How to select cells

Abstract

A method is provided for selecting cells and / or portions of cells in an EWOD or oEWOD device based on at least one characteristic, the method comprising the steps of: i. providing a test panel of microdroplets comprising at least culture medium or culture medium and at least one cell; ii. providing at least one reporter panel of microdroplets containing one or more reporter entities; iii. merging the test panel microdroplets with at least one reporter panel microdroplet to form a panel of merged assay microdroplets; and iv. monitoring the panel of assay microdroplets with a detection system capable of detecting a change in said reporter entity based on the presence of at least one property; and v. selecting a subset of microdroplets from the panel based on the change in reporter entity, said subset containing the selected cells; Equipped with The test panel of microdroplets comprising at least medium is made by dividing a panel of microdroplets containing at least one cell and medium, and further creating a reference panel of microdroplets containing at least one cell; and / or the test panel of microdroplets comprising at least one cell is further divided, before or after any of steps (i)-(v), into at least two panels of microdroplets, the two panels being a test panel of microdroplets comprising at least medium or medium and at least one cell, suitable for merging with the microdroplets of the reporter entity panel, and a reference panel of microdroplets containing at least one cell.

Description

[Technical Field]

[0001] The present invention relates to a method of using a device to select cells based on desirable properties. These selected cells can then be scaled up for therapeutic or manufacturing applications. This method is useful for adoptive cell therapy (ACT), particularly cellular immunotherapy, but also for other applications, particularly characterization of cells from a population, generally when the cells are genetically engineered. Furthermore, this method can also be used to select cells based on their ability to produce desired molecules, such as immunoglobulins or immunotherapeutic drugs. Synthetic biology is a rapidly developing field in which cells can be used to produce medicines, fuels, food, and beverages, or to produce enzymes that degrade plastics and other pollutants. The device of the present invention is in the field of microfluidics and relates to a microfluidic device capable of manipulating small droplets. [Background technology]

[0002] Rapid, high-throughput processing of cells is a significant challenge in research, development, and manufacturing. This is particularly true for the generation of biological therapeutics (biologics), such as cell therapies and cell-derived therapeutics, which require cell identification and selection while also needing to preserve cells for further expansion or testing. Existing methods for sorting, assaying, and selecting cells remain slow and cumbersome, despite the development of microfluidic devices.

[0003] As approvals for cell-based therapies increase, it is of utmost importance to ensure that the cells infused into patients have the desired characteristics and do not have any characteristics that could pose any risks to the patient. For many diseases, including genetic disorders of the immune system, hemoglobinopathies, metabolic disorders, and cancer, cells removed from the patient are genetically engineered (gene therapy) and then infused back into the patient. Another known application of cell-based gene therapy is for diseases and disorders of the eye.

[0004] In some cases, T cells are genetically engineered to contain a cell surface receptor known as a chimeric antigen receptor (CAR). These CAR-T cells have the important advantage of being able to bind to cancer cells via their expressed cell surface markers and elicit specific antitumor responses. While currently approved treatments involve autologous reinfusion of T cells, the use of allogeneic T cells is also possible in the future. However, it is crucial to use CAR-T cells with a stable phenotype, expressing the correct CAR on their surface without modification, and ensuring that these CARs do not exhibit "on-target / off-tumor" or even "off-target" binding, such as targeting of healthy cells in the patient. Additionally, it is beneficial to determine the efficacy of CAR-T cells prior to infusion. The two currently approved CAR-T cell therapies have numerous known deficiencies related to dosage consistency, patient safety, and the impact of the manufacturing process. These deficiencies are understood to be shared across the fields of adoptive cell therapy and gene therapy.

[0005] Important underlying factors include: Extreme variability in yields across treatments and manufactured doses makes efficacy in patients unpredictable. Known off-target effects (e.g., CNS off-targets and cardiac targets) result in serious side effects in some patients. The variability in yields from leukocyte depletion requires maximal cell viability and careful selection of input cell subpopulations. · Contaminants (e.g., B cells) can ruin the culture. · The lack of a simple, automated method to measure T cell activity against tumor targets makes it difficult or impossible to predict patient response.

[0006] Additionally, cells can be employed to create or manufacture products for many uses, including therapeutic indications, assays, research, food and beverage, agriculture, and as raw materials, effectively using cells to produce products that are harvested from the cells as secreted products, such as immunoglobulins, or by lysing the cells to recover intracellular products, such as drug molecules.

[0007] Of particular interest is the production of immunoglobulins from hybridomas and lymphocytes. It is often desirable to identify cells that produce immunoglobulins with specific antigen-binding capabilities, so that the cells can be expanded and analyzed once the specific antigen-binding properties have been determined. Such immunoglobulins are typically antibodies, such as monoclonal antibodies, and are used therapeutically or in assays and research. Selecting cells with such characteristics may require screening thousands of cells. Because intermediate steps between harvesting and identifying cells that produce the desired product can result in losses and / or reduced performance, it is best to load the cells directly onto the device, reducing handling steps. Without prior FAC sorting, the ratio of desired cells to undesired cells is significantly reduced. Here, large droplet handling and screening capabilities are required.

[0008] Selecting cells with desirable product-producing properties can result in cells being damaged or destroyed during the test, which is undesirable when selecting cells that produce the desired product.

[0009] Therefore, there is a need for a robust method that streamlines the labor-intensive process of screening cells for desirable properties, allowing for the selection and subsequent expansion or further testing of such cells, while rejecting specific cells with undesirable properties, reducing overall time and cost. Therefore, it is important that cells can not only be tested but also recovered. Microfluidic platforms have been proposed to address this need, but practical and reliable methods have yet to be proven in clinical settings. In particular, the ability to perform multiplexed assays on multiple cells has proven elusive, as has the ability to process large numbers of cells simultaneously and at high throughput. Indeed, much of the research on such devices has been dominated by the use of fixed structures such as isolation pens, which lack the flexibility to perform multiple assays on cells. Some platforms do not allow for interrogation at the individual cell level, instead evaluating multiple cells. Furthermore, inflexible systems mean that only a single assay can be performed on cells, as they lack mechanisms to enable sequential evaluation or to retain cells for further expansion once evaluation is complete. Assay miniaturization reduces the amount of reagents consumed in the assay process and lowers consumable costs.

[0010] The present disclosure provides a method for cell selection where the cells are contained in microdroplets, wherein the actuation mechanism for manipulating the cell-containing microdroplets on the surface of a microfluidic chip is electrowetting on a dielectric (EWOD), optionally optically mediated electrowetting on a dielectric (oEWOD).

[0011] Such chip-based devices advantageously enable manipulation of droplets over a wide range of sizes, providing dynamically reprogrammable manipulation steps under digital control. This device architecture allows for more sophisticated and integrated workflows compared to traditional approaches, such as independent droplet control, and allows for greater droplet density control across the entire surface area of ​​a microfluidic chip. Summary of the Invention [Problem to be solved by the invention]

[0012] The present disclosure provides a method that utilizes the flexibility of EWOD or oEWOD microfluidic chips to enable high-throughput processing of microdroplets, thus solving the need for multiplexing in screening applications. The methods of selecting cells claimed in the present invention include the cells, cell parts and / or cell-derived species disclosed herein.

[0013] Provided herein are methods for selecting cells, particularly for therapeutic applications or manufacturing, and the disclosed methods are particularly useful in adoptive cell therapy, gene therapy, and cell-based manufacturing. The selected cells may be a subset of a larger cell population, where the cells are generally of the same type or species. The selected cells do not have to be of the same type or species. Cells may be selected to provide heterogeneity within the cell population. The cells are genetically identical, but certain genes may be switched on or off.

[0014] Provided herein are methods for selecting cells to study their interactions with other entities, such as (bio)chemical / (bio)molecular entities, or other cells (cell-cell interactions). For example, the methods can include targeted cell-cell interactions when two or more selected cells are brought into close proximity.

[0015] Provided herein are methods for selecting cells to examine secretions from single cells or cell-cell interactions, for example, the methods can include targeted cell-cell interactions when two or more selected cells are brought into close proximity. [Means for solving the problem]

[0016] According to one aspect of the present disclosure, there is provided a method for selecting cells in an EWOD or oEWOD device, the method comprising the steps of: i. providing a test panel of microdroplets comprising at least culture medium, or culture medium and at least one cell; ii. providing at least one reporter panel of microdroplets containing one or more reporter entities; iii. merging the test panel microdroplets with at least one reporter panel microdroplet to generate a panel of merged assay microdroplets; and iv. monitoring the panel of assay microdroplets with a detection system capable of detecting a change in said reporter entity based on the presence of at least one characteristic; and v. selecting a subset of microdroplets from the panel based on a change in reporter entity, said subset containing the selected cells; Equipped with.

[0017] Here, a test panel of microdroplets comprising at least medium is prepared by dividing a test panel of microdroplets comprising at least one cell and medium, and further preparing a reference panel of microdroplets containing at least one cell.

[0018] And / or further, the test panel of microdroplets comprising at least one cell is divided into at least two panels before or after any of steps (i) to (v), said two panels being a test panel of microdroplets comprising at least one medium or medium and at least one cell suitable for merging with the microdroplets of the reporter panel, and a reference panel of microdroplets containing at least one cell.

[0019] Thus, cells can be selected based on one or more properties.

[0020] The methods described herein can include at least one splitting operation, such that, prior to step (i), a panel of microdroplets is first split to provide a test panel of microdroplets containing at least medium and a reference panel of microdroplets containing at least one cell.

[0021] If the microdroplet panel in step (i) contains one or more cells, there is no need to perform a splitting operation before step (iii).

[0022] Alternatively or additionally, one or more further division operations can be included at any suitable step of the procedure, which can generate test panels of microdroplets comprising at least medium and / or test panels of microdroplets comprising at least medium and at least one cell.

[0023] The splitting operation can be performed to include any secreted product in the medium if the cells are capable of producing it. The splitting step can be performed to generate multiple daughter droplets with different concentrations of the secreted product. Alternatively or additionally, the splitting operation can be performed at different times so that the daughter droplets contain different concentrations of the secreted product.

[0024] The splitting operation may result in the formation of one or more reference panels of microdroplets containing at least one cell, and one or more test panels of microdroplets that either contain at least one cell or do not contain at least one cell.

[0025] The method contemplates including, depending on the property of interest, a test panel of microdroplets containing at least one cell and a microdroplet containing at least a medium.

[0026] The purpose of such a splitting operation is to generate a reference panel of microdroplets containing at least one cell.

[0027] Alternatively, there is provided a method for selecting cells in an EWOD or oEWOD device, the method comprising the steps of: i. providing a panel of microdroplets comprising one or more cells and medium; ii. dividing the panel of microdroplets into at least two panels of microdroplets: a reference panel of at least one microdroplet comprising one or more cells, and a test panel of at least one microdroplet comprising at least the medium; iii. providing a reporter panel of at least one microdroplet containing one or more reporter entities; iv. merging the test panel microdroplets with at least one reporter panel microdroplet to form a panel of merged assay microdroplets; and v. monitoring the panel of assay microdroplets with a detection system capable of detecting a change in said reporter entity based on the presence of at least one property; and vi. selecting a subset of microdroplets from the reference panel based on the change in reporter entity in the corresponding assay microdroplet panel, said subset comprising the selected cells; Equipped with.

[0028] The methods of the present disclosure include detecting or determining one or more characteristics of a cell. As used herein, a cellular characteristic can be any suitable characteristic, such as one or more of the following: the presence of one or more cell surface molecules, the presence of one or more cellular activities, cell morphology, the presence of one or more cellular secretions, and / or the presence of one or more intracellular products.

[0029] The properties of the cells can be determined by any suitable means according to the methods described herein. The methods can use one or more reporter entities that can determine or detect the presence of the property. Such reporter entities can be provided within microdroplets so that they merge with the microdroplets being tested for the property.

[0030] The methods described herein may relate to cell selection, in which a panel of microdroplets containing at least one cell is divided to generate a panel of microdroplets containing at least one cell, which is defined herein as a reference panel. This division step or division operation may be performed at any suitable time or step in the procedure, i.e., the reference panel is constructed at a suitable time. This reference panel may be constructed at different time / steps in the procedure depending on the cells being selected. Thus, the reference panel may include a subset of the cells originally considered.

[0031] The division step also results in the generation of at least one test panel of microdroplets. The test panel of microdroplets may include culture medium from precursor microdroplets. If the cells have secretory capacity, the culture medium may also include secretions from the cells. The test panel and the reference panel are correlated, such that the identification result of the test panel and the selection of the desired property result in the selection of a corresponding reference microdroplet containing at least one cell. In this way, the identification result of the test panel is associated with the precursor cell and the same is selected.

[0032] Prior to the division step, the panel of microdroplets containing at least one cell can be cultured under conditions that allow cell division, and optionally clonal expansion. Such conditions can include providing additional medium to the cells by merging a microdroplet panel containing medium with a microdroplet panel containing at least one cell. Other conditions include providing supplements to the cells, such as growth factors, nutrients, cell signaling molecules, chemicals, etc., by merging a microdroplet panel containing supplemented medium with a microdroplet panel containing at least one cell.

[0033] If division of cells within the microdroplet panel is permitted, the reference panel and test panel of microdroplets can be divided, with the reference panel or microdroplet containing at least one clonal copy of a cell corresponding to a cell contained in the test panel of microdroplets, and thus the identification results of the test panel are correlated with the progenitor cells and the same are selected.

[0034] According to one embodiment, a test panel of microdroplets containing at least one cell can be merged with a panel of microdroplets containing an agent for lysing said cell. Such a step is necessary when the cellular property to be determined is the presence of an intracellular product. Alternatively, the intracellular product can be detected directly using a reporter entity that can enter the cell.

[0035] Thus, cells in the microdroplets forming the panel may be allowed to divide before being exposed to a particular reporter entity. Once the cells divide, each microdroplet in the panel can split, forming at least two daughter microdroplet panels, each containing at least one cell. At least one microdroplet panel can be reserved as a reference panel, and at least one other panel can be used in the methods of the invention (a "test panel"). Thus, the reference panel and the test panel contain corresponding or correlated cells, and the results of the test panel can result in the identification of the corresponding cells in the reference panel. The test panel of microdroplets and the reference panel of microdroplets each contain cells derived from original progenitor cells, and can therefore be described as genetically identical, or clonal.

[0036] According to any aspect of the present disclosure, cells in any panel of microdroplets containing at least one cell may be examined for their morphological characteristics, which may be used to select a subset of cells. This examination may be performed before step (ii) of the method, or before or after any step of the method. Morphological characteristics may include size, shape, adhesion state, cell membrane features such as blebs, and / or the presence of intracellular features such as vacuoles.

[0037] Selection of cells by any aspect means defining a subset of cells and discarding or excluding the remaining cells. Selection can be positive (selection of cells based on the presence of a characteristic) or negative (selection of cells based on the absence of a negative characteristic). Discarded / rejected microdroplets can be removed from the device or can remain in the panel and be removed from consideration.

[0038] According to some embodiments, prior to step (i), the panel of microdroplets comprising at least one cell may be evaluated with at least one assay to determine a characteristic of the cell, said assay being performed by merging said panel of microdroplets comprising at least one cell with at least one panel of microdroplets containing at least one reporter entity and determining a change in at least one reporter entity based on the presence of said characteristic. Thus, initial testing or identification can be performed prior to any splitting step.

[0039] According to one embodiment, once selected, the cells are removed from the device for expansion or further testing.

[0040] According to another aspect of the present disclosure, there is provided a method for selecting cells by use of an EWOD or oEWOD device, the method comprising the steps of: i. providing a panel of microdroplets comprising at least one cell and a medium; ii. providing a panel of at least one reporter microdroplet containing one or more reporter entities; iii. merging the microdroplets of the at least one cell-containing panel with the microdroplets of the at least one reporter panel to form a panel of combined microdroplets; iv. monitoring the panel of merged microdroplets using a detection system capable of detecting a change in said reporter entity based on one or more properties of the cells and selecting a subset of microdroplets based on said change in reporter entity to form a selected panel of microdroplets comprising cells; v. harvesting a panel of selected microdroplets containing cells, and optionally repeating steps (ii) to (iv) one or more times, wherein one or more different reporter entities are used in step (ii); vi. Selecting cells based on the final panel of microdroplets selected in step (v); Equipped with Prior to step (iii) and / or step (v), the panel of microdroplets containing at least one cell is divided into at least two panels of microdroplets, said panels being as follows: a. a test panel comprising at least a medium and suitable for merging with a microdroplet of a reporter entity panel; and b. A reference panel of microdroplets containing at least one cell is.

[0041] In one embodiment, prior to dividing a panel of microdroplets containing at least one cell, the cells can be cultured to allow cell division so that the test panel of microdroplets contains cells, optionally clonal cell copies of cells from the reference panel.

