9.25 inch format. The screens’ pixel pitch of 34.4 um is too large for single flow cellPATENT 15 CORLA.P001PCT resolution (roughly 5 PBMCs fit into one pixel at a one-to-one scale). Accordingly, due to the relatively large pixel size compared to a single cell, the mask is optically shrunk down using a 250 mm condensing lens, and columnated by a 25 mm plano-convex lens (Edmond Optics, stock numbers 67-199 and 45-278). This resulted in a diffraction limited mask, sharing the optical path of an Iris 15 camera (Teledyne Model: 01-IRIS-15-PCIE-M-16-C) to provide visual designation, data, and image recording for quality control and operative control with reporting. This combination of optical paths, enabled by a Longpass Dichroic Mirror (ThorLabs, DMLP550R), enables the device to mask or illuminate any pixel observable in the field of view of the camera.

[0074] The illuminating system requires light to be evenly dispersed, somewhat uniform in brightness, and columnated as it passes through the LCD. For this reason, an Anycubic Photon Mono M3 Plus 3D Printer UV LED Light Source was retrofitted with 2-color LEDs from Lumixtar Electronic Technology Co. (Part Number WL-6P3535EP120C2b1-UVG). The arrangement apparatus was mounted via custom 3D structural printed parts, such that, as illustrated, the LED passed through the Anycubic compound columnating lens, then the LCD, then the 250 mm condensing lens, then the 25 mm plano-convex lens, and is then reflected up by the ThorLabs dichroic mirror, and finally into a Zeiss 40X A-Plan objective optic (Model Number 421060-9900- 000). This ultimately results in a field of views, of roughly 0.5 mm, with an imaging resolution of 5056 x 2960 and a somewhat higher resolution mask. The resolution of the proposed system, both in camera resolution and mask resolution enable the inventive precise morphology based analysis of cells within the field of view of the camera and precise polymer capture. The microscope setup supporting this device was manufactured by Zaber, Inc., based on the Nucleus® MVR inverted fluorescence microscope platform.

[0075] Additionally referring now to Figs.4, 5, 6, and 7, wherein Fig.4 provides an illustrative view of synthetic DNA production, regarding nucleotide (NTP) additions as noted herein and above, wherein the system sought to build DNA oligomers one nucleoside at a time, starting from a functionalized surface that enables the oligomer to remain attached to the internal surface of the flow cell. The addition of a nucleotide can be blocked by polymerizing a small volume within the larger volume. The polymer is impenetrable to terminal transferase, which is an enzyme that can be used to add a nucleotide to an oligomer. The areas of the flowcell, or multiple flow cells, which are not protected by polymerization are exposed, and chemistry processing can proceed. In the case of organic synthesis with terminal transferase, the reaction can be quenched by the additionPATENT 16 CORLA.P001PCT of Ethylenediaminetetraacetic acid (EDTA). Once the reaction has extended the oligomer and the reaction has been quenched, the polymer can be dissolved by addition of DTT. Dissolving the polymer clears the device state and enables a new round of polymerization to protect a new set of positions. In this way cycles of nucleotide (NTP) additions enable the device to add exactly one NTP per round to the selected positions. For oligo synthesis using the 4 canonical bases (A, T, G, C), extending all oligomers by one NTP would require 4 cycles – each cycle involving one of the 4 NTPs.

[0076] Additionally referring additionally now to illustrative Figs. 5 and 7, wherein the illustrative system 700 provides an application space for cell sorting, wherein cells must be loaded into the flowcell in a monolayer. This is typically done by diluting the population to achieve a dispersed poisson distribution and allowing the cells to settle due to density. Non-adherent cells may require some surface treatment to remain in place on the flowcell (Poly-L-Lysine). The monolayer of cells can be observed and assessed via a variety of metrics (a common library for this is Cell PoseTMor skimage regionprops). When a cell of interest is visually identified, the respective coordinate or address can be established by referencing the xy-stage encoder and converting pixel space in the image to coordinates and encoded in a masking device. When the coordinate of the cell of interest is known, the space can be polymerized to hold the cell in place. A wash buffer can then flow through the flowcell, intended to carry away cells that are not blocked by polymer. After the wash, the target cells can be freed from polymer enclosures by dissolving the material with DTT or other reagent capable of dissolving the crosslinker. This sorting mechanism acts on all cells simultaneously within system 700 for the method noted herein.

