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 the device to select cells based on desired properties. These selected cells can be scaled up for therapeutic or manufacturing applications. The method is useful for adoptive cell therapy (ACT), especially cellular immunotherapy, but also for other applications, especially characterization of cells from a population, generally when the cells are genetically engineered. Furthermore, the 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, where 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 and high-throughput processing of cells is a significant challenge in research, development, and manufacturing. This is especially true for the generation of biological therapeutics (biologics), such as cell therapies and cell-derived therapeutics, where cell identification and selection are required while retaining the 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] With the increasing number of cell-based therapies being approved, it is paramount to ensure that the cells injected into the patient have the desired properties and do not have any properties that may lead to any risks to the patient. For many diseases, such as genetic disorders of the immune system, hemoglobinopathies, metabolic disorders, and cancer, it is known to genetically engineer cells taken from the patient and then return them to the patient. Other known applications of cell-based gene therapy are diseases and disorders of the eye.

[0004] In one example, T cells are genetically engineered to have a cell surface receptor known as a chimeric antigen receptor (CAR). Such CAR-T cells have the important advantage of being able to bind to cancer cells via the expressed cell surface marker and induce a specific antitumor response. Currently approved therapies involve autologous reinfusion of T cells, but in the future, allogeneic T cells may be used. However, it is very important to use CAR-T cells that have a stable phenotype and express the correct CAR on their surface without modification, and that the CAR does not exhibit "on target / off-tumour" or even "off target" binding capabilities, such that healthy cells of the patient are targeted. In addition, it is useful to determine that the CAR-T is efficacious on target before infusion. The two currently approved CAR-T cell therapies have many known deficiencies in terms of dosing consistency, patient safety, and the impact of the manufacturing process. It is understood that these deficiencies are shared throughout 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-target and cardiac targets) lead to severe side effects in some patients. The variability in yields from leukoreduction requires that cell viability be maximized and that input cell subpopulations be carefully selected. · Contaminants (e.g. B cells) can ruin the culture. · There are no simple, automated methods to measure T cell activity against tumor targets, making it difficult or impossible to predict patient response.

[0006] Additionally, the cells can be employed to make or manufacture products for many applications, including therapeutic indications, assays, research, food and beverage, agriculture, and as raw materials, effectively using the 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 a particular antigen-binding capacity, so that the cells can be expanded or analyzed if the particular antigen-binding properties are determined. Such immunoglobulins are usually antibodies, such as monoclonal antibodies, and are used therapeutically or in assays and research. To select cells with such properties, it may be necessary to screen thousands of cells. Since intermediate steps between harvesting the cells and identifying the cells that produce the desired product can cause losses and / or poor performance, it is best to load the cells directly onto the device, reducing handling steps. Without prior FAC sorting, the ratio of desired to undesired cells is greatly reduced. Here, large droplet handling and screening capabilities are required.

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

[0009] Thus, there is a need for a robust method that streamlines the labor-intensive process of screening cells for desirable properties, allows for the selection and subsequent expansion or further testing of such cells, and rejects certain cells with undesirable properties, reducing overall time and costs. It is therefore important that cells can not only be tested but also recovered. Microfluidic platforms have been proposed to address this need, but methods have yet to be proven practical and reliable in the clinical setting. 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 work with such devices has been dictated by the use of fixed structures such as isolation pens, which do not allow for interrogation at the individual cell level, instead providing evaluation of multiple cells. Furthermore, inflexible systems mean that only a single assay can be performed on a cell, with no mechanism to allow for sequential evaluation or to retain the cells for further expansion once evaluation is complete. Miniaturization of the assay reduces the amount of reagents consumed in the assay process, lowering the cost of consumables.