[0042] Alternatively, according to one aspect, the present invention provides a method for selecting cells by use of an EWOD or oEWOD device, the method comprising the steps of: i. providing a first panel of microdroplets containing at least one cell; ii. providing at least one panel of reporter microdroplets containing one or more reporter entities; iii. merging the microdroplets of the first panel with the microdroplets of at least one reporter panel to form a first panel of merged assay microdroplets; iv. monitoring the panel of assay microdroplets using a detection system capable of detecting a change in said reporter entity based on one or more cellular properties and selecting a first subset of microdroplets based on said change in reporter entity to form a first selected panel of microdroplets; v. Providing at least one reporter panel of microdroplets containing one or more reporter entities; vi. merging microdroplets from the selected panel of microdroplets with microdroplets from a reporter panel of additional microdroplets to form a second panel of merged assay microdroplets; vii. monitoring a second panel of assay microdroplets using a detection system capable of detecting a change in said reporter entity based on one or more cellular characteristics and selecting a second subset of microdroplets based on said change in reporter entity to form a second selected panel of microdroplets, said subset containing selected cells; Equipped with Prior to step (iii) and / or step (v), the panel of cell-containing microdroplets is divided into at least two panels of microdroplets, said two panels comprising: a. a panel of microdroplets comprising at least a medium and suitable for merging with the microdroplets of the reporter panel; and b. A reference panel of microdroplets containing at least one cell is.

[0043] The following features may be applied to any variation of the methods disclosed herein.

[0044] The division of the microdroplets may be uniform or uneven, with one microdroplet being divided into two or more microdroplets, each of which may be the same size or different sizes.

[0045] In one embodiment, prior to dividing a panel of microdroplets containing at least one cell, the cells can be cultured to allow cell division so that the test panel of microdroplets contains cells, optionally clonal cell copies of cells from the reference panel.

[0046] According to one embodiment of the method disclosed herein, the panel of microdroplets in step (i) may contain at least one cell, or may contain two or more cells if the original cell has undergone cell division. The purpose of the method is to characterize a particular cell type, where cell characteristics may vary across a homogenous cell population being assayed. The method may also include a step of discarding or disposing of microdroplets containing multiple different cells of the same type, where the cells have not resulted from cell division.

[0047] According to one embodiment of the method disclosed herein, the panel of microdroplets in step (i) can contain at least culture medium. The microdroplets containing at least culture medium have previously been split or divided from microdroplets containing cells in the culture medium. Prior to the splitting step, the panel of microdroplets containing at least one cell can be merged with a panel of microdroplets containing culture medium, which can optionally contain signaling molecules or cells, causing the cells to produce secretions. Thus, the culture medium in the test panel of microdroplets can contain secretions from the cells, and the secretions are exposed to a reporter entity to define the characteristics of the cells. The method can also include a step of discarding or disposing of microdroplets that do not contain the secretions of interest. The droplets containing culture medium can have a different volume than the droplets containing the cells.

[0048] Further optionally, the reporter panel used to select cells allows for the identification of cells with desired properties, e.g., cell surface properties such as expression of particular cell surface markers or secretion of desired molecules. The reporter entities in such rounds can be entities that bind to or interact with cell surface or secreted molecules.

[0049] Optionally, a selection step can identify desirable cells, cells with the cell interaction properties, such as cell binding, activation, cell fusion, cell uptake, cell death, etc. The reporter in such a round of selection can be a cell, such as a tumor cell.

[0050] Optionally, the selection step can include assays for viability, activation and / or proliferation properties or potential.

[0051] Optionally, if cells are present in the test panel of microdroplets, they are lysed to release the cellular contents. Such a step is considered if the cells have already undergone a cell division event and, in addition, a reference panel of microdroplets each containing at least one cell has been created.

[0052] Microdroplets can be removed from the panel at any point during the analysis process if it is determined that further analysis is not necessary, for example, microdroplets can be discarded if the cells do not contain the desired properties, do not undergo cell division, or do not possess the desired properties, such as promoting cell death of reporter cells or releasing antibodies capable of binding to antigens.

[0053] While it may be desirable to analyze any type of cell using the methods of the present invention, the cells may be of the same type, e.g., B cells or T cells (lymphocytes), and the methods of the present invention can be used to select cells with specific characteristics, which may be inherent in the cells or genetically engineered into the cells. Cells are also referred to as biological cells. It may also be desirable to analyze different types of co-cultures of cells in droplets, such as a combination of reporter cells and primary cells, or a culture of epithelial cells of different phenotypes combined to form a tissue-like structure. Cells may be natural. Cells may be artificial. Cells may be microcells. The methods of the present invention may be cell-free or may use parts of cells, such as nuclei and / or mitochondria. The methods of the present invention may also use liposomes.

[0054] The methods of the present invention can be used to select cells with desirable properties for therapeutic use.

[0055] The methods of the present invention can be used to select cells with desirable properties for use in research.

[0056] The methods of the present invention can be used to select cells with desired properties for use in the manufacture of a desired product such as a molecule / compound / chemical, etc. In such uses, the cells may be genetically engineered bacterial cells, etc. [Brief explanation of the drawings]

[0057] [Figure 1] An exemplary method is shown, in which blood is drawn from the patient, and T cells are isolated, transduced, sorted, and selected prior to mass expansion and reinfusion into the patient. Also shown are the sections within this manufacturing process that our CAR-T workflow covers. [Figure 2] This figure outlines the early steps in an exemplary CAR-T workflow, prior to dosimetry and profiling. T cells isolated from a patient's blood are emulsified into microdroplets, sorted, and transduced. Unwanted microdroplets are discarded, and a multi-reporter cell surface marker assay is performed on the cells in the remaining microdroplets. Based on this assay, a panel of microdroplets is selected. [Figure 3] This figure shows an overview of later steps in an exemplary CAR-T workflow, in which cells from a selected microdroplet panel are evaluated for cell viability, activation, and exhaustion behavior following a multi-reporter cell surface marker assay. The microdroplet panel can be divided so that CAR-T cells are retained in a reference panel and clonal CAR-T cells are used in further selection steps. Activation assessment, for example, involves mixing T-cell-containing microdroplets with cancer cell-containing microdroplets and quantifying their cancer cell-killing activity. A final panel of microdroplets is then selected, and the T cells are dispensed from the device for mass expansion and reinfusion into the patient. [Figure 4]An exemplary immuno-oncology workflow is outlined, comprising: A) modifying CHO cells to produce an immunotherapeutic drug and loading it into microdroplets; B) splitting the microdroplets into child droplets containing equal or different doses of drug depending on the time of splitting; C) merging T cell-containing microdroplets with cancer cell-containing microdroplets in a first merging operation, and then merging the merged microdroplets with microdroplets containing a dose of drug in a second merging operation; D) determining positive hits by quantifying cancer cell death and selecting CHO cells corresponding to the positive hits; and E) dispensing the selected CHO cells into well plates and growing them. [Figure 5] An exemplary preparation protocol for generating microdroplets containing either T cells or tumor cells and a TCR drug is shown. [Figure 6] A panel of microdroplets is shown, each containing T cells and tumor cells at various T cell / tumor cell ratios. For each droplet, the number of tumor cells is indicated to the left of each box, and the number of T cells is indicated to the right of each box. Droplets I, K, and L are negative controls containing only tumor cells. [Figure 7] Figure 1 shows a plot of the percentage of cancer cells that underwent apoptosis upon contact with T cells versus time (hours). Comparison with tumor-only controls is shown. In this case, apoptosis acts as a reporter of T cell anti-tumor killing activity under the test conditions and can be used as a selection criterion for subsequent T cell expansion or sampling for further analysis. [Figure 8] Figure 8A shows bright-field images of microdroplets containing T cells and two tumor cells taken periodically over a 36-hour period, demonstrating the sequential killing of two tumor cells by the T cells. Figure 8B confirms this tumor cell killing activity by quantifying the caspase fluorescence intensity of each tumor cell over the same time period. [Figure 9]Figure 9A shows close-up bright-field and Deep Red-stained images of microdroplets containing T cells and two tumor cells taken periodically over the first 13 hours of a 36-hour experiment, with caspase fluorescence intensity quantified over the same time period. Figure 9B shows close-up and Deep Red-stained images of the same microdroplets taken periodically over the last 10 hours of the experiment, with caspase fluorescence intensity quantified over the same time period. The continuous tumor cell-killing activity of T cells is visualized here. [Figure 10] Hybridoma cell viability is shown as a percentage over time, with cells in the device or droplets. Cell viability remains 100% for up to 5.3 hours in droplets and 3.6 hours in the device, and remains above 90% for up to 20.3 hours in droplets and 18.6 hours in the device. [Figure 11] On the left are images of panels of microdroplets containing hybridoma cells and beads, beads only, or hybridoma cells only. Beads are indicated by squares, and cells are indicated by circles. C1 and C2 each contain beads only, C3 contains cells only, D1–D3 contain two cells and one bead, respectively, and D4 and D5 contain one cell and one bead, respectively. On the right are images of the same panels, also showing bead fluorescence. Fluorescent aggregates appeared to varying degrees in D1–D5. No fluorescence was detected in microdroplets containing only cells or only beads (controls). [Figure 12] A graph of filtered AF488 fluorescence is shown on the Y-axis versus free anti-target antibody concentration on the X-axis. On the far right, the average filtered fluorescence is plotted for the irrelevant and target hybridoma populations. It can be seen that fluorescence close to the maximum level observed with free anti-target antibody was measured with secreting target hybridoma cells. [Figure 13]Graph showing data from a multiplex spiking assay, with filtered AF488 fluorescence of target beads on the Y-axis and time on the X-axis. Filtered AF488 fluorescence of target beads was measured over time for a population of microdroplets, each containing target or irrelevant hybridoma cells and reporter. A negative control was also included. D2, containing irrelevant hybridoma cells, remained dark and indistinguishable from the negative control. All target hybridomas showed positive signals. [Figure 14] Fluorescence and brightfield (insert) images of SolR1, R3, and R5 beads are shown using a 10x objective. The graph shows filtered fluorescence measurements to allow easy differentiation of SolR1, R3, and R5 bead populations within multiple beads. [Figure 15] The top figure shows data from a multiplexed bead assay, with the filtered fluorescence of the beads on the Y-axis and the filtered fluorescence of AF488 on the X-axis. Filtered AF488 fluorescence is measured for each bead in a population of microdroplets containing target or irrelevant hybridoma cells and reporters. The beads are decoded by the detection system (R5 human target, R3 off-target, and R1 cyno target). Positive (25 nM free target antibody) and negative (anti-mouse AF488 only) control microdroplets are also assayed. The negative control microdroplets remain dark, while the positive control microdroplets confirm that the free target antibody binds to both target antigens (R1 and R5). Microdroplets containing secreted antibody also bind to both target antigens. The bottom figure shows a bar graph with filtered AF488 fluorescence on the Y-axis and microdroplet categories on the X-axis, each of which is further divided into subcategories: R1, R3, and R5. [Figure 16]An exemplary monoclonal antibody discovery workflow is outlined, comprising the steps of: B cells being added to microdroplets and merging with a microdroplet containing cell culture medium of matching volume to facilitate a subsequent division step; the merged microdroplets dividing into daughter droplets to obtain multiple doses of secreted antibody produced by each cell; sorting the B cell-containing microdroplets into a reference panel while sorting the antibody dose-containing microdroplets into a test panel; pairing the test panel with a reporter panel of microdroplets and merging them; determining positive hits in the merged reporter-mAb panel using an optical detection system; and dispensing the B cell-containing reference panel microdroplets corresponding to the positive hits into well plates for mass expansion. [Figure 17] The paper outlines an exemplary bacterial cell workflow, comprising: adding bacterial cells to microdroplets and merging them with a microdroplet containing a matching cell culture medium for the added volume to facilitate a subsequent division step; allowing the merged microdroplets to undergo cell division and split into clonal colonies; retaining one set of microdroplets containing clonal bacterial cells as a reference panel while sorting an additional set of microdroplets containing clonal bacterial cells or sets of microdroplets containing secretions of said bacterial cells into test microdroplet panels; pairing and merging the test microdroplet panels with reporter microdroplet panels; lysing the bacterial cells to release their contents; determining positive hits in the merged reporter-bacterial cells using an optical detection system; and dispensing the reference panel microdroplets containing bacterial cells corresponding to the positive hits into well plates for mass propagation. [Figure 18] The structure of oEWOD is shown below. [Figure 19A] 10 is an image of a droplet merging operation where a pair of droplets merge horizontally. [Figure 19B] This is an image of paired microdroplets moving together before merging. [Figure 19C] 1 is a merged image of a microdroplet. [Figure 20A] 1 shows a droplet splitting operation where the droplet splits horizontally. [Figure 20B] 1 shows a droplet being elongated during a splitting operation. [Figure 20C] A droplet is shown splitting into two daughter droplets. [Figure 21] This is an image of a single-cell-occupied S. cerevisiae bioparticle (heat-inactivated yeast) encapsulated in a microdroplet. [Figure 22] Images of cytokine-secreting cells co-encapsulated with cytokine reporter beads within microdroplets in an emulsion of cell culture medium in oil. In the presence of secreted cytokine, the reporter beads fluoresce using a sandwich ELISA with fluorescently labeled antibodies. The bright-field image (a) shows the distribution of cells and beads within the droplets. Fluorescence images show (b) which reporter beads test positive in the presence of secreted cytokine, (c) the location of all cells with intact membrane integrity, (d) the location of all beads, and (e) dead cells as indicated by propidium iodide staining. [Figure 23] Flexible and unique workflow manipulation of microdroplets, including microdroplet operations such as injection, sorting, merging, and splitting operations. Programmable decisions can be made at any point throughout the workflow to deviate from the original instructions and / or apply operations to only selected droplets. [Figure 24] Antibody-secreting cells are co-encapsulated with reporter beads within droplets in an emulsion of cell culture medium in oil. In the presence of secreted antibodies, the reporter beads fluoresce using a sandwich ELISA mechanism with fluorescently labeled antibodies. The bright-field image (a) shows the distribution of cells and beads within the droplets. The fluorescence image shows b) which reporter beads react positively in the presence of secreted antibodies, c) the location of all cells with intact membrane integrity, and d) dead cells. [Figure 25] Various detection modes for monitoring cell secretions or cell-cell interactions. [Figure 26]A workflow illustrating typical microdroplet manipulation. Microdroplets containing cells can be manipulated, including splitting, merging, and sorting. Microdroplets can also contain reporters. [Figure 27] A workflow describing the sorting options and / or combinations of merge and dispense operations. [Figure 28] A–D, Various images of cells encapsulated in microdroplets incubated in the presence of an intracellular calcium dye. DETAILED DESCRIPTION OF THE INVENTION

[0058] The inventors have discovered that the deficiencies of prior art cell selection methods may be overcome using the methods disclosed herein, which result in better product quality, predictability, and more favorable cell selection.

[0059] The disclosed method is particularly unique because it proposes testing cells for multiple properties, and selecting cells with the desired properties and the best profile, which from a cell therapy perspective, represents the best safety profile. The disclosed method allows for the selection of cells with the desired properties in a format that allows for further expansion or testing after selection. Desirable cell properties include, but are not limited to, the presence or absence of specific cell surface molecules, the presence or absence of secreted products, the presence or absence of intracellular products, cell morphology, viability, proliferation capacity, and / or the presence of desired cell-cell interaction activities.

[0060] The disclosed method is performed on a microfluidic device capable of manipulating microdroplets, and at least one cell or part of a cell is present. The cell may be natural. The cell may be artificial. The cell may be a microcell. At least one liposome may be present.

[0061] [Device] The present invention relates to methods that can be performed on a microdroplet manipulation device, such as an EWOD or oEWOD device. Any suitable device capable of manipulating microdroplets according to the methods described herein can be employed.

[0062] Devices for manipulating droplets or magnetic beads have been previously described in the art; see, for example, U.S. Pat. No. 6,565,727, U.S. Patent Publication No. 2013-0233425, and U.S. Patent Publication No. 2015-0027889. In the case of droplets, this is typically accomplished by passing the droplets through a microfluidic channel defined by two opposing walls of a cartridge or microfluidic tube, for example, in the presence of an immiscible carrier fluid. Embedded in the walls of the cartridge or tube are electrodes covered with a dielectric layer, each connected to an A / C bias circuit that can be rapidly switched on and off at intervals to modify the electrowetting properties of the layer. This generates localized, directional capillary forces that can be used to propel the droplets along a predetermined path.

[0063] Electrowetting on dielectrics (EWOD) is a well-known effect in which applying an electric field between a liquid and a substrate causes the liquid to wet a surface more easily than it would naturally. The electrowetting effect can be used to manipulate (e.g., move, split, or change shape) fluids by applying a series of spatially varying electric fields across a substrate, increasing the surface's wettability according to the series of spatial variations. Droplets manipulated in electrowetting-based devices are typically sandwiched between two parallel plates and actuated by digital electrodes.