[0077] Additionally referring additionally to Figs. 6, 7, in combination, the current method, system, and apparatus or device for reversible mechanical volumetric protection and deprotection addresses several of the present concerns. This system enables precise control over the protection and deprotection process through the photo-catalyzation of polymer monomers into polymer blocks as directed by precise light exposure, and dissolving polymerized blocks via degradable polymer crosslinkers.

[0078] The system comprises a method that selectively exposes an aqueous volume to the appropriate wavelength of light with sufficient intensity to photo-catalyze polymer monomers in the aqueous reagent. The polymerization results in inclusions of materials that are then protected from the unpolymerized volume. Subsequent solvent exchange is excluded from the polymerizedPATENT 17 CORLA.P001PCT volume. In this way, unpolymerized areas can be exposed to reagents while polymerized zones are protected. The polymer can be degraded and removed by the addition of chemicals that break polymer cross-linkers. This results in resetting the features, exposing the entire surface area to the aqueous volume. This process can be done cyclically, allowing any address in the volume to be protected and unprotected as needed. This device can be finely controlled to ensure accurate and reversible protection, making it ideal for applications in synthetic DNA production, single cell manipulation, pharmacological discovery, and cell-free systems. The system enhances efficiency, reduces processing time, and maintains the integrity of the biological samples.

[0079] Referring additionally to Figs. 7, and 8-10 a system 700, and method 100, for the proposed method for reversible mechanical volumetric protection (photopolymerization of target cells) and deprotection (de-photopolymerization of target cells) includes the following components and steps as discussed herein. As generally noted in Fig. 8, descriptively steps are discussed as follows: An initial Step A wherein cells are mixed with a carrier fluid to form an aqueous cell slurry, a further Step B wherein the cell slurry is transferred to the input well of a flow cell and placed into the reaction chamber 100 of system 700, a further Step C conducted on the stage 100 where electronic control unit 500 operates the related systems and units to determine target cells, and to conduct photopolymerization steps; and a later, following Step D, where the non- polymerized carrier fluid is washed away removing the nontarget cells from the output well of the flow cell 300.

[0080] A reaction chamber 100 that may be within an auto sampler 701 having a reagent supply 200: Reaction chamber 100 is designed to hold the biological sample in one or more flow cells 300. For some applications, surface treatments may be optionally applied to fix the biological samples to the chamber with a stage 101. The chamber 100 is operative linked with an auto- sampler 701 that can deliver a variety of reagents from a reagent supply 200, including aqueous polymer components, and is operative for application specific reagents to carry out chemistry, and wash buffers to dissolve polymer blocks as is discussed herein. There is a waste opening (not illustrated) in chamber 100 connected to a waste container (not illustrated) to collect material that has passed through the chamber. A bypass valve (not shown) may be added to the outlet for reaction chamber 100 to enable collection of material that ought to be separated or collected for additional processing.PATENT 18 CORLA.P001PCT

[0081] Electronic Control Unit (ECU) for system 700: A system controller and electronic control unit (ECU) 500 having a process control unit (shown) controls light exposure on the reaction chamber 100 and operates related apparatus and system 700 in controlled manners, with user inputs as will be discussed herein. This can be done through digital masking with a monochromatic liquid crystal display (LCD) with a masking device 401, dynamic light projection, or mechanical rastering of an illumination source 402. Electronic control unit 500 (also electronic system control 500) further includes processing systems 501 for recording image data, event data, and operational data and reporting systems 502 for summary delivery of result reports and verification of quality controls. Electronic control unit 500, in combination with processing systems 501 and reporting systems 502 allows operative management of system 700 and the elements and systems therein during the method as will be noted herein allowing for the method, the high-resolution imaging with inline verification, and the selective immobilization and recovery of viable cells via reverse photopolymerization. A visualization system 400 with a camera system 401A, mirror element, and a microscopic objective system 401B that allows a zoom-in view of cell slurry in a flow cell with mask overlay (as shown in Fig. 9). Visualization system 400 identifies cells based on morphological or fluorescent features and directs the illumination pattern via the electronic control unit 500 and masking device 401 in combination with other elements herein. After photopolymerization, follow-up imaging is used to confirm that the desired target cells were successfully retained or excluded, enabling repeat processing, quality control, and operation and production at single-cell resolution.

[0082] Polymerization Mechanism: Polyacrylamide is a common polymer used in biological applications. The polymerization of polyacrylamide can be catalyzed by free radicals, which can be introduced by exposing TiO2 nanoparticles or Dimethoxy-2-phenylacetophenone (DMPA) to UV light. Any other suitable polyacrylamide suitable for the intended method, system, device, and apparatus may be used herein without departing from the scope and spirit of the present invention.