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

[0011] The use of such chip-based devices advantageously allows for the manipulation of microdroplets over a wide range of sizes and provides dynamically reprogrammable manipulation steps that are digitally controlled. This device architecture allows for more sophisticated and integrated workflows compared to traditional approaches, such as independent control of microdroplets, and allows for the control of higher densities of microdroplets over the entire area of ​​a microfluidic chip surface. Summary of the Invention [Problem to be solved by the invention]

[0012] The present disclosure provides a method that allows high throughput processing of microdroplets by utilizing the flexibility of EWOD or oEWOD microfluidic chips, thus solving the need for multiplex processing 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 is a method for selecting cells, particularly for therapeutic applications or manufacturing, and the disclosed method is 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. The cells may be selected to provide heterogeneity within the cell population. The cells are genetically the same, but certain genes may be switched on or off.

[0014] Provided herein are methods of selecting cells for studying cellular interactions with other entities, such as (bio)chemical / (biological)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 a single cell 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 feature; 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.

[0017] Here, a test panel of microdroplets comprising at least medium is generated by dividing a test panel of microdroplets comprising at least one cell and medium, and further generating 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, before or after any of steps (i) to (v), into at least two panels: 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 on the basis of 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 of step (i) contains one or more cells, there is no need to perform a splitting operation prior to step (iii).

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

[0023] The splitting operation can be performed such that the medium contains any secretory product if the cells are capable of producing the secretory product. The splitting step can be performed such that multiple child droplets are generated having different concentrations of said secretory product. Alternatively or additionally, the splitting operation can be performed at different times such that the child microdroplets contain different concentrations of the secretory product.

[0024] The splitting operation may lead to 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 both microdroplets containing at least one cell and microdroplets containing at least 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 of one or more cells and medium; ii. dividing said 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 said 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 the 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 characteristic of a cell can be any suitable characteristic, such as 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.

[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 capable of determining or detecting the presence of the property. Such reporter entities can be provided in microdroplets, as they merge with the microdroplets being tested for said properties.

[0030] The method described herein may be directed to cell selection, where 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 times / steps of the procedure depending on the cell 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 comprise culture medium from precursor microdroplets. If the cells have secretory capacity, the culture medium may also comprise secretions from said 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 results in the selection of a corresponding reference microdroplet comprising at least one cell. Thus, 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 the division of cells within the microdroplet panel is permitted, the reference panel and the 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 associated 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 property of the cell 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 may split, forming at least two child microdroplet panels, each of which contains at least one cell. At least one microdroplet panel may be reserved as a reference panel, and at least one other panel may be used in the methods of the invention (the "test panel"). Thus, the reference panel and the test panel contain corresponding or correlated cells, and the results of the test panel may 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 an original progenitor cell, and may 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). The 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, an initial test 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 medium; ii. providing a panel of at least one reporter microdroplet comprising one or more reporter entities; iii. merging a microdroplet of the at least one cell-containing panel with a microdroplet of 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 a cell and selecting a subset of the microdroplets based on said change in the 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 It 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 such that the test panel of microdroplets contains cells, optionally clonal cell copies of cells from a 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 at least one microdroplet of the 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 cell 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 microdroplets containing the cells is divided into at least two panels of microdroplets, said two panels being 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. It 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 such that the test panel of microdroplets contains cells, optionally clonal cell copies of cells from a 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 more than one cell if the original cell has undergone cell division. The objective of the method of the invention is to characterize a particular cell type, where the characteristics of the cell across the homogenous cell population being assayed may vary. The method may include discarding or disposing of microdroplets containing multiple different cells of the same type, where the cells have not arisen by cell division.

[0047] According to one embodiment of the method disclosed herein, the panel of microdroplets in step (i) may contain at least culture medium. The microdroplets containing at least culture medium have previously been split or divided from the microdroplets containing cells in the culture medium. Prior to the splitting step, the panel of microdroplets containing at least one cell may be merged with the panel of microdroplets containing culture medium, which may optionally contain signaling molecules or cells, causing the cells to produce secretions. Thus, the culture medium in the test panel of microdroplets may contain secretions from the cells, which are exposed to a reporter entity to define a characteristic of the cells. The method may include a step of discarding or disposing of the microdroplets that do not contain the secretions of interest. The droplets containing culture medium may 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 a particular cell surface marker or secretion of a desired molecule. The reporter entities in such rounds can be entities that bind or interact with cell surface or secreted molecules.