[0064] Variations on this approach based on optically mediated electrowetting are disclosed, for example, in U.S. Pat. No. 6,958,132, U.S. Patent Publication No. 2015-0298125, and U.S. Pat. No. 9,815,056. An improved version of this approach, enabling the simultaneous manipulation of thousands of microdroplets, typically in the sub-10 μm size range, is described in International Publication No. 2018 / 234445, incorporated herein by reference. In this oEWOD device, microdroplets travel through a microfluidic space defined by containing walls, such as a pair of parallel plates with a microfluidic space between them. At least one of the containing walls contains what are referred to as "virtual" electrowetting electrode locations, which are generated by selectively illuminating regions of a semiconductor layer embedded within the wall. By selectively illuminating the layer with light from a separate light source, a virtual path of virtual electrowetting electrode locations can be transiently generated along which the microdroplets can be moved. Additionally, a suitable oEWOD device is described in WO 2020 / 104769, which is also incorporated by reference, and may be particularly relevant to the present method as it is particularly relevant when the microdroplets contain cells. Another oEWOD device is described in WO 2020 / 169965, which is incorporated by reference herein.

[0065] Another disclosure of oEWOD is a single-sided open-configuration platform described in non-patent literature (Park, Sung-Yong, Michael A. Teitell, and Eric PY Chiou, "Single-sided continuous optoelectrowetting (SCOEW) for droplet manipulation with light patterns," Lab on a Chip 10.13 (2010): 1655-1661).

[0066] A device suitable for manipulating microdroplets optionally has the following features: A first compound wall comprising: a first transparent substrate; a first transparent conductor layer on said substrate having a thickness in the range of 70 to 250 nm; a photoactive layer activated by electromagnetic radiation with a wavelength in the range of 400 to 1000 nm on said conductor layer, said photoactive layer having a thickness in the range of 300 to 1000 nm; a first dielectric layer on the conductor layer, the first dielectric layer having a thickness in the range of 120 to 160 nm; the first composite wall comprising: A second compound wall comprising: a second substrate, a second conductor layer on the substrate, the second conductor layer having a thickness in the range of 70 to 250 nm; and an optional second dielectric layer on the conductor layer, having a thickness in the range of 120 to 160 nm; the second composite wall comprising: may include one or more of The exposed surfaces of the first and second dielectric layers are spaced at least 10 μm apart, preferably 50-100 μm apart, to define a microfluidic space adapted to contain microdroplets, and one of the dielectric layers is coated with a biocompatible coating or an antifouling coating.

[0067] Optionally, an intervening layer of silica between the dielectric layer and the antifouling / biocompatible coating, the intervening layer having a thickness of 1 to 10 nm; an A / C power source that provides a voltage across first and second composite walls connecting the first conductor layer and the second conductor layer; at least one second electromagnetic radiation source having an energy higher than the bandgap of the photoactive layer adapted to impinge on the photoactive layer to induce corresponding virtual electrowetting electrode locations on the surface of the first dielectric layer; There may also be means for manipulating the point of impact of the electromagnetic radiation on the photoactive layer to change the configuration of the virtual electrowetting electrode locations, thereby forming at least one optically mediated electrowetting path along which the microdroplets can move.

[0068] Methods and apparatus for high-throughput microdroplet manipulation are described in co-pending application PCT / GB2021 / 050148, and such apparatus and techniques may be suitable for implementing the methods of the present disclosure. The apparatus disclosed therein includes two independently controllable optical assemblies, both of which are capable of generating an array of fixed and switchable light spots on the surface of an oEWOD chip. This configuration is optimized to facilitate high-throughput, flexible microdroplet loading and processing. A first optical assembly can form multiple oEWOD traps on the surface of the chip, causing multiple microdroplets on the surface of the chip to form an array of microdroplets corresponding to the first array of oEWOD traps, and a second optical assembly can form a second array of oEWOD traps on the surface of the chip. One or more of the oEWOD traps in the second array are aligned with the oEWOD traps in the first array. The method may include inspecting the contents of the array of microdroplets and adjusting the first optical assembly while one or more of the microdroplets are held in place by the second array of oEWOD traps. PCT / GB2021 / 050148 is incorporated herein by reference.

[0069] In some embodiments, there is provided an apparatus for manipulating microdroplets using optically mediated electrowetting, the apparatus comprising: A first compound wall comprising: First board a first conductor layer on the substrate; an optically active layer of said conductor layer; the first composite wall comprising a first dielectric layer on the optically active layer, the first dielectric layer having a thickness of less than 20 nm; A second compound wall comprising: Second board a second conductor layer on the substrate; the second composite wall comprising a second dielectric layer on the conductor layer, the second dielectric layer having a thickness of less than 20 nm; Equipped with.

[0070] The first and second dielectric layers may be continuous layers. The first dielectric layer may be deposited on the photoactive layer by atomic layer deposition. Additionally or alternatively, the second dielectric layer may be deposited on the photoactive layer.

[0071] Surprisingly, it has been discovered that providing the first and / or second dielectric layers as continuous layers with a thickness of less than 20 nm makes the droplets more stable, thus allowing the droplets to rest on the substrate. In contrast, the inventors have found that increasing the thickness of the first and / or second dielectric layers to a thickness greater than 20 nm results in less controlled droplet movement on the substrate and therefore increases the likelihood of the droplets exhibiting uncontrolled movement that deviates from the illumination area. As a result, uncontrolled droplets can make accurate and efficient oEWOD operations, such as droplet merging and splitting, more difficult.

[0072] In some embodiments, the first and / or second dielectric layer may be 1 nm to 20 nm thick, or may be 2 nm to 20 nm, 3 nm to 20 nm, 4 nm to 20 nm, 5 nm to 20 nm, 6 nm to 20 nm, 7 nm to 20 nm, 8 nm to 20 nm, 9 nm to 20 nm, 10 nm to 20 nm, 12 nm to 20 nm, 14 nm to 20 nm, 15 nm to 20 nm, or 18 nm to 20 nm, or may be 1 to 15 nm, 1 to 10 nm, 1 to 5 nm, 5 to 10 nm, 5 to 15 nm, or 10 to 15 nm.

[0073] The first substrate and first conductor layer, and / or the second substrate and second conductor layer may be transparent.

[0074] The apparatus may further include an A / C source for supplying a voltage across the first and second composite walls connecting the first and second conductor layers, at least one electromagnetic radiation source having an energy higher than the bandgap of the photoactive layer adapted to impinge on the photoactive layer to induce corresponding transient electrowetting locations on the surface of the first dielectric layer, and a microprocessor for manipulating the impingement point of the electromagnetic radiation on the photoactive layer to change the location of the transient electrowetting locations and form at least one electrowetting path capable of moving microdroplets.

[0075] The device may further include an intervening layer of silicon oxide. The intervening layer has the advantage of being usable as a bonding layer for either the antifouling layer or the non-antifouling layer. The intervening layer is disposed between the dielectric layer and the hydrophobic layer. The thickness of the intervening layer may be between 0.1 nm and 5 nm. The thickness of the intervening layer may be 0.1, 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, or 4.5 nm or more, or may be 5 nm, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.75, 0.5, or 0.25 nm or less.

[0076] The exposed surfaces of the first and second dielectric layers can be spaced apart by less than 200 μm to define a microfluidic space adapted to contain microdroplets. The width of the microfluidic space can be between 2 and 50 μm. In some embodiments, the microfluidic space is 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48 μm or greater. In some embodiments, the microfluidic space can be less than 50, 48, 46, 44, 42, 40, 38, 36, 34, 32, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 8, 6, or 4 μm.

[0077] The exposed surfaces of the first and second dielectric layers can include one or more spacers to hold the first and second walls a predetermined distance apart, defining a microfluidic space suitable for containing microdroplets. The physical shape of the spacers can be used to aid in the splitting, merging, and elongation of the microdroplets within the device. In some embodiments, the microdroplets can contain one or more cells. The microdroplets can also contain a medium, such as cell culture medium and / or a buffer solution.

[0078] The A / C source may be configured to apply a voltage between 0 V and 100 V across the first and second composite walls connecting the first and second conductor layers. In some embodiments, the A / C source may be configured to apply a voltage greater than 0, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, or 90 V, or less than 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 V.

[0079] The first and second composite walls may further include first and second antifouling layers on the first and second dielectric layers, respectively. The antifouling layer on the second dielectric layer may be hydrophobic. The electromagnetic radiation source may form a pixelated array of light reflected from or transmitted through such an array. The electrowetting locations may be crescent-shaped in the direction of movement of the microdroplets. The device may further include a photodetector located midstream or downstream of the device for detecting an optical signal in the microdroplets. The optical signal may be a fluorescent signal. The device may further include an upstream inlet for generating a medium comprising an emulsion of aqueous microdroplets in an immiscible carrier fluid. The device may further include an upstream inlet for directing a flow of the medium comprising an emulsion of aqueous microdroplets in an immiscible carrier fluid through the microfluidic space via the inlet to the microfluidic space.

[0080] The first and second composite walls defining the microfluidic space therebetween can form the periphery of a cartridge or chip. The device may further include a plurality of first electrowetting paths running concurrently with one another. The device may further include a plurality of second electrowetting paths adapted to intersect with the first electrowetting paths to form at least one microdroplet coalescence site.

[0081] The device may further include an upstream inlet for introducing microdroplets into the microfluidic space, wherein the diameter of the microdroplets is at least 20% greater than the width of the microfluidic space.

[0082] The second composite wall may further include a second photoexcitable layer, and the electromagnetic radiation source may also impinge on the second photoexcitable layer to form a second pattern of transient electrowetting locations that may also be altered.

[0083] The electromagnetic radiation source may be an LED light source, with a power of 0.005-0.1 Wcm -2 In some embodiments, the electromagnetic radiation source can provide a power level of 0.005 to 0.1 Wcm. -2 or at a level of 0.005, 0.0075, 0.01, 0.025, 0.05 or 0.075 Wcm -2 In some embodiments, the electromagnetic radiation source may have a power of 0.1, 0.075, 0.05, 0.025, 0.01, 0.0075, 0.005, or 0.0025 Wcm -2 It may be at a level less than that.

[0084] The first transparent conductor layer on the substrate can have a thickness in the range of 70 to 250 nm. The photoactive layer is activated by electromagnetic radiation in the wavelength range of 400 to 1000 nm on the conductor layer, and the conductor layer can have a thickness in the range of 300 to 1000 nm. In some embodiments, the photoactive layer can be made of amorphous silicon.

[0085] In some embodiments, microdroplets can pass through a microfluidic space defined by two opposing walls, each wall including a dielectric layer with a sufficiently low voltage applied across the dielectric layer to be below the breakdown voltage of the dielectric layer. The use of two dielectric layers with a sufficiently low voltage across the dielectric layer not only prevents destructive ionization of conductive droplets, but also substantially eliminates the adverse effects on the droplets of dielectric pinhole defects, resulting in unexpected performance improvements despite the reduced electrowetting force due to the use of two dielectric layers. As a result, optically mediated electrowetting can achieve powers of, for example, 0.01 W / cm for simultaneously manipulating thousands of droplets. 2 This can be achieved using low power illumination sources such as LEDs that generate low power such as 10,000 droplets or more. For embodiments comprising large area microfluidic devices greater than 1 cm x 1 cm, the devices are suitable for simultaneous manipulation of 10,000 droplets or more, and for ultra-large area devices, 50,000 droplets or more, 100,000 droplets or more, or 1,000,000 droplets or more.

[0086] In some embodiments, large area devices can be utilized to handle thousands of droplets. The inventors previously attempted to create larger devices using a single dielectric layer to simultaneously handle droplets, but encountered defective areas where droplets could not be moved. Through experimentation and testing, the inventors discovered that pinhole defects become a significant limitation in device performance, especially as devices become larger.

[0087] The dielectric layer always has scattered pinhole defects that make it conductive in small isolated areas. In optimized known processes, 2 This results in a pinhole density of approximately 38 per square inch. Pinhole defects trap droplets, preventing them from moving. The effect is even more severe when using droplets of conductive media, such as buffer solutions.

[0088] In some embodiments, a two-dielectric layer structure is provided that can be used below breakdown. When used below the breakdown voltage, the two-sided dielectric layer structure offers the novel advantage of nearly eliminating the effects of pinhole defects. Because dielectrics are located both above and below the droplet, a conductive path is formed only if a pinhole defect in the first dielectric layer is directly aligned with a pinhole defect in the second dielectric layer. The probability of this occurring is extremely small. This pinhole reduction property, achieved by the presence of the second dielectric layer, is key to enabling the simultaneous manipulation of thousands of droplets over a relatively large area.

[0089] For large-area or ultra-large-area devices suitable for simultaneously manipulating over 100,000 droplets, or even over 1,000,000 droplets, the number of pinhole defects becomes a significant limitation on device performance, as the probability of a single droplet coming into contact with a pinhole defect becomes very high. A single droplet trapped in a pinhole defect can block the movement of other droplets within the device, potentially impairing or disrupting system operation. Thus, the benefit of the present invention in countering the effects of pinhole defects is especially important in the operation of ultra-large-area devices containing many small droplets.

[0090] FIG. 18 illustrates a microfluidic device, specifically an oEWOD device 100. The oEWOD device illustrated in FIG. 18 comprises a first composite wall 102 including a first substrate 104, which can be made of glass; a first transparent conductor layer 106 on the substrate 104, the first transparent conductor layer 106 having a thickness in the range of 70 to 250 nm; a photoactive layer 108 activated by electromagnetic radiation having a wavelength in the range of 400 to 850 nm on the conductor layer 106, the photoactive layer 108 having a thickness in the range of 300 to 1500 nm; and a first dielectric layer 110 on the photoactive layer 108. The first dielectric layer 110 is formed as a continuous layer having a thickness of less than 20 nm. The lower limit of the layer thickness is determined, at least in part, by the methodology for providing such a thin layer, which must be continuous. However, theoretically, the thickness can be between 0.1 nm and 20 nm.

[0091] The device 100 also includes a second composite wall 112 including a second substrate 114, which can be made of glass, and a second transparent conductor layer 116 on the substrate 114. The second conductor layer 116 may have a thickness ranging from 70 to 250 nm. A second dielectric layer 118 may be on the transparent second conductor layer 116, with the second dielectric layer 118 having a thickness of less than 20 nm. Like the first dielectric layer, the second dielectric layer must be continuous, and its practical lower thickness is determined by manufacturing constraints, but can be between 1 nm and 20 nm. The exposed surfaces of the first dielectric layer 110 and the second dielectric layer 118 are spaced 20 to 180 μm apart and define a microfluidic space 121 adapted to contain a microdroplet 122. The photoactive layer 108 is made of amorphous silicon. The first and second conductor layers are made of ITO.

[0092] The intervening tie layer 124 is disposed on the first dielectric layer 110 and may also be disposed on the second dielectric layer 118. The thickness of the intervening tie layer may be between 0.1 nm and 5 nm. The thickness of the intervening layer may be 0.1, 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, or 4.5 nm or more, or may be 5 nm, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.75, 0.5, or 0.25 nm or less. The advantage of the intervening layer is that it can be used as a tie layer for either an antifouling layer or a non-antifouling layer, and it can be hydrophobic.

[0093] A hydrophobic layer 126 is disposed on the intervening tie layer 124. An example of a hydrophobic layer is fluorosilane. The intervening tie layer 124 is optional; the channel walls 120 can be made of SU8 or can be part of a glass structure. The intervening tie layer 124 is disposed between the dielectric layers 110, 118 and the hydrophobic layer 126.

[0094] 18, incident light 130 can be used to impart a light sprite pattern 131 that illuminates a portion of photoactivator 110 and holds microdroplets 122 in a stationary position within microfluidic space 121. An oil-based carrier phase 134 can be delivered to microdroplets 122 through holes 136 in the device to replenish important nutrients and components to keep the contents within microdroplets 122, such as one or more cells, alive and healthy. In some cases, oil phase 134 can provide nutrients, medium, and components important for cell growth, viability, and / or productivity.

[0095] The first and second substrates 104, 114 are made of materials that have mechanical strength. For example, the first and second substrates can be formed from glass, metal, or engineering plastic. In some embodiments, the substrates may have some flexibility. In some embodiments, the first substrate is silicon, fused silica, or glass. In some embodiments, the second substrate is fused silica and glass.

[0096] The first and second conductor layers 106, 116 are located on one surface of the first and second substrates 104, 114 and typically have a thickness ranging from 70 to 250 nm, preferably 70 to 150 nm. At least one of these layers is made of a transparent conductive material such as indium tin oxide (ITO), an ultrathin film of a conductive metal such as silver, a conductive polymer such as PEDOT, or the like. These layers may be formed as a continuous sheet or a series of discrete structures such as wires. Alternatively, the conductor layer may be a mesh of conductive material, with electromagnetic radiation irradiated through the gaps in the mesh.