[0083] Depolymerization Mechanism: Using N,N′-Bis(acryloyl)cystamine cross links acrylamide with thiol bonds that can be degraded by Dithiothreitol (DTT). Addition of a chain length terminator, like sodium formate, assists in the degradation speed and allows the polymer to be broken down into smaller moieties. The present invention envisions other chain length terminators that may be adaptive used without departing from the scope and spirit of the present invention.PATENT 19 CORLA.P001PCT

[0084] Sensors and Feedback System: A range of sensors and feedback systems in a designated sensor system 600 may be provided interlinking the operative elements of system 700 depending on the application. For DNA synthesis, the system 700 can progress through a series of states without deviating from the preprogrammed series controlled by system controller / electronic control unit 500 – and no sensor feedback is required. To operate on select cells in system 700 on flow cell 300, a computer image feedback conducted by electronic system control 500, in combination with other steps, is needed to identify targets of interest and trigger a program decision tree based on its state and position stored within electronic system control 500. It is envisioned that the computer vision feedback devices 400, masking device 401, light source 401, and system controls 500 includes all suitable devices, sensors, linking systems and devices, programing code and operational systems suitable for such steps without departing from the scope and spirit of the present invention.

[0085] Referring additionally further to Fig. 10, in combination with the system 700 inclusive herein, and the related discussions and disclosures, an exemplary method of precise cell selection via reversible light-induced photopolymerization 1000 is provided. Method 1000 is operative with system 700 and includes, at an initial but optional step 1001 placing pre-packaged reagent supplies 200 in device 700 with operative autosampler 701 and a mixing step 1002 of mixing cells with a carrier fluid to form an aqueous monomer cell slurry. A step 1003 transfers the cell slurry to one or more flow cells 300 having input wells and output wells, which may, in an alternative step 1003A include loading one or more flow cells into a reservoir for multiple flow cells in device 700 for operation of method 1000 across multiple flow cells. In step 1004, which follows either or both of step 1003 and 1003A, the one or more flow cells are in the system 700, and positioned on stage 101 for further analysis. As discussed previously electronic control unit 500 with integrated sensors, visualization system 400 with related elements conducts a visualization step 1005 of visualization of the target cells in the flow cell(s) and determination of identifiable features, stores requisite initial data, and transmits instructions to masking device 401 that conducts a step of masking 1006 target regions, following by an illumination step 1007 of non-masked non-targeted regions with flow cells(s) by a first wave length(s) causing an immobilization step 1008 by photopolymerization / protection of the non-masked targeted regions of the flow cell(s) (and the cells therein) as discussed herein. In a following step of washing 1009, the flow cell is washed and the mobile suspended nonpolymerized (masked) targeted region / cells are washed from thePATENT 20 CORLA.P001PCT output wells of flow cell(s) 300 for separate storage and analysis in system 700. Steps 1005-1009 may be conducted in repeated series until system 700 and visualization image analysis confirms that mobile cells suspended in solution are removed from the flow cell. In a second illumination step 1010 an illumination of the previously photopolymerized immobilized target region(s) / cells in the flowcell(s) are targeted with a second wavelength that is different from the first wavelength causing a remobilization step 1011 (a depolymerization / deprotection) of previously photopolymerized / protected / immobilized cells returning into an aqueous state. It will be recognized that steps 1005, and subsequent imaging and data analysis steps 1005A may adaptively and selectively target cells in masking step 1006 for initial photopolymerization by illumination step 1007 with the first wavelength and to arrange for operation of masking device 401 to separately target or additionally target or re-target the targeted regions within flow cell(s) for exposure to the second wavelength. In a further wash step 1012, a wash is conducted to remove the remobilized cells from the output well(s) from the flow cell(s) 300. As indicated by the process arrows regarding steps 1005-1012, and linking with visualization step 1005 and related imaging and data analysis step 1005A, the method 1000 may be conducted in one or more serries and verification steps to cause complete separation for inline verification and reporting in a step 1013 or a series of steps, so that a method for selective immobilization and recovery of selected viable cells from a heterogenous carrier fluid may be achieved. As will be further understood, based upon a designation of a target region in step 1006 a first-wash of the flow cell may separate desired or un-desired mobile cells, depending upon a user’s intended step (such that a first-step of masked or unmasked may separate the cell slurry based on one or more cellular features (including morphological features and fluorescent features) in either desired order (first or second)).