[0049] Optionally, a selection step can identify cells that have desirable 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 further, 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, do not have 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 method of the invention, the cells may be of the same type, e.g. B cells or T cells (lymphocytes), and the method of the invention may be used to select cells with specific characteristics, which may be inherent to the cells or may be genetically engineered into the cells. Cells are also referred to as biological cells. It may also be desirable for the cells in the droplet to analyze different types of co-cultures, such as a combination of reporter cells and primary cells, or a culture combining epithelial cells of different phenotypes to form a tissue-like structure. The cells may be natural. The cells may be artificial. The cells may be microcells. The method of the invention may be cell-free and may use parts of cells, e.g. nuclei and / or mitochondria. The method of the invention may use liposomes.

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

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

[0056] The methods of the 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 description of the drawings]

[0057] [Figure 1] An exemplary method is shown in which blood is drawn from the patient and the T cells are isolated, transduced, sorted, and selected prior to mass expansion and reinfusion back into the patient, and the sections within this manufacturing process that our CAR-T workflow covers are also shown. [Diagram 2] Figure 1 shows an overview of the early steps in an exemplary CAR-T workflow, prior to dosimetry and profiling, where 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. [Diagram 3] An overview of later steps in an exemplary CAR-T workflow is shown, where following a multi-reporter cell surface marker assay, cells from a selected microdroplet panel are assessed for cell viability, activation, and exhaustion behavior. The panel of microdroplets can be split such that CAR-T cells are retained in the reference panel and clonal CAR-T cells are used in further selection steps. Activation assessment can include, for example, mixing microdroplets containing T cells with microdroplets containing cancer cells and quantifying the cancer cell killing activity. A final panel of microdroplets is then selected and T cells are dispensed from the device for mass expansion and reinfusion into the patient. [Figure 4]An exemplary immuno-oncology workflow is outlined, which includes A) modifying CHO cells to produce an immunotherapeutic drug and loading them into microdroplets; B) splitting the microdroplets into child droplets containing equal or different doses of drug depending on the time of splitting; C) merging the microdroplets containing T cells pairwise with microdroplets containing cancer cells in a first merging operation, and the merged microdroplets are then merged with microdroplets containing a dose of drug in a second merging operation; D) positive hits are determined by quantifying cancer cell death and CHO cells corresponding to the positive hits are selected; and E) dispensing the selected CHO cells into well plates and growing. [Diagram 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 shown to the left of each box, and the number of T cells is shown 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 undergoing apoptosis when contacted with T cells versus time (hours). Comparison to tumor-only controls is shown. In this case, apoptosis acts as a reporter of anti-tumor killing activity of T cells under the test conditions and can be used as a selection criterion for subsequent expansion of T cells or for sampling them for further analysis. [Figure 8] Figure 8A shows brightfield images of a microdroplet containing a T cell and two tumor cells taken periodically over a 36-hour period, demonstrating the sequential killing of two tumor cells by the T cell. 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 staining images of a microdroplet containing a T cell 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 axis. Figure 9B shows close-up bright-field and Deep Red staining images of the same microdroplet taken periodically over the last 10 hours of the experiment, with caspase fluorescence intensity also quantified over the same time axis. The continuous tumor cell killing activity of the T cells is now visualized. [Figure 10] Hybridoma cell viability as a percentage over time, with the cells either in the device or in the droplets. Cell viability remains 100% for up to 5.3 hours in the droplets and up to 3.6 hours in the device, and remains above 90% for up to 20.3 hours in the droplets and up to 18.6 hours in the device. [Figure 11] On the left are images of panels of microdroplets containing hybridoma cells and beads, or beads only, or hybridoma cells only. Beads are shown as squares and cells as circles. C1 and C2 each contain beads only, C3 contains cells only, D1-D3 contain two cells and one bead each, and D4 and D5 contain one cell and one bead each. On the right are images of the same panels, also showing the fluorescence of the beads. In D1-D5, fluorescent aggregates appeared to different degrees. 