[0097] The photoactive layer 108 is formed from a semiconductor material capable of generating localized regions of charge in response to stimulation by a source of electromagnetic radiation. For example, it may be a hydrogenated amorphous silicon layer having a thickness in the range of 300-1500 nm. In some embodiments, the photoactive layer is activated by the use of visible light. The dielectric properties of this layer are preferably 10 7These include high breakdown voltages of greater than V / m and dielectric constants of greater than 3. In some embodiments, the dielectric layer is selected from alumina, silica, hafnia, or thin non-conductive polymer films.

[0098] Alternatively, at least the first dielectric layer, and preferably both, may be coated with an antifouling layer to help establish the desired microdroplet / carrier fluid / surface contact angle at the various virtual electrowetting electrode locations. The antifouling layer additionally serves the purpose of preventing the microdroplet contents from adhering to the surface and dissipating as the microdroplet moves through the chip. For optimal performance, the antifouling layer should help establish the microdroplet / carrier fluid / surface contact angle, which should be in the range of 50°C to 180°C, measured at the air-liquid-surface three-point interface at 25°C. In some embodiments, these layers have a thickness of less than 10 nm and are typically formed as monolayers. Alternatively, these layers can comprise polymers of acrylic esters, such as methyl methacrylate, or their dielectrics substituted with hydrophilic groups (e.g., alkoxysilyl). Either or both of the antifouling layers may be hydrophobic to ensure optimal performance. In some embodiments, an intervening layer of silica less than 20 nm thick may be inserted between the antifouling coating and the dielectric layer to provide a chemically compatible bridge.

[0099] The first and second dielectric layers, and thus the first and second walls, have a width of at least 10 μm, preferably in the range of 20-180 μm, defining a microfluidic space in which the microdroplets are contained. Preferably, prior to encapsulation, the microdroplets themselves have a characteristic diameter that is 10% or 20% larger than the width of the microdroplet space. Therefore, upon entering the chip, the microdroplets are compressed, resulting in a spherical deformation of the microdroplets, which can lead to improved electrowetting performance (e.g., better microdroplet merging force). In some instances, the first and second dielectric layers can be coated with a hydrophobic coating, such as fluorosilane.

[0100] In some embodiments, the microfluidic space includes one or more spacers to hold the first and second walls a predetermined distance apart. Spacer options include beads or pillars, ridges formed from an intermediate resist layer created by photopatterning, and other materials. Alternatively, spacers can be formed using evaporated materials such as silicon oxide or silicon nitride. Alternatively, layers of film, including flexible plastic films with or without adhesive coatings, can be used to form the spacer layer. Various spacer shapes can be used to form narrow channels, tapered channels, or partially enclosed channels defined by lines of pillars. With careful design, these spacers can be used to aid in the deformation of microdroplets, leading to subsequent microdroplet splitting and effective manipulation of the deformed microdroplets. Similarly, these spacers can be used to physically separate zones of the chip to prevent cross-contamination between droplet populations and to facilitate the correct direction of droplet flow when the chip is loaded under hydraulic pressure.

[0101] The first and second walls are biased using an A / C power supply attached to the conductor layer to provide a potential difference therebetween, suitably between 0 and 50 volts. Such oEWOD structures are typically employed in conjunction with an electromagnetic radiation source having a wavelength in the range of 400 to 850 nm, e.g., 550 nm, 620 nm, or 660 nm, and having an energy above the bandgap of the photoactive layer. Preferably, the photoactive layer is used with an incident intensity of radiation between 0.005 and 0.1 Wcm. -2 The electromagnetic radiation source is activated with a virtual electrowetting electrode position in the range of 0.005 to 0.1 Wcm. -2 or at a level of 0.005, 0.0075, 0.01, 0.025, 0.05 or 0.075 Wcm -2 In some embodiments, the electromagnetic radiation source can be 0.1, 0.075, 0.05, 0.025, 0.01, 0.0075, 0.005, or 0.0025 Wcm ―2It may be at a level less than that.

[0102] If the electromagnetic radiation source is pixelated, it is preferably supplied directly or indirectly using a reflective screen such as a digital micromirror device (DMD) illuminated with light from an LED or other lamp. This allows for very complex patterns of virtual electrowetting electrode positions to be rapidly created and destroyed on the first dielectric layer, thereby enabling microdroplets to be precisely moved along essentially any virtual path using tightly controlled electrowetting forces. Such electrowetting paths can be viewed as constructed from a continuum of virtual electrowetting electrode positions on the first dielectric layer.

[0103] The first and second dielectric layers may be composed of a single dielectric material or a composite of two or more dielectric materials, such as, but not limited to, Al2O3 and SiO2.

[0104] A structure may be provided between the first and second dielectric layers. The structure between the first and second dielectric layers may be made of, but is not limited to, epoxy, polymer, silicon, or glass, or a mixture or composite thereof, and may have straight, angled, curved, or microstructured walls / surfaces. The structure between the first and second dielectric layers may be connected to the upper and lower composite walls to create a sealed microfluidic device and define channels and regions within the device. The structure may occupy the gap between the two composite walls. Alternatively or additionally, the conductor and dielectric may be deposited on a shaped substrate already having walls.

[0105] [Microdroplet] The methods of the present invention require the manipulation of at least one microdroplet within the device, which generally refers to a droplet of liquid, such as an aqueous liquid, containing, for example, cells / cells, reporter entities, or a combination of the two. Microdroplets provide a partition that can separate biological species or reactions. They enable the possibility of single-cell manipulation and are suitable for high-throughput experiments. Microdroplets can encapsulate cells and / or reporter entities and keep them separate until merging is desired.

[0106] The microdroplets may be of any shape or size, preferably spherical or cylindrical. The size of the microdroplets may be between 20 and 600 μm, but may also be 20, 30, 40, 50, 60, 80, 100, 120, 140, 150, 160, 180, 200, 220, 240, 250, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 550, 560, or 580 μm or greater. In some embodiments, the size of the microdroplets may be less than 600, 580, 560, 550, 540, 520, 500, 480, 460, 440, 420, 400, 380, 360, 340, 320, 300, 280, 260, 250, 240, 220, 200, 180, 160, 150, 140, 120, 100, 80, 60, 50, 40, or 30 μm. Multiple microdroplets may be merged to form larger droplets. Alternatively, larger droplets may be split to form smaller sized microdroplets (daughter microdroplets).

[0107] If a droplet is too small relative to the height of the microfluidic device, it will not make contact with either sidewall within the microfluidic chamber and therefore will not be able to move by the oEWOD. In contrast, droplets that are large relative to the geometry of the device will move poorly and / or too slowly within the microfluidic device, or will simply obstruct or merge with other droplets of the correct size, disrupting other operations.

[0108] In general, microdroplets typically consist of an aqueous phase containing the contents of the microdroplet (cell(s), nutrients, reporter entities, assay reagents, hydrogel bead polymers, etc.). In some cases, microdroplets further consist of an internal phase of an immiscible fluid surrounding the aqueous material, forming multiple emulsions such as double emulsions and droplet-in-droplet emulsions.

[0109] In microfluidic devices, microdroplets are separated from each other by an immiscible carrier phase or fluid. Suitable carrier phases include oils such as silicone oil, mineral oil, or fluorocarbon oil. Exemplary fluorocarbon oils include HFE7500, HFE7700, or FC-40. The carrier phase may further contain surfactants and other additives to maintain the stability of the microdroplets. The surfactant localizes at the microdroplet / carrier interface, thereby stabilizing the microdroplets by reducing surface tension. When the weight ratio of aqueous microdroplets to carrier is low, the microdroplets tend to shrink over time, leading to a loss of reactivity within them. One way to counteract this effect is to use a hydrated carrier phase. Generally, because the above-mentioned carriers do not have a high capacity to dissolve water, hydration is preferably achieved by forming micelles or secondary microdroplets of water or aqueous buffer within the carrier phase. This buffer may be the same or different in composition from the microdroplets themselves. In some embodiments, these micelles or secondary microdroplets contain up to five times the salt content of the microdroplets themselves and can optionally contain glycerol. Typically, these micelles and secondary microdroplets are an order of magnitude smaller. In some circumstances, the carrier phase may contain lipophilic compounds that can diffuse in and out of the dispersion phase droplets.

[0110] The microdroplets used in the disclosed methods contain various components depending on which panel they are present in. The microdroplets are aqueous in nature and may contain additional components, such as components to provide a support structure, e.g., gels or coated microspheres. They may further contain cell culture media in formulations containing nutrients, energy sources such as carbohydrates, buffers, etc.

[0111] The disclosed method involves using microdroplets containing at least a single cell. The cell is the cell to be assayed or tested for selection, optionally based on the characteristics of the cell. Given that the target to be selected is a cell's characteristic, the initial microdroplet generally contains a single cell. Therefore, the disclosed method involves selecting only microdroplets for a panel containing a single cell and discarding empty or multi-occupied cells. After this step, the cell can divide, and the microdroplet now contains at least one cell.

[0112] The microdroplets containing at least one cell form a panel of microdroplets.

[0113] A microdroplet panel is formed by a plurality of microdroplets. A microdroplet panel is a collection, group, or array of microdroplets, each microdroplet having a similar content. For example, in a panel of microdroplets containing cells, each of the microdroplets contains at least one cell, optionally of the same type (e.g., B cells). A microdroplet panel may alternatively contain cell culture medium, reporter entities, selected cells, expanded cells, nucleic acids, etc. A microdroplet panel may contain, for example, 50 to 700,000 microdroplets.

[0114] In the methods of the present disclosure, a panel of microdroplets comprising at least one reporter entity is provided. Such microdroplets may contain at least one reporter entity as further defined herein.

[0115] The disclosed methods involve the selection of a subset of a panel of microdroplets, which may then form a new panel of microdroplets for further use in the disclosed methods.

[0116] The microdroplets are manipulated within the device using real or virtual electrowetting electrodes. The microdroplets may be manipulated into arrays to facilitate arranging merging, splitting, and detection events. The merging and splitting events may be performed using real or virtual electrowetting electrodes. Any suitable layout may be used to manipulate the microdroplets to achieve the desired method. The microdroplets may also be manipulated using other methods, such as acoustic methods.

[0117] Microdroplets are generally prepared by encapsulating the desired components (e.g., cells, reporter entities) in an aqueous formulation. Microdroplets are formed by emulsification techniques. In one example, the emulsification is vortex emulsification. In another example, the emulsification is step emulsification. Microdroplets may be generated on or adjacent to a microfluidic device.

[0118] [cell] The disclosed methods are for selecting cells with specific or desired properties. For the avoidance of doubt, the cells are biological cells. The cells may be natural. The cells may be artificial. The cells may be microcells. The methods of the present invention may be cell-free or may use parts of cells, such as the nucleus and / or mitochondria. The methods of the present invention may use liposomes. The cells may be obtained from any suitable source, such as a cell sample from a human or animal, plant, or microorganism.

[0119] The cell may be of human or animal origin, optionally mammalian. The cell may be a plant cell, insect cell, fungal cell, bacterial cell, or amoeba cell. It may also be a cell fusion such as a hybridoma.

[0120] The cells may be obtained from cell cultures, for example, cultures of stem cells, pluripotent cells, or genetically engineered cells.

[0121] If the cells are derived from a sample / biological sample, it may be a human, animal, environmental (natural, artificial, or modified), or food sample containing at least one type of cell. The sample / biological sample may be selected from stool, peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, earwax, breast milk, bronchoalveolar lavage fluid, semen, prostatic fluid, Cowper's fluid or pre-ejaculatory fluid, female ejaculate, sweat, feces, hair, tears, bladder fluid, pleural and peritoneal fluid, pericardial fluid, lymph, fibrillary fluid, chili, bile, interstitial fluid, menstrual fluid, pus, sebum, vomit, vaginal fluid, mammary gland secretions, mucosal secretions, stool, pancreatic juice, sinus lavage, bronchopulmonary aspirate, blastocyst fluid, and umbilical cord blood. Alternatively, the sample / biological sample may be obtained from a tissue specimen.

[0122] The cells may be isolated from a patient or individual, and the methods described herein can be used to screen for such cells and return them to the patient (autologous cell transplantation). Cells may also be isolated from an individual and selected for administration to the patient (allogeneic cell transplantation).

[0123] In some embodiments, a panel of microdroplets containing at least one cell contains cells of the same type, e.g., lymphocytes such as T cells. Thus, cells may be preselected prior to inclusion in the microdroplets. However, some contamination of any biological cell may occur, and a microdroplet may contain cells of a different type, e.g., B cells when T cells are the desired type. The disclosed method rapidly identifies such microdroplets and prevents them from being selected into a subset of microdroplets.

[0124] In some embodiments, the panel of microdroplets may contain a variety of cell types, such as when screening environmental samples for the presence of unknown bacterial cells.

[0125] The cells may be human or mammalian cells. The cells may be of any suitable type derived from any tissue, such as an organ or tissue of the body.

[0126] The cell may be an immune system cell, including monocytes, macrophages, osteoclasts, neutrophils (polymorphonuclear leukocytes), dendritic cells, microglial cells, mast cells, T cells (including helper T cells, regulatory T cells, cytotoxic T cells, and natural killer T cells), B cells, natural killer cells, hematopoietic stem cells, and the like.

[0127] The cells may be pluripotent cells or stem cells, isolated or prepared by culture techniques. Pluripotent stem cells may be reprogrammed to mature cell types.

[0128] The cells may be genetically engineered prior to encapsulation in the microdroplets. The cells may be genetically engineered after encapsulation in the microdroplets.

[0129] Genetic engineering of cells can be by any suitable method, including transduction (viral gene transfer), gene editing (using nucleases such as zinc finger nucleases, TALEN, CRISPR / Cas9 base and prime editing), non-viral gene transfer (such as nanoparticle transfer), gene knockdown using RNA, such as gene silencing or activation, gene knock-in and genetic manipulation, or optogenetics. Genetic engineering generally involves introducing genetic elements into cells by any suitable means.

[0130] Genetic engineering involves simply introducing a mutation-causing entity into the genome of a cell to see if the random mutation results in a desired cellular characteristic. The disclosed method allows for the screening of large numbers of cells with random or deliberate mutations, and the selection of cells with desired characteristics after mutation. This allows for the optimization of cell selection, particularly cell-based manufacturing. This can be referred to as "directed evolution" of cells.

[0131] The genetic element optionally includes a nucleic acid operably linked to a promoter, and a nucleic acid sequence encoding a desired product, such as a CAR.

[0132] The genetic element can be a viral vector containing a sequence of interest that is desired to be expressed or contained in the cell. The nucleic acid can be a non-viral "naked" vector, such as a minicircle, for expression in the cell. The nucleic acid can also contain gene editing components for editing genes in the cell. The genetic element can contain a desired sequence to allow for any desired genetic engineering of the cell, such as expressing a new cell surface molecule.

[0133] In one embodiment, cells may be encapsulated in microdroplets and genetically engineered. In this embodiment, a panel of microdroplets containing at least one cell and a panel of microdroplets containing at least one genetic element are prepared. The disclosed method may therefore comprise the further step of merging a microdroplet from the panel containing at least one cell with a microdroplet from the panel containing at least one genetic element to form a panel of merged microdroplets. Such microdroplets contain at least one genetically engineered cell according to the selection method described herein. The success of the genetic engineering is then determined by assay, and unmodified cells are discarded.

[0134] Genetic engineering involves providing a genetic sequence under an inducible promoter, which stimulates the engineered cells to produce the product encoded by the genetic sequence.

[0135] When preparing a panel of cell-containing microdroplets, some microdroplets may be empty because only the medium is encapsulated during the encapsulation step, or because cells die between the encapsulation and method steps. Alternatively, the segmentation step may result in microdroplets lacking cells. This process results in a population of droplets with encapsulated cells that have an occupancy rate described by Poisson statistics.

[0136] The disclosed methods can be used in conjunction with actively controlled droplet generation methods to improve the efficiency of single-cell encapsulation. Optionally, the encapsulation process produces panels of microdroplets containing single cells. Encapsulation of single cells allows for clonal expansion during the process.

[0137] However, some available encapsulation processes are inefficient, and in these systems, the number of cells encapsulated per droplet is described by Poisson statistics, reducing the percentage of droplets containing the desired number of cells and, consequently, the effective rate of encapsulating single cells. Therefore, the encapsulation process selected by the device user may result in a minority of droplets containing one or more cells. However, those skilled in the art are tolerant of this situation, and these empty microdroplets are subjected to the same splitting and merging operations and ignored for selection purposes. Thus, the selection method described herein is flexible and can be used in conjunction with a variety of cell encapsulation methods.

[0138] Thus, when the term "panel of microdroplets containing at least one cell" is used, those skilled in the art will understand that a proportion may lack one or more cells. The disclosed methods can rapidly identify such empty microdroplets where cells are needed and discard them from further consideration. Thus, in a panel of microdroplets containing at least one cell, substantially all of the microdroplets may contain at least one cell, but some or all may be empty. A panel of microdroplets may also intentionally include empty microdroplets as controls or position markers.