[0086] As will be noted, a programmable logic controller (PLC) or integrated industrial computer system is within electronic system controller 500 (e.g., one or more PLC controller system(s) 500 in related visualization system(s) 400, masking device 401, and data and reporting systems 501, 502) and operation are illustrated with generic input controls such as a keyboard, hand mouse controller, and graphic display (monitor) (all as shown) and is provided inter operably with data connection links (illustrated but not numbered) throughout system 700.

[0087] The discussion will now review selected additional features and details that will be appreciated by those of skill in this art having studied and understood the complete disclosure.PATENT 21 CORLA.P001PCT

[0088] De-Risking Methods: It will be understood that free radicals like O2⁻, H+, and -OH can damage organic material. As a result of the present invention, it is recognized that this risk can be mitigated by adding Superoxide Dismutase (SOD), and Catalase enzymes to the aqueous solution. These enzymes are specific to O2⁻ and H2O2. This is understood to slightly reduce the effectiveness of Dimethoxy-2-phenylacetophenone (DMPA), but the resulting free radicals of the homolytic cleavage of DMPA are larger molecules that are not suitable substrates for these enzymes. Glutathione Peroxidase (GPx) may be less effective due to non-specific actions. Those of skill in the art having studied and appreciated the present disclosure will recognize that other de-risking methods will be understood as within the scope of the invention during the preparation of the carrier fluid and the cell slurry.

[0089] As discussed herein, according to the present invention, the method and process begins by placing the biological sample into the flow cell of a designated chamber. The sample is suspended in an aqueous volume, and it may interact with the surface of the chamber or related structures by settling due to density differences, or may stochastically interact due to Brownian motion. Alternatively, the flow cell or chamber may mechanically constrain the sample vertically, and have a large length and width causing constraints, so that the sample is in range of the polymerization mechanism. The control unit 500, visualization system 400, and masking device 401, then exposes selected areas to light, which activates the polymerization mechanism. This mechanical protection can be finely adjusted via exposure time or exposure area to ensure optimal protection without damaging the sample.

[0090] To cause depolymerization (e.g., to ‘deprotect’) in one alternative and adaptive sample, the system controller 500 prompts the auto-sampler 701 to begin solvent exchange with a buffer containing DTT or other chemical solvent, thereby dissolving all polymers in the flow cell and reversing the protection process. It is worth noting that photodegradable linkers, like Nitrobenzyl (NB) based linkers, can be used to degrade the polymer and polymerization process. In a photodegradable example, a 2nd wavelength of light may be used to control degradation. For example, a 410 nm light can be used to polymerize the monomers, and a 350 nm light could be used to separately degrade and break down the polymer. It will be understood by those of skill in the art having studied the present disclosure that variations of the control unit, programing parameters, light filters, light applications and linkers etc., will be within the scope of the present invention.PATENT 22 CORLA.P001PCT

[0091] The proposed integrated system 700 and method 1000 are particularly advantageous in high-throughput settings and applications requiring repeated cycles of photopolymerization and depolyermization (e.g., protection and deprotection) of targeted cells in a flow cell. The system and methods herein offer a high degree of precision and repeatability that may rely on commonly available consumer electronics like LCD panels. The number of features on a device is determined by the number of pixels on the LCD panel, or the raster accuracy of dynamic light projection. This cost effective method of selective protection enables the device to control millions of discrete features, making it a valuable tool in various biotechnological applications.

[0092] In view of the above, those of skill in the art, having studied and appreciated the above disclosure, will further recognize that adaptations and alternatives may be readily extrapolated including, but not limited to: i. Adaptive applications in non-aqueous environments with hydrophobic polymers ought to be feasible. ii. Multiple polymers may be used to improve performance with diverse properties being acted upon by a variety of interfaces or radiation wavelengths. iii. Multiple degradable cross linkers that can be selectively acted on by compounds in solution or external inputs, such as radiation of multiple wavelengths, can modulate the properties of the polymer for performance advantages. iv. Use of photocatalysts that can be acted on by various forms and modalities of radiation including visible, non-visible, or energetic particles which can be additionally inclusive to the present inventive system and method. v. Aqueous partitions in non-aqueous media, i.e. an emulsion, may be manipulated by dynamic polymer generation and degradation in multiple steps (e.g., photopolymerization and depolymerization in portions or along target cells or for other purposes such as creating partitions for further processing of target cells. vi. Physical structures may be dynamically generated to affect fluidic behaviors within the flowcell.PATENT 23 CORLA.P001PCT