[0139] The panel of microdroplets may contain control microdroplets, such as microdroplets containing cells with known properties or known products / molecules such as antibodies, which may provide positive controls for the reporter entities. Negative controls may also be included, which may be cells or products / molecules that are not expected to produce a positive result with the reporter entity, or may be empty droplets or droplets of medium.

[0140] Similarly, when the term "a panel of microdroplets containing one or more reporter entities" is used, those skilled in the art will understand that a proportion may lack one or more reporter entities. Although the methods of the present disclosure identify such empty microdroplets and remove all that do not have the required reporter entities, droplets without the required reporter entities may still be present in the assay. In some cases, droplets without reporter entities may be introduced into a subset of the panel to act as negative controls. Substantially all of the panel may contain a reporter entity.

[0141] [Cell characteristics] The selected cells are selected based on one or more characteristics.

[0142] Such characteristics include the presence and / or absence of any one or more of the following: These include cell surface molecules, activation profile, proliferation capacity, secretion capacity, affinity of secreted products, functional behavior of secreted products, ability to make intracellular products, viability, morphology, and / or cell-cell interaction capacity (such as cell killing, cell activation, or cell aggregation).

[0143] Cells can be selected based on any desired combination of properties and can be tested substantially simultaneously (multiple times) or sequentially. Thus, one or more reporter entities may be required, or one or more reporter microdroplet panels containing at least one reporter entity may be required. Successive selection rounds can select a subset of microdroplets containing at least one cell.

[0144] In one embodiment, the characteristic to be determined is the presence or absence of one or more cell surface molecules or markers. All cells express characteristic molecular markers (e.g., protein, lipid, glycan markers, etc.), which help distinguish between cell types. Specific combinations of cell surface molecules or markers can be used to identify specific cells.

[0145] In one embodiment, the cell surface marker is a CD molecule. CD (cluster of differentiation) stands for cluster of differentiation (also known as cluster of specification or classification determinants, often abbreviated as CD). Cell phenotyping is possible using CD molecules. Such markers are often associated with specific immune functions, but are not limited to these. For example, various T cells can be identified by the presence of CD3, CD4, or CD8, B cells by CD19 or CD20, and NK cells by CD56.

[0146] In one embodiment, the cell surface molecule or marker is a cell surface receptor, a cell surface transporter, a cell adhesion protein, a cell surface signaling molecule, or a cell surface molecule involved in cell-cell interactions.

[0147] In one embodiment, the cell surface marker is an artificial, synthetic, or chimeric cell surface marker that the cells are genetically engineered to produce.

[0148] Optionally, the artificial cell surface marker is a chimeric antigen receptor (CAR). CARs may consist of four domains: an antigen recognition domain, an extracellular hinge domain, a transmembrane domain, and an intracellular signaling domain. The antigen recognition domain is exposed on the outside of the cell and interacts with potential target molecules, allowing the modified cell to target any cells expressing the appropriate molecule. Antigen recognition domains are typically derived from the variable regions of monoclonal antibodies linked as single-chain variable fragments (scFv), but can also be derived from engineered single domains. Antibody-free approaches have also been used to develop CARs, such as using ligand / receptor pairs that typically bind to each other.

[0149] Cell surface markers (natural or modified) may be used to select cells during selection to define a first subset of cells, or a second subset of cells, or further subsets of cells.

[0150] In one embodiment, cell selection may be based on a cell surface marker or molecule, and thus the reporter entity may interact with a cell surface marker or molecule.

[0151] In one embodiment, the characteristic to be determined is the presence or absence of one or more secretions from a cell. Many cells are capable of secretion; for example, mammalian cells constantly excrete proteins into the extracellular matrix. Different specialized cells have specialized secretions; for example, immune cells express secreted proteins such as cytokines, immunoglobulins, ligands, and receptors. Cells secrete proteins, including glycoproteins or lipoproteins, chemicals, nucleic acids, polymers, or molecules. Secretion is the process of moving a substance from the inside to the outside of a cell, and can use various pathways depending on the cell type. For example, extracellular vesicles may be involved in any of the embodiments of the present invention. Bacterial cells may rely on secretory capabilities to adapt and survive.

[0152] The secreted product may be a natural secretion of the cell, e.g., an immunoglobulin such as an antibody from a B cell, or the secreted product may be the result of genetic engineering of the cell by introducing a gene or coding sequence for the secreted product. In one embodiment, the cell is a B cell or a hybridoma and the secreted product is a monoclonal antibody.

[0153] The secreted product may or may not have a desired property, such as binding ability, which can be assayed by one or more reporter entities.

[0154] In one embodiment, cell selection may be based on secretions from the cells. Thus, the reporter entity interacts with the cell secretions. The cell secretions can be separated from the cells by dividing the microdroplets into a reference microdroplet containing at least one cell and a test microdroplet containing the medium in which the secretions are present.

[0155] In one embodiment, the characteristic to be determined is the presence or absence of one or more intracellular products produced by the cell. Most, if not all, cells can be used to produce proteins, including glycoproteins and lipoproteins, but some cells, particularly bacteria, can make chemicals, nucleic acids, and other polymers. Genetically engineered cells can be modified to make a desired product, which may be retained intracellularly rather than secreted, but must be lysed to release it.

[0156] The intracellular product may or may not have a desired property, such as catalytic activity, which can be assayed by one or more reporter entities.

[0157] The product may be a native product of the cell or may be the product of genetic engineering of the cell by introducing a gene or coding sequence for said product.

[0158] In one embodiment, cell selection is based on the detection of an intracellular product. Accordingly, the reporter entity may interact with said product. The product may be detected in situ by the reporter entity entering the cell, or the product may be separated from the cell by cell lysis within the test microdroplet.

[0159] The intracellular product may be any suitable biological or chemical molecule. The intracellular product may be a protein, such as a hormone, an enzyme, a cell signaling molecule, a signal transduction molecule, or an immunoglobulin. The intracellular product may be a nucleic acid, such as RNA, DNA, or a hybrid thereof. The intracellular product may be a chemical, such as a pesticide, a toxin, an antibiotic, a fuel, a pharmaceutical, a vaccine, or an antiviral.

[0160] In one embodiment, cells are selected based on their proliferation potential. Such selection can be determined by detecting the presence of a cell division event (an increase in the number of cells in the microdroplet) or based on proliferation markers. Proliferation markers can be detected using reporter entities specific to the markers.

[0161] In one embodiment, cells are selected based on their morphology. Such selection can be determined, for example, by visual inspection of the cells using appropriate optical detection means. Cell morphology can include one or more of the following: size, shape, presence or absence of cell adhesion, presence or absence of cell membrane protrusions such as blebs, and presence or absence of intracellular entities such as vacuoles.

[0162] In one embodiment, the cells are selected based on the result of interaction with a reporter entity, where the reporter entity is a cell. The interaction occurs between the cell and the reporter entity, or between the secreted product and the reporter entity. The reporter entity cells can be any cell type, such as tumor cells, cell lines, tumor-like cells, bacterial cells, or immune system presenting cells (e.g., antigen-presenting cells). In response to the reporter entity cells, the selected cells can be activated, induced to secrete molecules, and / or promote cellular activation or death of the reporter entity cells, or the secreted product can bind to them.

[0163] Selection of cells based on the outcome of direct or indirect (via secreted products) interactions with reporter cells can determine properties relevant to therapeutic guidance.

[0164] Cells can be selected based on any number of desirable characteristics and / or the absence of undesirable characteristics, which may be based on any combination of the characteristics described herein.

[0165] [Reporter] The methods described herein involve the use of at least one panel of microdroplets containing at least one reporter entity. Any suitable number of panels may be employed, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, each panel containing one or more reporter entities and each panel being different.

[0166] Any panel of microdroplets containing at least one reporter entity may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more reporter entities.

[0167] The reporter entity is any suitable entity that can interact with a cell, a cell product, or a cell secretion to produce a detectable change. The reporter entity may be any one or more of the following entities or derivatives thereof: i. Antibodies, ii. Antigen, iii. receptors, iv. ligands, v. Substrate; vi. enzymes, vii. Ligands, viii. Diffusion, ix. Cells, x. part of a cell, xi. extracellular vesicles, xii. liposomes, xiii. polymers, xiv. Chemicals, xv. Drugs, xvi. FRET reporter, xvii. Chemiluminescent substances, xviii. tissue samples; xix. Viruses or bacteriophages, xx. cytokines, and / or xxi. Protein It can be any one or more of:

[0168] The reporter entity can interact with a cell or a part thereof or a derivative thereof (such as a product or secretion), and this interaction results in a detectable change in the reporter entity. Such changes can be binding, annealing, aggregation, separation, amplification, conformation, activation, and / or destruction. The reporter entity is suitable for directly monitoring the detectable change, such as in the case of a reporter cell. The reporter entity can be associated / conjugated to a protein-complementary ligand, such as a bead or a label, such as a visible, colorimetric, luminescent, fluorescent, phosphorescent label, split-fluorescent, split-luminescent, or fluorogenic label.

[0169] Alternatively or additionally, the change in the reporter entity may be detected by a second entity, labeling it (visible, luminescent, fluorescent, or phosphorescent label). The second entity, for example, detects binding of the reporter entity to the cell and can be a secondary antibody or derivative thereof. Alternatively or additionally, the reporter entity may be conjugated or linked to an enzyme, and a substrate for that enzyme may be added as a second entity to elicit a visual, colorimetric, or fluorescent signal. Those skilled in the art will be aware of numerous assay means for determining the presence of a particular analyte.

[0170] Typically, the reporter entity is an antibody or a fragment or derivative thereof, such as an antigen-binding fragment (Fab), a single-chain variable fragment (scFv), a miniaturized antibody, or an affibody.

[0171] In one embodiment, the test panel of microdroplets is contacted with a panel of microdroplets containing multiple reporter entities. Thus, the cells are referred to as "multiple element reporters." These may be assessed in a "multi-element reporter assay" or "multi-reporter cell-surface marker assay," which is an assay in which many of the features are examined by a system of reporter entities, each capable of producing an individual detectable change.

[0172] In one embodiment, at least one of the reporter entities comprises at least one reporter cell. The cell may be any suitable cell type, such as a tumor cell, a cancer cell, a cell line, a tumor-like cell, a presentation cell, an accessory cell, or an "off-target" cell with which interaction is undesired. A change in the reporter cell is detected. Such a change may be cell death or destruction, cell activation, or cell aggregation. These changes may be observed visually (under appropriate magnification) or through a second entity that detects the presence of a suitable analyte in the microdroplet, such as an internal cell component, a cell signaling molecule such as a cytokine, or the increased presence of a cell surface molecule. Alternatively or additionally, the reporter cell may be genetically engineered to contain at least one reporter gene associated with an event, such as receptor activation. When this event occurs, an intracellular signaling cascade activates the reporter gene, generating a measurable signal (e.g., a luciferase gene produces light when the required substrate is provided).

[0173] In one embodiment, at least one of the reporter panels comprises at least one or more of the following: i. antigen- or ligand-binding beads and fluorescent dye-conjugated secondary antibodies; ii. secondary antibody-conjugated beads and fluorescent dye-conjugated antigen or ligand, or iii. Antigen or ligand bound to the carrier surface and fluorescent dye-conjugated secondary antibody beads It includes at least one of the following.

[0174] Such reporter entities are useful for detecting molecules that have binding capacities such as antibodies, receptors, enzymes, etc. One skilled in the art of assay design will know all the possible combinations of reporter entities and how properties can be detected and communicated.

[0175] For CAR-T cells, the following entities are suitable characteristics: CD4, CD8, CD19, scFv domain markers (for specificity and / or affinity screening), B cell markers (or markers of other unwanted contaminating cells), and / or differentiation markers (to select for relevant subpanels).

[0176] [detection] The change in the reporter entity can be detected. As mentioned above, detection can be simple observation of a visible event, albeit under magnification (microscope). Thus, the detection event can be visual detection of a change.

[0177] In some embodiments, detection is optical, e.g., the labels employed are visual, chromogenic, luminescent, fluorescent, or phosphorescent. Any suitable detection technique can be employed.

[0178] The detection system can be used to screen the signals of each individual, independent reporter entity in each microdroplet. In one example, a panel of merged microdroplets is imaged, and any changes thereto are detected and quantified. The results of such assays can be used to determine the characteristics (phenotype) of cells, allowing individual cells to be screened for the markers they express, the activities they possess, or the products they may produce or secrete.

[0179] The detection system enables high-quality imaging over a wide area, for example, using a highly sensitive camera. In some embodiments, the detection system supports multi-channel fluorescence detection, enabling dark-field imaging, bright-field imaging, and detection of cell morphology. High-quality imaging is supported by multiple objective lenses, including high-NA (60x) lenses. Signals of different reporter entities are detected and spatially quantified for intensity for individual microdroplets, facilitating high-throughput screening of cells.

[0180] [choice] The methods disclosed herein enable the selection of a subset of cells depending on their characteristics. Cells in a microdroplet panel are selected based on specific characteristics, such as cell surface markers / molecules, intracellular products, or secreted products from the cells. The characteristics may involve the specificity / activity of the secreted or intracellular products. The selected or sorted cells form a subset of the microdroplet panel. The remaining microdroplets containing at least one cell are discarded or ignored. This selected subset of microdroplets may be subjected to further steps in which the characteristics of the cells are further investigated or assayed, resulting in the selection of a further subset. When the final step of the method described herein is performed, a final subset of microdroplets containing at least one cell contains the selected cell.

[0181] The selected cells are then dispensed from the device. Once selected and dispensed from the device, the cells may be subjected to further assays or may be cultured to promote cell maintenance and / or proliferation. After dispensing, the cells may optionally be prepared for infusion or re-infusion into a patient. After dispensing, the cells may be expanded for use in the production of therapeutics such as immunoglobulins or pharmaceuticals.

[0182] In one embodiment, the selected cells are present in a reference panel of microdroplets containing at least one cell. The reference panel includes microdroplets that correlate with the microdroplets in the test panel because they originate from the same precursor microdroplet. In one embodiment, division of the precursor microdroplets produces reference panel microdroplets containing at least one cell and test panel microdroplets containing at least medium. The medium may contain secretions from the cell, which is a characteristic of the cell. In an alternative embodiment, the precursor microdroplets produce reference panel microdroplets containing at least one cell and test panel microdroplets containing one cell. The reference panel of microdroplets thus contains cells that are clones or clonal copies of the panel of microdroplets containing at least one cell being evaluated in the assay to determine the cell's characteristic. The selection step may involve lysing the cells in the test panel, which is undesirable when selecting cells for further expansion.

[0183] In this way, in either embodiment, the results obtained from the assay of the microdroplet test panel can be directly correlated with the cells of the reference panel, and it is the cells from the reference panel that have been selected. The advantage of such a step is that the cells are not exposed to some reporter entities that may be harmful to the cells or that may be undesirable in the preparation of cells to be used for therapeutic purposes, such as exposure to tumor cells.

[0184] Cells may be selected based on the presence of a property, the absence of a property, or a mixture of both. Thus, cells, extracts, or secretions therefrom can be examined or assayed for multiple properties using the methods described herein. In one embodiment, cells are tested for multiple properties simultaneously in a single step. Such multiplex testing is feasible when changes in reporter entities are detected using different means, such as fluorescent, luminescent, or phosphorescent labels or events. Detection means may also include protein complementation events, such as split-fluorescence, split-luminescence, or fluorogenic ligands.

[0185] The selected cells may be further dispensed from the device and cultured to undergo cell division before further use. In one embodiment, the cells are for infusion into a patient. In some embodiments, after dispensing a panel of selected microdroplets, the cells contained therein are treated to expand the population and / or culture clones.

[0186] After dispensing from the device, the cells and / or non-cellular bodies, such as secretions, metabolites, etc., contained in a subset of the microdroplets may be one or more of the following: i. Mass spectrometry; ii. Surface plasmon resonance; iii. High performance liquid chromatography, and / or iv. genetic analysis, including nucleic acid sequencing and PCR amplification; Further analyses may be imposed, including but not limited to any one or more of the following:

[0187] [Culture medium] For the method of the present disclosure, cells are encapsulated in microdroplets. Cells according to any embodiment are generally provided in a medium, usually an aqueous medium. The medium is simply a substance for culturing cells. This medium may be a minimal medium containing sufficient substances to support the cells, and the contents of such a minimal medium depend on the type of cells to be encapsulated. The required components vary depending on the type of cell being studied, and those skilled in the art will understand how to identify a medium suitable for the task.