[0093] Additionally, in view of the above; those of skill in the art, having studied and appreciated the above disclosure, will further recognize that adaptations and alternatives modes of exposing a liquid volume of a flowcell to light may be readily extrapolated including: i. The use of dynamic light projection to selectively expose the substrate to light across the entire flowcell or with a field of view from the perspective of an optical system. ii. Rastering lasers or other masking systems may expose a volume of a flowcell, given that the gaussian distribution of the light can be arbitrarily large or small, and will be directed at the specified volume within a flow cell so that a multiple heterogenous cells of multiple types may be separated using the proposed method and system herein. iii. The mode of the laser may be altered to achieve a variety of practical differences in the behavior of the polymer, including applying a gradient of parameters across the space of the flowcell or over the time of the experiment. iv. The excitation and / or emission of substrate or additives may be interpreted as data, as well as bright field - or other image modalities. v. The data acquired from the system may be immediately actionable and used to improve the performance of the system in situ (during the process), or in a longer term and persistent manner to accumulate deep and high- dimensional data for post processing, quality control and verification. vi. The systems and methods herein may operate on live cells and therefore may additionally include thermal controls, buffers and solvent exchanges, dissolved gas systems, and other engineering controls appropriate for the long-term viability of living cells.

[0094] According other aspects of the present invention, there is provided a goal to selectively block or unblock chemistry from occurring within an aqueous volume by the use of photopolymerization. Generally, it will be understood that the present method and system provides a physical block (polymerization) that may be dissolved (depolymerized) and this may happen in cycles, where: i. A flowcell has spots selectively blocked;PATENT 24 CORLA.P001PCT ii. Then chemistry is applied in bulk; iii. The chemical agents are washed away; iv. The polymer blocks are dissolved away with a buffer; and v. The steps may be repeated.

[0095] The proposed method and system with operative device selectively masks a surface on a volume containing photopolymerizable volume and may optionally involve the use of a mSLA 3D printer and related devices known to those of skill in the art.

[0096] Adaptively, the proposed system and method may additionally include chemistry used with adaptive features: such as (i) the photocurable polymer is polyacrylamide (which is biocompatible), but other polymers may also be used; (ii) The polymer uses biscyctamine as a crosslinker, which allows the polymer to be dissolved by DTT or just low pH (reducing the thiol). Other methods of dissolving the polymer are possible including photo-degradable crosslinkers, and (iii) The proposed photocatalyst is TiO2, which makes the polymer tend to solidify from the bottom up.

[0097] In summary, the device, apparatus, and system operates with within the scope and spirit of the present invention and the skill of those in this art regarding electronics, chemistry, computer science established methods and existing data tools. The improvement further includes, without limitation, the combination of photopolymerization, degradation, and applying the system to selectively apply chemistry.

[0098] The proposed system, method, and device has many adaptive applications as will be appreciated by those of skill in the arts, and these include: 1) Synthetic DNA synthesis. i. DNA can be synthesized by using a Terminal Transferase (TdT) enzyme conjugated with a nucleotide, wherein a proposed polymer plugs will exclude the enzyme conjugate from accessing the growing strand to block the bulk nucleotide identity from contributing to that strand. In other places the lack of a plug will allow chemistry. This would be one addition round that would add a nucleotide, like adenine, to the available strands. The next round would have a new mask applied, and a different nucleotide, like thymine, will be added. In this way, the complex pool of oligomers willPATENT 25 CORLA.P001PCT grow according to the preprogrammed set of masks and result in a pool of oligomers that match the intended identities. 2) Cell sorting ii. Cells can be flowed into the system suspended in the monomeric form of the polymer. Cells settle a short distance to rest on the bottom surface of the flowcell. A visualization system captures the position of cells on the system and data is used to select where polymerization can capture target cells. The non-captured cells are washed from the system. The polymer is dissolved, then the released cells flow off to a new collection site. 3) Pharmaceutical testing panels iii. Target cells can be fixed to the flowcell as described above. An alternative chemistry that enables photodegradation can be used to unblock a single cell or a set of cells, exposing it to the selected test conditions. A visualization system can be used for data collection, and the target cells can flow off in selective cohorts to enable downstream analysis.