[0188] Generally, cell culture media may contain a carbon source (e.g., a sugar such as glucose), water, one or more salts, a pH buffer, an amino acid source, and nitrogen. The media may be supplemented, if necessary, with molecules or agents that support or promote cell division or cell survival. This may include appropriate nutrients, growth factors, signaling molecules (e.g., cytokines), vitamins, salts, serum proteins, coenzymes, carbohydrates, gases, trace elements, and antibiotics.

[0189] An exemplary medium is Dulbecco's modified Eagle's medium (DMEM), which is a basal medium and does not contain proteins or growth promoters. Therefore, it requires supplementation to be "complete." It is typically supplemented with 5-10% fetal bovine serum (FBS). DMEM has proven to be widely applicable to mammalian cell culture and can also be used to culture hybridomas. An alternative medium is Eagle's minimum essential medium (EMEM). EMEM contains balanced salt solution, non-essential amino acids, and sodium pyruvate. Because EMEM is a non-complex medium, it is generally enriched with additional supplements or higher serum concentrations to make it compatible with a wide range of mammalian cell types. RPMI is a versatile medium with broad applicability to mammalian cells, particularly hematopoietic cells. RPMI is an improved version of McCoy's 5A, developed for long-term culture of peripheral blood lymphocytes and supporting the growth of a variety of cells in suspension. When supplemented with serum or serum substitutes, this medium has a wide range of applications for mammalian cells, including fresh human lymphocyte culture, fusion protocols, and the growth of hybrid cells such as mouse hybridoma cells for antibody preparation. It is also often used for the storage / suspension of peripheral blood mononuclear cells. Many cell culture media are available, and the one chosen should optimize the survival of the cell type of choice.

[0190] The disclosed method may involve dividing a panel of microdroplets containing at least one cell into a reference panel of microdroplets containing at least one cell and a test panel of microdroplets containing at least medium. Those skilled in the art will understand that some of the test panel microdroplets may also contain cells, particularly if cell division has occurred in a precursor microdroplet containing at least one cell.

[0191] In one embodiment, the microdroplet containing at least one cell has already been subjected to a division operation to create a reference panel of cells containing at least one microdroplet. This preserves clonal copies of the cells, allowing cells to be selected from the reference panel or microdroplet even if any subsequent steps stimulate the cells of the test panel, damaging them. In particular, this damage may occur during the process of lysing the cells to access genetic material or intracellular products.

[0192] When such division is employed to separate cells and medium, precursor microdroplets containing at least one cell are cultured under conditions that allow secretions to accumulate in the medium. Such conditions include providing a medium containing a secretory agent, such as a stimulatory or signaling molecule (e.g., cytokine), to promote secretion, or including cells, such as antigen-presenting cells, that interact with the cells to induce secretion. Alternatively, if the secretory product is genetically engineered to be produced by the cells, manipulations can be applied to condition the expression of the secretory product, such that expression is activated by the addition of a chemical, such as lactose. Such an appropriate signal is provided to initiate expression.

[0193] Once the cells are cultured and secretion is promoted, the panel of microdroplets can be split. The splitting operation may be performed using real or virtual electrowetting electrodes. In one embodiment, each microdroplet in the microdroplet panel splits to form at least two panels of daughter droplets. These daughter droplets may be approximately the same size, or the splitting may be asymmetric, with the microdroplets in one panel being larger in size. Once the microdroplets split, one daughter droplet may receive at least one cell, and such microdroplet forms a reference panel.

[0194] The presence of secretions in the medium can be confirmed as described herein. It can be useful to employ a series of merging and splitting events on the microdroplets of the test panel, such as merging one or more panels of aqueous microdroplets, so that the secretions in the test panel of microdroplets can be diluted or washed away.

[0195] The microdroplets containing the cells and medium can be maintained under conditions such as controlled environment and temperature that are optimal for cell maintenance, which involves providing a flowing stream of an immiscible carrier liquid containing saturating levels of one or more of nutrients, buffers, nitrogen, oxygen, and / or carbon dioxide, as described in WO 2020 / 104769.

[0196] Those skilled in the art will understand that a microdroplet containing at least one cell inherently also contains culture medium, as the microdroplet is aqueous in nature.

[0197] [Cell division] The disclosed methods may include allowing a panel of microdroplets containing at least one cell to undergo cell division. Cell division is the process by which a parent cell divides into two or more daughter cells. In this context, each daughter cell is genetically identical to the parent cell (in eukaryotes, the cell has undergone mitosis). Prokaryotes (bacteria and archaea) may undergo binary division, in which genetic material is evenly separated into two daughter cells. These cells are sometimes described as clones because their genetic material should be identical.

[0198] In one embodiment, the panel of microdroplets containing at least one cell is capable of undergoing clonal expansion, a process by which daughter cells arise from parent cells, a term generally applied to certain types of cells such as immune cells (e.g., NK cells, B and T lymphocytes).

[0199] In the disclosed methods, cell division or clonal expansion is permitted or encouraged within the panel of microdroplets. Cells may be provided with appropriate conditions to allow for said cell division, such as provision of an appropriate culture medium within the microdroplets, addition of relevant auxiliary molecules such as nutrients, cytokines, vitamins, activators, hormones, or growth factors. Those skilled in the art will recognize that the conditions required for cell division will vary depending on the cell type in question.

[0200] In one embodiment, the cells present in the panel of microdroplets containing at least one cell are immune cells, such as lymphocytes (NK cells, B cells, or T cells). Such immune cells may require activation to induce clonal expansion. Activation may involve signaling molecules, such as specific cytokines. Activation may alternatively or additionally involve binding to specific cell surface receptors on the immune cells. This can be achieved by binding with antibodies directed against these receptors. Alternatively, activation can be achieved using appropriate cells (antigen-presenting cells, APCs, etc.).

[0201] In one embodiment, a panel of microdroplets containing at least one T cell can include or be merged with a panel of microdroplets comprising at least one T cell activator, such as an anti-CD3 antibody or an anti-CD28 antibody.

[0202] In one embodiment, the cells may be minimally stimulated / activated so that minimal cell division occurs within the microdroplets.

[0203] If a cell does not activate or divide, the microdroplet may not be selected due to the lack of generation of further copies of that cell (clone). Thus, activation and / or division capacity may be properties that are selected for in the methods of the present disclosure.

[0204] Once the cells are cultured to promote cell division, the panels or microdroplets can be split. The splitting operation may be performed using real or virtual electrowetting electrodes. In one embodiment, each microdroplet in a microdroplet panel splits to form at least two panels of daughter droplets. These daughter droplets can be approximately the same size, or the split can be asymmetric, with the microdroplets in one panel being larger in size. Once the microdroplets split, each daughter droplet receives at least one cell. Microdroplets without at least one cell can be discarded or excluded from selection. The cells in the split microdroplets are substantially genetically identical, or clonal.

[0205] In one embodiment, after cell division, the panel of microdroplets containing at least one cell is divided into two panels: a reference panel of microdroplets and a panel of test microdroplets that can be further used in the methods described herein. The methods of the present disclosure allow for the selection of cells in the reference panel of microdroplets based on assays and / or tests for properties of cells in the panel of microdroplets containing at least one cell, as both panels contain genetically identical cells.

[0206] The methods described herein refer to "culturing" cells to allow for cell division. Such techniques simply involve growing cells in a microdroplet environment. As discussed herein, suitable cell culture media, nutrients, and other conditions favorable for growth are used. Those skilled in the art will be aware of cell culture techniques that promote cell growth and division.

[0207] [Identifying and Tracking Microdroplets] The microdroplets of the present disclosure can be suitably manipulated on a device such as that described herein. Control of the manipulation of the microdroplets can be performed by a computer. The location of the microdroplets on the device allows for identification of correlated or corresponding microdroplets, such as those derived from a fission event of a precursor microdroplet. This allows for the selection of a microdroplet from the reference panel when a reporter entity indicates a result in the test panel. Additionally, if one of the microdroplets generated from the fission event is recovered from the microfluidic chip and dispensed into an external microwell plate, the original precursor microdroplet of the fission event (maintained on the chip of the reference panel) can correspond to the results of an off-chip assay, such as DNA sequencing, PCR, or mass spectrometry, performed on the associated daughter droplet.

[0208] In one embodiment, there is no need to label or barcode the microdroplets on any of the panels.

[0209] The location of the microdroplet within the panel can determine the identity of the microdroplet. The location of the microdroplet and its identity can be controlled by software. Once a cell's characteristics are determined within a microdroplet merged with a reporter entity microdroplet, the results can be correlated with the corresponding reference panel microdroplet associated with the same cell.

[0210] Software control of the assay steps described above allows the sequence of operations to be adapted or modified depending on the exact requirements of a particular cell type or reporter assay. For example, for slow-secreting cells, the incubation period for reporter panel droplets to merge can be adjusted. Software algorithms can be implemented to implement assay steps that respond to time-dependent events occurring on the chip. In the case of cell division, image recognition software can be used to count the number of cells in a microdroplet and trigger a process to split the droplet once the cell number reaches a certain level. In some embodiments, the microdroplet is a droplet of fluid contained within a microstructure trap, such as a miniature valve structure or a fluid-holding pen within a sealed chamber structure in a microfluidic chip. In some embodiments, the process of splitting the droplet is performed by applying a mechanical structure or deforming the microstructure. In some embodiments, the droplet is split using acoustic manipulation.

[0211] In some embodiments, the process of merging the microdroplets is performed by colliding the microdroplets with each other through the use of mechanical actuation, electrophoresis, acoustic waves, or centrifugal force.

[0212] [method] The present disclosure relates to methods that allow for the selection of cells, and in particular to methods of selecting cells that allow for further use of the cells after selection is complete.

[0213] In one embodiment, the method is as follows.

[0214] According to one aspect of the present disclosure, there is provided a method for selecting cells in an EWOD or oEWOD device, the method comprising the steps of: i. providing a test panel of microdroplets comprising at least medium or medium and at least one cell; ii. providing at least one reporter panel of microdroplets containing one or more reporter entities; iii. merging the test panel microdroplets with at least one reporter panel microdroplet to form a panel of merged assay microdroplets; and iv. monitoring the panel of assay microdroplets with a detection system capable of detecting a change in said reporter entity based on the presence of at least one property; and v. selecting a subset of microdroplets from the panel based on a change in reporter entity, said subset containing the selected cells; Equipped with a test panel of microdroplets containing at least medium is prepared by dividing a panel of microdroplets containing at least one cell and medium, and further preparing a reference panel of microdroplets containing at least one cell; and Further, the test panel of microdroplets comprising at least one cell is divided into at least two panels of microdroplets before or after any of steps (i) to (v), the two panels being a test panel of microdroplets comprising at least medium or medium and at least one cell, the test panel of microdroplets being suitable for merging with the microdroplets of the reporter entity panel, and a reference panel of microdroplets containing at least one cell.

[0215] Thus, cells may be selected based on one or more properties.

[0216] Alternatively, there is provided a method for selecting cells in an EWOD or oEWOD device, the method comprising the steps of: i. providing a panel of microdroplets comprising one or more cells and medium; ii. dividing the panel of microdroplets into at least two panels of microdroplets: a reference panel of at least one microdroplet comprising one or more cells, and a test panel of at least one microdroplet comprising at least the medium; iii. providing a reporter panel of at least one microdroplet comprising one or more reporter entities; iv. merging the test panel microdroplets with at least one reporter panel microdroplet to form a panel of merged assay microdroplets; v. monitoring the panel of assay microdroplets with a detection system capable of detecting a change in said reporter entity based on the presence of at least one property; vi. Selecting a subset of microdroplets from the reference panel based on changes in reporter entities in the corresponding assay microdroplet panel, said subset comprising the selected cells. Equipped with.

[0217] According to another aspect of the present disclosure, there is provided a method of selecting cells by use of an EWOD or oEWOD device, the method comprising the steps of: i. providing a panel of microdroplets comprising at least one cell and a medium; ii. providing a panel of at least one reporter microdroplet containing one or more reporter entities; iii. merging microdroplets of at least one cell-containing panel with microdroplets of at least one reporter panel to form a panel of merged microdroplets; iv. monitoring the panel of merged microdroplets using a detection system capable of detecting a change in said reporter entity based on one or more properties of the cells and selecting a subset of microdroplets based on said change in reporter entity to form a selected panel of microdroplets comprising cells; v. harvesting a selected panel of cell-containing microdroplets and repeating steps (ii)-(iv) one or more times, wherein one or more different reporter entities are used in step (ii); vi. Selecting cells based on the final panel of microdroplets selected in step (v); Equipped with wherein, prior to step (iii) and / or step (v), the panel of microdroplets containing at least one cell is divided into at least two panels of microdroplets, said two panels comprising: a. a test panel of microdroplets comprising at least a medium and suitable for merging with the microdroplets of the reporter entity panel; and b. A reference panel of microdroplets containing at least one cell is.

[0218] The following features are applicable to any of the methods described herein.

[0219] The microdroplets of any panel may be subjected to multiple merging and splitting operations to dilute or "wash" the contents of the microdroplets. Such operations include merging the panel of microdroplets with a panel of microdroplets containing medium, buffer, or a suitable aqueous solution, and splitting the merged microdroplets into at least two panels of daughter microdroplets. This step may be performed sequentially to further dilute the contents.

[0220] In one embodiment, the microdroplets in the various panels are merged in pairs, where a pair (two) of microdroplets merge to form a new microdroplet. This pairing step may be performed more than once, with multiple microdroplets combining to form a merged microdroplet. Thus, the step of merging microdroplets may ultimately involve 2, 3, 4, 5, 6, 7, 8, 9, 10, or more microdroplets, each merged sequentially or nearly simultaneously.

[0221] When a set of microdroplets is merged, one microdroplet from each panel is merged at approximately the same time to form a new merged panel of microdroplets. The set of microdroplets for this merge operation may include 2, 3, 4, 5, 6, 7, 8, 9, 10 or more microdroplets, each from a different panel.

[0222] When a panel of microdroplets containing at least one reporter entity is prepared, each panel may contain microdroplets containing at least one reporter entity, where each reporter entity is used to determine the presence or absence of a particular cellular characteristic. Alternatively, a single panel of microdroplets containing multiple reporter entities may be employed in the merging step.

[0223] In one embodiment, step (ii) involves the use of reporter entities that determine cell surface properties of the cells, such as the presence or absence of particular cell markers or molecules. The test panel of microdroplets can then be merged with a further reporter panel of microdroplets, involving the use of reporter entities that determine properties of cell-cell interactions, such as the ability to activate or kill another cell type.

[0224] Alternatively, in another embodiment, these steps are reversed, with cells being characterized first with a reporter entity that determines a property of the cell-cell interaction, e.g., the ability to activate or kill another cell type, and second with a reporter entity that determines a cell surface property of the cell, e.g., the presence or absence of a particular cell marker.

[0225] When multiple steps of merging a test panel of microdroplets with a reporter panel of microdroplets are used, a splitting step can be employed before or after such multiple steps, and a reference panel of cell-containing microdroplets is formed at any appropriate point in the method, which may therefore represent a subset of the cells initially obtained.

[0226] In one embodiment, before merging the panel of microdroplets containing at least one cell with the panel of reporter entities containing at least one reporter entity, the microdroplets are split into at least two panels of microdroplets, with a reference panel of microdroplets being retained to contain at least one cell. These cells are not in contact with reporter entities that may be harmful to the cells or prevent further use of the cells, such as in therapy. The other panel of constructed microdroplets (the test panel) can contain culture medium or culture medium and at least one cell. Prior to the splitting step, the cells can be cultured under conditions that allow either cell division or cell secretion.

[0227] In certain embodiments, the method comprises:

[0228] 1. A method for selecting immune cells, such as T cells, transfected with a nucleic acid encoding a CAR using an EWOD or oEWOD device, comprising the steps of: i. preparing a panel of microdroplets comprising at least one immune cell transfected with a nucleic acid encoding a CAR; ii. providing at least one panel of reporter microdroplets comprising one or more reporter entities specific for cell surface markers; iii. merging the microdroplets of the panel comprising at least one cell with the microdroplets of the reporter panel to form a merged panel of microdroplets; iv. monitoring the merged panel of microdroplets using a detection system capable of detecting a change in said reporter entity based on the presence of one or more cell surface markers and selecting a subset of microdroplets based on said change in reporter entity to form a selected panel of microdroplets comprising at least one cell; v. collecting a panel of selected microdroplets containing at least one cell and optionally repeating steps (ii) to (iv) one or more times, wherein step (ii) uses one or more different reporter entities; vi. Selecting cells based on the final panel of microdroplets selected in step (iv) or (v); Equipped with Prior to step (iii) and / or step (v), the panel of microdroplets containing at least one cell is cultured to allow cell division and then divided into at least two panels of microdroplets containing at least one cell, the two panels comprising: a. a test panel of microdroplets containing at least one cell; and b. A reference panel of microdroplets containing at least one cell is.