[0099] As will be further understood by those of skill in the art, the integrated data, as discussed herein, will include but is not limited to the sensor and visual data, recorded image data, analysis image data, cycle and operation system data, high-resolution imaging with light-induced cell selection and in-line verification data, wavelength and operational mode data, and other system data to allow controlling method calculations via controlling programs through that operate the method and operative components noted herein.

[0100] Another optional and adaptive embodiment of the present invention which will be appreciated by those of skill in this art having understood the entire invention, is operation of moisture removal and concentration for moisture rich products. The present invention offers precise and rapid moisture reduction by boiling under vacuum. It prevents overcooking, preserves desirable texture and flavors, and significantly reduces undesired browning and burning.

[0101] Those of skill in the related arts will understand that the linguistic use of phrases such as polymerization and depolymerization (as related to ‘protection’ or ‘immobilization’ of target cells and ‘deprotection’ or ‘mobilization’ of target cells for recovery of such target cells within a hydrogel related system) as discussed herein are understood in the context of the present method, apparatus, and systems as discussed herein and not in unrelated systems.PATENT 26 CORLA.P001PCT

[0102] The above disclosure is sufficient to enable one of ordinary skill in the art to practice the invention, and provides a mode of practicing the invention. While this is a full and complete disclosure of the preferred embodiments of this invention, it does not limit the invention to the exact construction, dimensional relationships, and operations shown and described. Various modifications, alternative constructions, changes and equivalents will readily occur to those skilled in the art and may be employed as suitable, without departing from the true spirit and scope of the invention. Such changes might involve alternative materials, components, structural arrangements, sizes, shapes, forms, functions, operational features or the like.

[0103] As an example, it will be understood that system electronic control unit 500 may be any combination of program logic controllers (PLC) or industrial computers each of which will include all needed route programing, memory (fixed and transitory) input controllers and display features necessary to operably conduct method 1000 disclosed herein for system 700. As non- limiting examples related keyboards, sensor I / Os, program chips (individual or a plurality) and related updates for such systems as will be understood from the art.

[0104] Throughout the disclosure above, the terms "top", "bottom", and "side" are used throughout in the discussion of certain devices, systems, or steps. In this context, and for purposes of this disclosure, these terms are descriptive, for the aid of those of skill in the art having studied and appreciated the disclosure and are not to be taken literally; and therefore for example, should a system, apparatus, device, or otherwise be physically inverted such that a ‘bottom’ becomes a ‘top’ the understanding will remain to the person of skill in this art as described.

[0105] The expressions such as "include" and "may include" which may be used in the present disclosure denote the presence of the disclosed functions, operations, and constituent elements, and do not limit the presence of one or more additional functions, operations, and constituent elements. In the present disclosure, terms such as "include" and / or "have", may be construed to denote a certain characteristic, number, operation, constituent element, component or a combination thereof, but should not be construed to exclude the existence of or a possibility of the addition of one or more other characteristics, numbers, operations, constituent elements, components or combinations thereof.

[0106] As used herein, the article "a" is intended to have its ordinary meaning in the patent arts, namely "one or more." Herein, the term "about" when applied to a value generally means within the tolerance range of the equipment used to produce the value, or in some examples, meansPATENT 27 CORLA.P001PCT plus or minus 10%, or plus or minus 5%, or plus or minus 1%, unless otherwise expressly specified. Further, herein the term "substantially" as used herein means a majority, or almost all, or all, or an amount with a range of about 51% to about 100%, for example. Moreover, examples herein are intended to be illustrative only and are presented for discussion purposes and not by way of limitation.

[0107] As used herein, to "provide" an item means to have possession of and / or control over the item. This may include, for example, forming (or assembling) some or all of the item from its constituent materials and / or, obtaining possession of and / or control over an already-formed item.

[0108] Unless otherwise defined, all terms including technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure pertains. In addition, unless otherwise defined, all terms defined in generally used dictionaries may not be overly interpreted. In the preceding, details are set forth to provide a more thorough explanation of the embodiments. However, it will be apparent to those skilled in the art that embodiments may be practiced without these specific details. In other instances, well- known structures and devices are shown in block diagram form or in a schematic view rather than in detail in order to avoid obscuring the embodiments. In addition, features of the different embodiments described hereinafter may be combined with each other, unless specifically noted otherwise. For example, variations or modifications described with respect to one of the embodiments may also be applicable to other embodiments unless noted to the contrary.

[0109] Further, equivalent or like elements, systems, or components with equivalent or like functionality are denoted in the preceding description with equivalent or like reference numerals. As the same or functionally equivalent elements are given the same reference numbers in the figures, a repeated description for elements provided with the same reference numbers may be omitted. Hence, descriptions provided for elements having the same or like reference numbers are mutually exchangeable.

[0110] It will be understood that when an element is referred to as being "connected" or "coupled" to another element, it can be directly connected or coupled to the other element or intervening elements may be present. In contrast, when an element is referred to as being "directly connected" or "directly coupled" to another element, there are no intervening elements present. Other words used to describe the relationship between elements should be interpreted in a like fashion (e.g., "between" versus "directly between," "adjacent" versus "directly adjacent," etc.).PATENT 28 CORLA.P001PCT

[0111] In the present disclosure, expressions including ordinal numbers, such as "first", "second", and / or the like, may modify various elements. However, such elements are not limited by the above expressions. For example, the above expressions do not limit the sequence and / or importance of the elements. The above expressions are used merely for the purpose of distinguishing an element from the other elements. For example, a first box and a second box indicate different boxes, although both are boxes and may be understood to function as a combined box or a combined step. For further example, a first element could be termed a second element, and similarly, a second element could also be termed a first element without departing from the scope of the present disclosure as would be understood by one of skill in this art having appreciated the enclosed description.

[0112] A sensor refers to a component which converts a physical quantity to be measured to an electric signal, for example, visual image signal to a data-signal for graphical illustration purposes, or a current signal or a voltage signal. A physical quantity may, for example, comprise electroimagry of cellular representations (e.g., photons received of light passing through a flow cell from an LCD), or a sensed radiation, magnetic field, or electric field, that a process controller (PC) converts to data field under operating control software (e.g., program) to display visual images, graphic results, or count-indicators.

[0113] Use of the phrases "capable of," "capable to," "operable to," "configured to," or "programmed to" in one or more embodiments, refers to some apparatus, logic, hardware, and / or element designed in such a way to enable the use of the apparatus, logic, hardware, and / or element in a specified manner. The subject matter of the present disclosure is provided as examples of apparatus, systems, methods, circuits, and programs for performing the features described in the present disclosure. However, further features or variations are contemplated in addition to the features described above. It is contemplated that the implementation of the components and functions of the present disclosure can be done with any newly arising technology that may replace any of the above-implemented technologies.

[0114] Also, the inventors intend that only those claims which use the specific and exact phrase "means for" are intended to be interpreted under 35 USC 112. The structure, device, and arrangement herein is noted and well supported in the entire disclosure. Moreover, no limitations from the specification are intended to be read into any claims, unless those limitations are expressly included in the claims.PATENT 29 CORLA.P001PCT

[0115] Having described at least one of the preferred embodiments of the present invention with reference to the accompanying drawings, it will be apparent to those skills that the invention is not limited to those precise embodiments, and that various modifications and variations can be made in the presently disclosed system without departing from the scope or spirit of the invention. Thus, it is intended that the present disclosure covers modifications and variations of this disclosure provided they come within the scope of the appended claims and their equivalents.

[0116] Although only a few embodiments have been disclosed in detail above, other embodiments are possible and the inventors intend these to be encompassed within this specification. The specification describes certain technological solutions to solve the technical problems that are described expressly and inherently in this application. This disclosure describes embodiments, and the claims are intended to cover any modification or alternative or generalization of these embodiments which might be predictable to a person having ordinary skill in the art.

[0117] Therefore, the above description and illustrations should not be construed as limiting the scope of the invention, which is defined by the claims set out herein.

Claims

PATENT 30 CORLA.P001PCT WHAT IS CLAIMED IS:

1. A method, for cell selection via reversible light-induced polymerization, comprising the steps of: preparing an aqueous cell slurry containing a heterogenous group of cells in a photopolymerizable carrier fluid; the heterogenous group of cells having identifiable cellular features including morphological features or fluorescent features; transferring the cell slurry to a flowcell; visualizing the flow cell with a visualization system; designating a targeted region and a nontargeted region of the flow cell based upon the identifiable cellular features of the heterogenous group of cells in the cell slurry visualized by the visualization system; masking the targeted region of the flow cell with a masking device and preventing a light- induced photopolymerization of the masked targeted region thereby maintaining a mobility of cells in the cell slurry of the targeted region of the flowcell; illuminating the unmasked nontargeted regions of the flow cell with a first wavelength of light causing a light-induced polymerization and immobilization of the cell slurry in the unmasked nontargeted regions; washing the flowcell and collecting mobile cells remaining in the cell slurry from the masked targeted region that are not light-induced polymerized; illuminating the unmasked nontargeted regions of the flow cell with a second wavelength of light causing a light-induced depolymerization and remobilization of the cell slurry in the unmasked nontarget region; and washing the flowcell and collecting remobilized cells from the unmasked nontargeted region of the flow cell.