[0229] The immune cells may be T cells. Suitable detectable markers for T cells include any one or more of the following: CD4, CD8, CD19, scFv domain markers (for specificity and / or affinity screening), B cell markers (or markers of other unwanted contaminating cells), and / or differentiation markers (to select relevant subpanels), Examples include:

[0230] Cells that do not divide, become activated, or do not have the necessary cell killing properties (T cells) are discarded.

[0231] In a different embodiment, the method provides: 1. A method for selecting genetically modified hematopoietic stem cells using an EWOD or oEWOD device, comprising the steps of: i. providing a panel of microdroplets containing at least one genetically modified hematopoietic stem cell; ii. providing a panel of at least one reporter microdroplet containing one or more reporter entities; iii. merging the microdroplets of the panel comprising at least one cell with the microdroplets of the reporter panel to form a panel of merged microdroplets; iv. monitoring the panel of merged microdroplets using a detection system capable of detecting a change in said reporter entity based on one or more properties of the cell and selecting a subset of microdroplets based on said change in reporter entity to form a panel of selected microdroplets comprising at least one cell; v. collecting a panel of selected microdroplets containing at least one cell and optionally repeating steps (ii) to (iv) one or more times, wherein step (ii) uses one or more different reporter entities; vi. Selecting cells based on the final panel of microdroplets selected in step (iv) or (v); Equipped with Prior to step (iii) and / or step (v), a panel of microdroplets containing at least one cell is cultured to allow cell division and then divided into at least two panels of microdroplets containing at least one cell, said two panels being as follows: a. a panel of microdroplets comprising at least one cell and suitable for merging with a microdroplet of a reporter panel; and b. A reference panel of microdroplets containing at least one cell is.

[0232] In a different embodiment, the method provides: 1. A method for selecting cells based on immunoglobulin production in an EWOD or oEWOD device, said method comprising the steps of: i. providing a test panel of microdroplets comprising at least medium or medium and at least one immune cell; ii. providing a reporter panel of at least one microdroplet containing one or more reporter entities; iii. merging the test panel microdroplets with at least one reporter panel microdroplet to form a panel of merged assay microdroplets; iv. monitoring the panel of assay microdroplets using a detection system capable of detecting a change in said reporter entity based on the presence of at least one immunoglobulin; and v. selecting a subset of microdroplets from the panel based on a change in reporter entity, said subset containing the selected cells; Equipped with A test panel of microdroplets comprising at least medium is prepared by dividing a panel of microdroplets comprising at least one cell and medium, and a reference panel of microdroplets containing at least one cell is prepared; and / or further, the test panel of microdroplets comprising at least one cell is divided into at least two panels of microdroplets before or after any of steps (i) to (v), said two panels being a test panel of microdroplets comprising at least medium or medium and at least one cell and suitable for merging with the microdroplets of the reporter entity panel, and a reference panel of microdroplets containing at least one cell.

[0233] In a different embodiment, the method provides: 1. A method for selecting genetically engineered cells based on the production of a pharmaceutical product in an EWOD or oEWOD device, said method comprising the steps of: i. providing a test panel of microdroplets comprising at least a medium or a medium and at least one genetically engineered cell; ii. providing a reporter panel of at least one microdroplet containing one or more reporter entities; iii. merging the test panel microdroplets with at least one reporter panel microdroplet to form a panel of merged assay microdroplets; and iv. monitoring the panel of assay microdroplets with a detection system capable of detecting a change in said reporter entity based on the presence of at least one agent; and v. selecting a subset of microdroplets from the panel based on a change in reporter entity, said subset containing the selected cells; Equipped with a test panel of microdroplets comprising at least medium is prepared by dividing a panel of microdroplets comprising at least one cell and medium, and further preparing a reference panel of microdroplets containing at least one cell; and / or, the test panel of microdroplets comprising at least one cell is divided into at least two panels of microdroplets before or after any of steps (i) to (v), the two panels being a test panel of microdroplets comprising at least medium or medium and at least one cell and suitable for merging with the microdroplets of the reporter entity panel, and a reference panel of microdroplets containing at least one cell.

[0234] The following features may be applied to any of the methods described herein.

[0235] Manipulation of microdroplets within any panel is accomplished using real or virtual electrowetting electrodes. Microdroplet merging involves the coalescence of multiple individual microdroplets to form a single microdroplet.

[0236] Optionally, the method can include the further step of allowing differentiation on the device prior to any merging step. Reporter entities can determine the activity of engineered transgenes, or can look for specific cell surface markers, cell products, or cell secretions.

[0237] In an alternative embodiment, the method involves selecting specific genetically modified cells based on the expression of a genetically added transgene, which can be used to produce a product (e.g., a chemical or protein).

[0238] 1. A method for selecting genetically engineered cells using an EWOD or oEWOD device, comprising the steps of: i. providing a panel of microdroplets containing at least one genetically engineered cell; ii. providing a panel of at least one reporter microdroplet containing one or more reporter entities; iii. merging the microdroplets of the panel comprising at least one cell with the microdroplets of the reporter panel to form a panel of merged microdroplets; iv. monitoring the panel of merged microdroplets using a detection system capable of detecting a change in said reporter entity based on one or more properties of the cell and selecting a subset of microdroplets based on said change in reporter entity to form a panel of selected microdroplets comprising at least one cell; v. collecting a panel of selected microdroplets containing at least one cell and repeating steps (ii)-(iv) one or more times, wherein one or more different reporter entities are used in step (ii); vi. selecting cells based on the final panel of microdroplets selected in step (v); Equipped with wherein, prior to step (iii) and / or step (v), a panel of microdroplets containing at least one cell is cultured to allow cell division and then divided into at least two panels of microdroplets containing at least one cell, said two panels comprising: a. a test panel of microdroplets containing at least one cell; and b. a reference panel of microdroplets containing at least one cell; is.

[0239] Optionally, the method can further comprise lysing the cells in the microdroplets of the panel before merging with the reporter panel of microdroplets. Such a step is generally performed after establishing a reference panel of microdroplets, each containing at least one cell.

[0240] The cells may be any type of cell suitable for genetic modification, such as mammalian, insect, plant, bacterial, or fungal.

[0241] Any combination of the methods disclosed herein is contemplated, and the steps of the methods and the order in which the methods are performed will depend on the nature of the cells selected and the properties desired.

[0242] Microdroplets are sometimes referred to as droplets. [Example]

[0243] Certain aspects and embodiments of the present invention will now be described, by way of example and with reference to the figures mentioned above. The methods described herein were carried out on a proprietary microfluidic device (Lightcast Discovery LTD, UK).

[0244] Example 1—Representative CAR-T Workflow This workflow targets specific areas of the CAR-T cell manufacturing process, starting with genetic modification of T cells and concluding with mass expansion of selected T cells prior to their reinfusion into the patient (as depicted in Figure 1).

[0245] Essentially, a two-part screen is used for the selection of CAR-T cells: 1) a multi-reporter cell surface marker assay, and 2) activation assessment, and these steps can be performed in either order.

[0246] Blood is drawn from a patient and unmodified T cells from the patient are isolated. These T cells are emulsified into microdroplets using conventional methods and introduced into a microfluidic platform. The T cells are then transduced, which involves merging the panel of microdroplets, each containing a T cell, with a panel of microdroplets, each containing a vector as a pair, to form a first panel of merged microdroplets. The vector contains a nucleic acid operably linked to a promoter, and the nucleic acid sequence encodes a chimeric antigen receptor. After transduction, the T cells undergo a first screening step. Empty or multiply occupied microdroplets are discarded, if desired (Figure 2).

[0247] Multi-component reporter assays can use a detection system that analyzes multiple markers on transduced T cells, and microdroplets are selected based on this. Reporter schemes can include, for example, the following markers: CD4 / CD8, CD19, scFv domain markers (for specificity and / or affinity screening), B cell markers (or markers for other unwanted cells), and / or differentiation markers (to select relevant subpanels). Analyzing multiple markers on T cells involves merging microdroplets containing transduced T cells with microdroplets containing reporters (such as primary antibodies and fluorochrome-conjugated detection antibodies). Suboptimal cells are removed as early as possible before expansion, providing the best possible input for subsequent expansion and profiling steps. From here, selected microdroplets proceed to the dosimetry and profiling stages (as depicted in Figure 2).

[0248] A reference panel of CAR-T cells can be constructed by dividing a panel of microdroplets containing at least one CAR-T cell after the cells have been activated and divided.

[0249] The cells in the selected microdroplets are deployed on the chip and evaluated for viability, activation, and attrition behavior. Activation screening includes, for example, screening for antitumor activity and efficacy. Detailed methods for evaluating antitumor cell activity are described in Example 3. Based on the results of these assays, unnecessary microdroplets are discarded, and a final panel of microdroplets is selected, which can serve as a subset of the reference panel. In on-chip activity assays, for example, microdroplets containing a dose of T cells are merged with microdroplets containing tumor cells or tumor-like substances at various T cell / tumor cell ratios to measure the cancer cell-killing activity of the T cells. The efficacy and safety profile of the dose can be evaluated at this stage. The genomes of these T cells can then be sequenced to obtain single-cell genomic data (Figure 3).

[0250] The final selected microdroplet subpanel of T cells can be dispensed from the device into well plates, expanded in large quantities, and then reinfused into patients (Figure 3). This method allows for the best possible consistency and viability of the selected cells, enabling quantification of efficacy and safety profile information.

[0251] Example 2—Immuno-Oncology Workflow Chinese hamster ovary (CHO) cells are modified to produce immunotherapeutic drugs (e.g., TCRs), emulsified into microdroplets, and then introduced into a microfluidic platform. Empty or multiply occupied microdroplets are discarded. The remaining microdroplets containing a single CHO cell are incubated on-chip to promote immunotherapeutic drug production. The CHO-containing droplets are then split to obtain multiple doses of the drug produced by each cell. T cells and target tumor cells are separately emulsified and introduced into the microfluidic platform in an array to adjust the cell occupancy of each microdroplet as desired. The T cells and tumor cells are then merged. The second merging step is used to add doses of immunotherapeutic drug and track which CHO cell each dose came from. The resulting assay is then incubated and the killing behavior of the T cells is monitored using a detection system by detecting caspase 3 / 7 fluorescence, a fluorescent marker of apoptosis. CHO cells that have produced an effective amount of the test drug can be dispensed from the device into well plates (Figure 4).

[0252] Example 3 - Pan T cell killing assay [Adjustment Protocol] Tumor model cell lines (passage 59+1) cultured to 80% confluence in EMEM (10% FCS, 1% glutamine, 1% penicillin / streptomycin) were then incubated at a density of 3e6 / mL in droplet medium (RPMI 1640 (10% FCS, 1% glutamine, 1% penicillin / streptomycin)) supplemented with 1 µM caspase 3 / 7, 50 µM Hoechst 33342, and 0.1 nM TCR drug at 5% CO and 37 °C for 2 h. Meanwhile, human donor-derived Pan T cells were thawed and washed with PBS before being incubated in droplet medium (RPMI 1640) at a density of 2e6 / mL, followed by incubation with 1 µM Deep Red at 5% CO and 37 °C for 30 min. Both cell suspensions were then emulsified into microdroplets (Figure 5).

[0253] [Droplet Panel] Microdroplets containing Pan T cells are sorted into the first panel, and microdroplets containing tumor cells are sorted into the second panel. The two panels are then merged in pairs to generate microdroplets containing both T cells and tumor cells at various tumor cell / Pan T cell ratios. As a negative control, microdroplets containing only tumor cells are also included (Figure 6). To confirm that tumor cells are not undergoing premature apoptosis, the merged microdroplets at t=0 time are scanned for caspase 3 / 7 fluorescence.

[0254] [result] The results showed that the 50% viability zone was approximately 28 hours, a sufficient time to confirm the distinct death behavior of cancer cells (Figure 7). To quantify time-dependent tumor cell apoptosis, caspase intensity was measured over time for each droplet. Bright-field microscopy was used to image T cells interacting with and continuously killing tumor cells within each droplet (Figure 8 and Figure 9).

[0255] Example 4—Antibody Discovery [Cell viability] Unrelated hybridoma cells were stained with Zombie stain, emulsified in microdroplets, and then introduced into the microfluidic platform. Cell viability was assessed by imaging the microdroplets (10x lens, Zombie Green / GFP (excitation: 457 / 50, emission: 520 / 28): 0.54 s exposure time, 100% lamp power, BF: 0.1 s exposure time). Heat-killed unrelated hybridoma cells were used as a positive control. Cell viability remained at 100% (33 cells) for 5.3 h in the droplets and 3.6 h in the device. Furthermore, cell viability remained above 90% for 20.3 h in the droplets and 18.6 h in the device (28 cells, 5 cells in one droplet were not retained throughout the assay). Thus, excellent viability was demonstrated throughout and after the assay period. This may be due in part to the gas resupply mechanism used in this system, which may contribute to the sustained viability observed (Figure 10).

[0256] [Negative control] Microdroplets containing hybridoma cells and microdroplets containing reporters (antigen-conjugated beads and secondary detection antibodies) were introduced into the Lightcast platform and merged in pairs, generating microdroplets containing 1 bead / 2 cells, 1 bead / 1 cell, beads only, and cells only (Figure 11). No fluorescence was detected from negative control microdroplets containing only hybridoma cells or reporters. Cells were also stained with Zombie Green dye—no cell death was detected during the experiment. The assay was run for up to 16 hours and 20 minutes. Fluorescent aggregates began to appear at t = 3 hours and 20 minutes in microdroplets containing hybridomas and reporters.

[0257] [Bulk assay] Hybridoma cells were emulsified into microdroplets and then introduced into the microfluidic platform. Hybridoma medium containing reporters (e.g., antigen-conjugated beads and AF488-conjugated secondary antibodies) was also emulsified into microdroplets and introduced into the device. Microdroplets containing hybridoma cells were sorted into the first panel, and microdroplets containing reporters were sorted into the second panel. The first and second panels were merged in pairs to generate microdroplets containing 0.5 μM / mL beads, 0.5 μM / mL cells, and 200 nM AF488-conjugated secondary antibodies in hybridoma medium. These microdroplets were then incubated at 37°C for 2 hours. The fluorescence of the beads was imaged for secreted anti-target antibodies (BB1, 10x objective, 2-second exposure time).

[0258] Additional microdroplets containing 2 μM / mL beads, 200 nM AF488-labeled secondary antibody, and 10-100 nM free anti-target antibody (anti-mouse AF488) were generated after incubation in hybridoma medium for 4.5 hours at room temperature. The fluorescence of the beads was imaged (BB1, 10x objective, 2-second exposure) over the range of free anti-target antibody concentrations tested, and a curve showing filter fluorescence (a.u.) versus anti-target antibody concentration was generated (Figure 12).

[0259] Microdroplets containing irrelevant hybridoma cells were consistently dark. In contrast, fluorescence approaching the maximum level observed with free anti-target antibody was measured in cells secreting target hybridoma cells (Figure 12). The detection system scans the beads for increased AF488 fluorescence intensity.

[0260] [Unrelated / target hybridoma] Cell Tracker Deep Red fluorescent staining was applied to unrelated hybridomas before emulsification and used to identify them on the microfluidic device. In this case, unrelated hybridomas are defined as hybridoma cells secreting proteins that should not be detected by the reporter molecule. Microdroplets containing hybridoma cells and microdroplets containing reporters were introduced into the microfluidic platform and merged in pairs to generate microdroplets containing a range of different unrelated or target hybridoma cell / bead ratios. Negative control microdroplets containing reporter alone were also prepared. Beads incubated with the negative control and cells identified as unrelated by Deep Red did not glow for the duration of the experiment (16 hours). Beads incubated with target hybridomas (hybridomas producing anti-target antigen primary antibodies) showed a clear AF488 signal 2 hours and 20 minutes after merging (Figure 13).

[0261] [Multiplexed spiking assay] A multiplexed spiking assay was used to separate a mixed population of microdroplets, each containing either unrelated or target hybridoma cells. This assay required a large number of microdroplets, all of which were imaged at over 10x resolution. Two separate cultures of hybridoma cells (target and unrelated) were grown and harvested in media. The target and unrelated hybridoma cells were then separately emulsified into microdroplets and introduced into a microfluidic platform. Microdroplets containing either target or unrelated hybridoma cells were sorted into the first panel. Media containing reporters (target antigen-conjugated beads and 200 nM [AF488-conjugated anti-mouse secondary antibody]) was also emulsified into microdroplets and sorted into the second panel. The first and second panels were merged in pairs to generate a panel of merged microdroplets. The filter fluorescence on the beads ((exc. ​​457 / 50 nm) / au) was measured over time for each merged microdroplet of the spiking run, and anti-target antibody secretion curves were generated for each hybridoma cell line (Figure 13). AF488 signals from irrelevant hybridoma cell lines were indistinguishable from negative controls. All target hybridomas showed positive signals. Aggregates began to form in the droplets after approximately 4 hours and began to interfere with the analysis after 9 hours.