2. The method, according to claim 1, wherein: the second wavelength of light is different than the first wavelength of light.

3. The method, according to claim 1, wherein: the first wavelength of light includes illumination with a 405 nm light; and the second wavelength of light includes illumination with a 505nm light.

4. The method, according to claim 1, wherein:PATENT 31 CORLA.P001PCT the carrier fluid includes a multiarmed PEG derivative with malemide groups functionalized with a protein prepared with Donpra145N.

5. The method, according to claim 1, wherein: the carrier fluid includes a photocatalyst.

6. The method, according to claim 1, wherein: the masking device is selected from one of a Liquid Crystal Display (LCD), a spatially controlled digital micromirror device (DMDs), and a precision laser scanning system.

7. The method, according to claim 1, wherein: the step of transferring the cell slurry to a flowcell further comprises: placing the flowcell in a chamber of an autosampler; and supplying the autosampler with access to a reagent supply.

8. The method, according to claim 1, wherein: the step of visualizing the flow cell with the visualization system, further comprises: designating a plurality of targeted regions and a plurality of nontargeted regions of the flow cell of the flow cell.

9. The method, according to claim 1, further comprises: repeating the steps of: visualizing the cell slurry in the flowcell, designating one or more targeted regions and nontargeted regions; masking the one or more targeted regions; illuminating the one or more nontargeted regions with the first wavelength; washing the flowcell and collecting mobile cells that are not light polymerized; illuminating the one or more nontargeted regions with the second wavelength; and washing the flowcell and collecting remobilized cells, whereby multiple cycles of the method allow for multiple cell selections from said heterogenous group of cells.

10. The method, according to claim 1, wherein: the first wavelength of light includes illumination with a wavelength selected from the wavelengths from 400 nm to 409 nm.

11. The method, according to claim 1, wherein: the second wavelength of light includes illumination with a wavelength selected from the wavelengths from 500 nm to 509 nm.PATENT 32 CORLA.P001PCT 12. The method, according to claim 1, wherein: the visualization system, further comprises: a controllable light source; a microscopic objective system; and at least one of a compound columinating lens, a plano-convex lens, and a dichroic mirror.

13. The method, according to claim 12, wherein: the visualization system, further comprises: a camera system in electronic communication with an electronic control unit receiving a visual image from said visualization system.

14. The method, according to claim 13, wherein: the flow cell is a plurality of flowcells in the chamber; and the step of transferring the cell slurry to the flowcell further includes a step of transferring the cell slurry to the plurality of flowcells.

15. A system, for conducting a method of cell selection via reversible light-induced polymerization, according to claim 1.

16. A system, for conducting a method of cell selection via reversible light-induced polymerization, comprising: a flowcell containing an aqueous cell slurry with a heterogenous group of cells in a photopolymerizezable carrier fluid; the heterogenous group of cells having identifiable cellular features including morphological features or fluorescent features; a visualization system for visualizing the aqueous cell slurry; the visualization system designating a targeted region and a nontargeted region of the flow cell based upon the identifiable cellular features of the heterogenous group of cells in the cell slurry visualized by the visualization system; a masking device that masks that masks the targeted region of the flow cell and prevents a light-induced photopolymerization of the masked targeted region thereby maintaining a mobility of cells in the cell slurry of the targeted region of the flowcell; a wavelength emitter that illuminates the unmasked nontargeted regions of the flow cell with a first wavelength of light causing a light-induced polymerization and immobilization of thePATENT 33 CORLA.P001PCT cell slurry in the unmasked nontargeted regions; whereby a washing of the flowcell collects mobile cells remaining in the cell slurry from the masked targeted region that are not light-induced polymerized; the wavelength emitter that illuminates the unmasked nontargeted regions of the flow cell with a second wavelength of light causing a light-induced depolymerization and remobilization of the cell slurry in the unmasked nontarget region; and whereby a washing the flowcell collects remobilized cells from the unmasked nontargeted region of the flow cell.

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