[0262] Single-cell secretion data were also collected, and single-cell secretion curves were generated to demonstrate the time dependence of antibody secretion from single cells. Beads were loaded at 128 nM, and bead fluorescence was measured over a 7-hour period, counting from the merging of the beads and antibody. This result was reproduced across multiple runs. To collect single-cell data, multiply occupied hybridoma cell microdroplets were discarded before merging the first and second panels.

[0263] [Multiplexed beads] We tested bulk imaging of hybridoma culture media on a microfluidic platform. Microdroplets containing high concentrations of beads (SolR1, R3, and R5 beads) were introduced into the device. We tested imaging at different magnifications (2x, 4x, and 10x) to determine the optimal imaging parameters while providing good separation of all intensity bands (R1, R3, and R5) and adequate resolution of the beads for intensity quantification. Good separation between all intensity bands was achieved at 10x magnification, and fair separation was obtained at 4x magnification.

[0264] Color-coded beads (solR1, R3, and R5) were conjugated with the cytoplasmic target antigen, off-target antigen, and human target antigen, respectively. Cell culture medium containing antigen-conjugated beads and anti-mouse AF488 detection antibodies was then emulsified into microdroplets and introduced into the Lightcast platform. Microdroplets, each containing a single target or unrelated hybridoma cell, were also obtained and split to generate a panel of microdroplets containing secreted products and a reference panel of microdroplets containing the corresponding hybridoma cell, tracking the origin of each volume of secreted antibody. Microdroplets containing reporters (beads and detection antibodies) were then paired and merged with microdroplets containing secreted antibodies. The characteristics of the merged and control microdroplets were monitored using the Lightcast detection system, which allows both decoding the intensity bands of the beads and quantifying the concurrent AF488 fluorescence. Negative control microdroplets containing no cells (reporter only) and merged microdroplets containing irrelevant hybridoma cells remained dark, confirming the absence of primary antibody. Positive control microdroplets containing reporter and free target antibody [25 nM] confirmed that the free target antibody bound to both target antigens (R1 and R5). Microdroplets containing secreted antibody also bound to both target antigens (Figure 15).

[0265] [Antibody discovery workflow] B cells are emulsified into microdroplets and introduced into a microfluidic platform. Empty or multiply occupied microdroplets are discarded. The remaining microdroplets containing single B cells are paired and merged with microdroplets containing cell culture medium to increase volume and facilitate subsequent division steps. The merged microdroplets are then incubated on-chip to promote monoclonal antibody production and split into daughter droplets to obtain multiple doses of antibody produced by each cell. Microdroplets containing B cells are sorted into a reference panel, while microdroplets containing antibody doses are sorted into a separate panel and paired and merged with a reporter panel of microdroplets containing antigen-binding beads and detection antibodies. An optical detection system is used to identify positive hits in the reporter-mAb panel. The reference panel microdroplets containing B cells corresponding to the positive hits are dispensed into well plates and expanded (Figure 16).

[0266] Example 5 - Bacterial workflow Bacterial cells are emulsified in microdroplets and introduced into the Lightcast platform. Empty or multiply occupied microdroplets are discarded, while the remaining microdroplets containing single bacterial cells are paired and merged with microdroplets containing cell culture medium to increase volume and facilitate subsequent division steps. The merged microdroplets grow and divide into clonal colonies. A set of microdroplets containing clonal bacterial cells is sorted into a reference panel, while another set of microdroplets containing clonal bacterial cells or a set of microdroplets containing secreted products of the bacterial cells is sorted into a microdroplet assay panel. The microdroplet assay panel is then paired and merged with a microdroplet reporter panel. If the assay components are inside the bacterial membrane, the bacterial cells can be lysed at any stage to release their contents. An optical detection system is used to determine positive hits from the merged reporter panel. The reference panel microdroplets containing bacterial cells corresponding to the positive hits are dispensed into well plates and grown (Figure 17).

[0267] Example 6 - Bright-field imaging of merging and / or splitting microdroplets 19A-19C are bright-field images of an exemplary merging operation. Pairs of droplets are merged in parallel across the device. FIG. 19A shows a pair of microdroplets selected for the merging operation. FIG. 19B shows the paired microdroplets in close proximity and ready to merge. FIG. 19C shows the merged microdroplets.

[0268] 20A-20C are bright field images of an exemplary splitting operation. A microdroplet splits into two pairs of daughter microdroplets in parallel. FIG. 20A shows a droplet in an array awaiting a splitting operation. FIG. 20B shows a microdroplet being elongated midway through a splitting operation. FIG. 20C shows a microdroplet that has split into a pair of daughter droplets. The operation preserves history information, allowing full information about the progeny droplets to be obtained.

[0269] Referring to FIG. 21, a bright field image of a S. cerevisiae bioparticle (heat-inactivated yeast) encapsulated in a droplet with single cell occupancy is shown.

[0270] Example 7 - Cells encapsulated with reporter beads in droplets in an emulsion of cell culture medium in oil Referring to Figure 22, images of cytokine-secreting cells co-encapsulated with cytokine reporter beads within microdroplets in an emulsion of cell culture medium in oil are shown. In the presence of secreted cytokine, the reporter beads fluoresce using a sandwich ELISA mechanism using fluorescently labeled antibodies. As shown in Figure 22A, a bright-field image shows the distribution of cells and beads within the microdroplets. The fluorescence image shown in Figure 22B indicates which reporter beads react positively in the presence of secreted cytokine. Figure 22C shows the location of all cells with intact membrane integrity. The fluorescence image shown in Figure 22D indicates the location of all beads, and Figure 22E shows dead cells indicated by propidium iodide staining.

[0271] Referring to Figure 24, images of antibody-secreting cells co-encapsulated with reporter beads within microdroplets in an emulsion of cell culture medium in oil are shown. In the presence of the secreted antibody, the reporter beads fluoresce using a sandwich ELISA mechanism using fluorescently labeled antibodies. The bright-field image in Figure 24A shows the distribution of cells and beads within the microdroplets. The fluorescent image shown in Figure 24B shows which reporter beads react positively in the presence of the secreted antibody. Figure 24C shows the location of whole cells with intact membrane integrity, while Figure 24D shows dead cells.

[0272] Example 8 - Modular design for microdroplet manipulation Figure 23 outlines a modular design of operations that can be flexibly combined in any order, in whole or in part, to build unique workflows. Programmable decisions can be made at any point throughout the workflow, based on decision-tree logic navigation, to deviate from the original order and / or apply operations only to selected droplets. In this way, assays can be dynamically reconfigured. Exemplary modules are shown, but are not limited to: droplet input and / or sorting, droplet merging, incubation, assay, splitting, and export. Examples of droplet sorting include, but are not limited to, sorting based on size, content, and / or occupancy. Droplet merging enables the delivery of reagents and / or reporters and the controlled introduction of cells to promote cell-cell interactions. Droplet splitting allows for the recovery of materials for downstream analysis or, in the case of cell recovery, further expansion.

[0273] The workflow shown in Figure 25 illustrates various detection modes available for monitoring cell secretion or cell-cell interactions. Detection includes, but is not limited to, fluorescence detection, luminescence detection, FRET detection, brightfield detection, etc. Intracellular imaging can reveal signals within a cell or part of a cell container, such as the nucleus, Golgi apparatus, and / or mitochondria.

[0274] Example 9—Synthetic Biology Workflow, Mammalian Cell Line Development and / or CRISPR Screening Figure 26 outlines an exemplary workflow suitable for, but not limited to, synthetic biology, mammalian cell line development, and / or CRISPR screening. Cells engineered to produce a desired product are introduced into the platform and contained in droplets, which can be sorted by occupancy to select single cells. The cells are incubated and allowed to grow on the platform for a period of time. Growth can be monitored to select healthy clones. Microdroplets can be optionally split to control droplet size or to collect either the product or cells for downstream analysis while retaining a population for alternative manipulations. Retaining a population is particularly useful when performing an assay step that may result in cell lysis on a daughter microdroplet while retaining live cells for further testing in a second daughter microdroplet. Merging allows for droplet size control and the provision of components to cell droplets, such as media and / or reporters to extend cell lifespan. Reporters can facilitate titer readouts using various detection modes to assess cell productivity. Microdroplets can be dispensed and collected from the platform. Cell recovery allows for further growth or downstream analysis. Product recovery, as one example, allows for evaluation of product quality. The order of operations can be freely changed. For example, droplets can be dispensed based on evaluations made during screening, such as top clones with high productivity and proliferation.

[0275] Example 10 - Microdroplet sorting workflow Referring to Figure 27, an exemplary workflow is provided that follows an example sequence of modular operations, illustrating potential points throughout the workflow where microdroplets can be optionally classified. Classification can be based on many factors, including droplet size, contents, occupancy, assay results, or a combination thereof. Brightfield, fluorescence, luminescence, and FRET are some examples of detection modalities for classifying where a signal can be localized to a reporter or cell within the microdroplet.

[0276] Example 11 - Bright-field imaging of cells encapsulated in microdroplets Referring to Figures 28A-28D, multiple images of cells encapsulated in microdroplets incubated in the presence of an intracellular calcium dye are provided. Figure 28A is a bright-field image of the droplets, showing the location and number of cells within each droplet. Figure 28B shows a fluorescent image demonstrating activated calcium metabolism. Figure 28C shows a fluorescent image of the nuclear staining of the cells, and Figure 28D shows dead cells indicated by propidium iodide staining.

[0277] Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

[0278] As used herein, "and / or" is considered to be a specific disclosure of each of the two specified features or components, with or without the other. For example, "A and / or B" is considered a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each were individually set forth herein.

[0279] Unless the context dictates otherwise, the feature descriptions and definitions set forth above are not limited to any particular aspect or embodiment of the invention, but apply equally to all aspects and embodiments described.

[0280] While the present invention has been illustratively described with reference to certain embodiments, it will be further understood by those skilled in the art that the invention is not limited to the disclosed embodiments, and that alternative embodiments may be constructed without departing from the spirit of the invention as defined in the appended claims.

Claims

1. 1. A method for selecting cells and / or parts of cells based on at least one property in an EWOD (Electrowetting on Dielectric) or oEWOD (Optically Mediated Electrowetting on Dielectric) device, comprising the steps of: i. providing a test panel of microdroplets comprising at least culture medium or culture medium and at least one cell; ii. Providing at least one reporter panel of microdroplets containing one or more reporter entities; iii. merging the test panel microdroplets with at least one reporter panel microdroplet to form a panel of merged assay microdroplets; and iv. monitoring the panel of assay microdroplets with a detection system capable of detecting a change in said reporter entity based on the presence of at least one property; and v. selecting a subset of microdroplets from the panel based on a change in reporter entity, said subset comprising the selected cells; Equipped with a test panel of microdroplets containing at least medium is prepared by dividing a panel of microdroplets containing at least one cell and medium, and further preparing a reference panel of microdroplets containing at least one cell; and / or wherein the test panel of microdroplets comprising at least one cell is divided into at least two panels of microdroplets before or after any of steps (i) to (v), said two panels being a test panel of microdroplets comprising at least medium or medium and at least one cell suitable for merging with the microdroplets of the reporter entity panel, and a reference panel of microdroplets containing at least one cell.

2. 2. The method of claim 1, wherein the cell characteristics are selected from any one or more of the following: the presence of one or more cell surface molecules, the presence of one or more cell activities, cell morphology, the presence of one or more cell secretions, and / or the presence of one or more intracellular products, and the cell characteristics are viability, cell activation ability, and / or cell killing ability.

3. 3. The method of claim 1 or claim 2, wherein prior to dividing the panel of microdroplets containing at least one cell, the cell is cultured under conditions that allow cell division; i. said conditions include merging a panel of microdroplets containing at least one cell with a panel of microdroplets containing medium; ii. the reference panel and test panel of microdroplets are divided so that the reference panel of microdroplets contains at least one clonal copy of a cell corresponding to a cell contained in the test panel of microdroplets; and / or iii. said test panel of microdroplets containing at least one cell is merged with a panel of microdroplets containing an agent for lysing said cells; method.

4. 4. The method of claim 1, wherein cells in any panel of microdroplets containing at least one cell are examined for their morphological properties, and said morphological properties can be used to select a subset of cells, said morphological properties including size, shape, adhesion state, cell membrane state, and / or the presence of intracellular features.

5. 5. The method of claim 1, wherein prior to step (ii), the panel of microdroplets containing at least one cell may be evaluated with at least one assay to determine a property of the cell, the assay being performed by merging the panel of microdroplets containing at least one cell with at least one panel of microdroplets containing at least one reporter entity and determining a change in the at least one reporter entity based on the presence of the property.

6. 6. The method of any one of claims 1 to 5, wherein after step (iv) at least one panel of aqueous microdroplets is merged to form a merged assay panel of microdroplets, and the merged panel of microdroplets is then split, and at any stage of the method unwanted or empty microdroplets are discarded.

7. 7. The method of any one of claims 1 to 6, wherein cells of a selected panel of microdroplets are dispensed from the device, and after dispensing the selected panel of microdroplets: i) treating the cells contained therein to expand the population and / or cultivate the clone; and / or ii) further analysis of the cells and / or non-cellular bodies, such as secretions, metabolites, etc. contained therein, including but not limited to analysis of any one or more of the following: i. Mass spectrometry; ii. Surface plasmon resonance; iii. High performance fluid chromatography, and / or iv. genetic analysis, including nucleic acid sequencing and PCR amplification; A method of imposing.

8. 8. The method of any one of claims 1 to 7, wherein the microdroplets of the test panel correspond to the microdroplets of the reference panel, and cells retained in the reference panel can be selected based on a change in the reporter entity in the test panel of microdroplets.

9. In the method according to any one of claims 1 to 8, wherein the cells are genetically engineered, the cells may be genetically engineered prior to step (i), which genetic engineering comprises the following steps: a providing a panel of microdroplets each containing at least one cell; b. providing a panel of modified microdroplets, each containing one or more genetic elements capable of modifying a cell; and c. merging the cell-containing microdroplet with one or more genetic element-containing microdroplets to form a panel of microdroplets each containing a genetically engineered cell; A method comprising:

10. The method according to any one of claims 1 to 9, wherein the cellular characteristics are the following products as intracellular products, cell surface molecules and / or secreted products that can be detected using reporter entities: i. proteins, including glycoproteins or lipoproteins; ii. Chemical substances, iii. polymers, iv. Nucleic acid, v. Compound, A method that can produce one or more of the following:

11. The method according to any one of claims 1 to 10, wherein the at least one reporter entity is one of the following entities: i. Antibodies, ii. Antigen, iii. receptors, iv. Substrate; v. enzymes, vi. Ligands, vii. Nucleic acids, viii. cells, ix. Part of a cell; x. extracellular vesicles, xi. liposomes, xii. polymers, xiii. Chemicals; xiv. Drugs, xv. FRET reporter xvi. Chemiluminescent substances, xvii. tissue samples; xviii. Virus or bacteriophage, xix. cytokines, and / or xx. Protein, The method is any one or more of the following:

12. 12. The method of any one of claims 1 to 11, wherein the reporter entity is labeled to directly detect the change and / or also includes a second labeled entity that can detect and report the change in the reporter entity, and the label is a protein-complementary ligand such as visible, luminescent, fluorescent, phosphorescent, split-fluorescent, split-luminescent, or fluorogenic.

13. The method according to any one of claims 1 to 12, wherein the cells are of the following types: i. immune cells, ii. bacteria, iii. fungal cells, iv. Insect cells, v. pluripotent cells, vi. cancer cells, vii. hybridoma cell fusions; viii. Cell lines, ix. plant cells, x. Artificial cells, xi. Microcells, xii. Part of a cell, xiii. extracellular vesicles, or xiv. Liposomes, wherein the cells are capable of producing an immunotherapeutic agent.

14. 14. The method of any one of claims 1 to 13, wherein the cells are lymphocytes; a) the cells are B lymphocytes and are capable of secreting immunoglobulins; b) the reporter entity is a reporter cell, and the desired immunoglobulin can bind to the reporter cell and cause a change; or c) The one or more reporter entities are: i. antigen-conjugated beads and fluorescent dye-conjugated secondary antibodies; ii. secondary antibody-conjugated beads and fluorescent dye-conjugated antigen, or iii. antigen and fluorescent dye-conjugated secondary antibody beads bound to a carrier surface; is selected from The method, wherein monitoring the properties of the immunoglobulin comprises assaying antigen binding activity.

15. 15. The method according to any one of claims 1 to 14, wherein the cells are i. the cells are genetically engineered to express either a cell surface receptor or a T cell engaging molecule; ii. an immune cell, wherein the cell surface receptor is a chimeric antigen receptor, and the method is for selecting CAR-T cells; and / or iii. isolated from a patient.

16. 16. The method of any one of claims 1 to 15, wherein the microdroplets are surrounded by an immiscible liquid, one or more reporter entities are present in each microdroplet of the reporter panel, and step (v) is a multiplex assay detection.

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