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 approaching 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 is measured versus time for a population of microdroplets, each containing target or irrelevant hybridoma cells and reporters. A negative control is also included. D2, containing irrelevant hybridoma cells, remains dark and cannot be distinguished from the negative control. All target hybridomas showed positive signals. [Figure 14] Fluorescence and bright field (insert) images of SolR1, R3, and R5 beads are shown using a 10x objective. The graph shows filtered fluorescence measured such that populations of SolR1, R3, and R5 beads can be easily distinguished within the multiple beads. [Figure 15] The top figure shows a graph of 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 for a population of microdroplets containing target or irrelevant hybridoma cells and reporter. 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, whereas the positive control microdroplets show that the free target antibody has bound to both target antigens (R1 and R5). Microdroplets containing secreted antibodies also bind to both target antigens. The bottom figure shows a bar graph with the filtered AF488 fluorescence on the Y-axis and the categories of microdroplets on the X-axis, with each category further divided into subcategories R1, R3, and R5. [Figure 16]An exemplary monoclonal antibody discovery workflow is outlined, comprising: B cells are loaded into microdroplets and merged with a microdroplet containing cell culture medium that is matched to the volume loaded to facilitate a subsequent division step; the merged microdroplets split into daughter droplets to obtain multiple doses of secreted antibody produced by each cell; the microdroplets containing B cells are sorted into a reference panel while the microdroplets containing the antibody doses are sorted into a test panel; the test panel is paired and merged with a reporter panel of microdroplets; positive hits in the merged reporter-mAb panel are determined using an optical detection system; and the microdroplets of the reference panel containing B cells corresponding to the positive hits are dispensed into well plates for mass expansion. [Figure 17] An exemplary bacterial cell workflow is outlined, comprising: bacterial cells are added into microdroplets and merged with a microdroplet containing a matching cell culture medium for the added volume to facilitate a subsequent division step; the merged microdroplets are allowed to undergo cell division and split into clonal colonies; one set of microdroplets containing clonal bacterial cells is kept as a reference panel while a further set of microdroplets containing clonal bacterial cells or a set of microdroplets containing secretions of said bacterial cells are sorted into a test panel of microdroplets; the test panel of microdroplets is paired and merged with a reporter panel of microdroplets; 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 of microdroplets containing bacterial cells corresponding to the positive hits into a well plate for mass propagation. [Figure 18] The configuration of oEWOD is shown. [Figure 19A] 13 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 illustrates 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 child droplets. [Figure 21] 13 is an image of S. cerevisiae bioparticles (heat-inactivated yeast) encapsulated in microdroplets with single cell occupancy. [Figure 22] Images of cytokine-secreting cells co-encapsulated with cytokine reporter beads in microdroplets in an emulsion of cell media in oil. In the presence of secreted cytokine, the reporter beads fluoresce using a sandwich ELISA with fluorescently labeled antibodies. Bright field image (a) shows the distribution of cells and beads within the droplet. Fluorescent images show b) which reporter beads react positively 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. [Diagram 23] Flexible and unique workflow manipulation of micro-droplets, including micro-droplet 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 media in oil. In the presence of secreted antibodies, the reporter beads fluoresce using a sandwich ELISA mechanism with fluorescently labeled antibodies. Bright field image (a) shows the distribution of cells and beads within the droplet. Fluorescence images show 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. [Diagram 25] Various detection modes for monitoring cell secretions or cell-cell interactions. [Figure 26]A workflow showing typical microdroplet manipulations. Microdroplets containing cells can be manipulated, such as splitting, merging, and sorting operations. 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 PREFERRED EMBODIMENTS

[0058] The inventors have discovered that the deficiencies of prior art methods of cell selection 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 unique, in particular because it proposes to test cells for multiple properties, and the cells with the desired properties and the best profile are selected, which from a cell therapy perspective is 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 carried out 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. Pat. Pub. No. 2013-0233425, and U.S. Pat. Pub. 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 creates localized, directional capillary forces that can be used to drive the droplets along a predetermined path.

[0063] Electrowetting on dielectrics (EWOD) is a well-known effect in which the application of 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 over a substrate, which in turn increases the wettability of the surface following 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 of 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, which allows for the simultaneous manipulation of thousands of microdroplets, typically in the size range below 10 μm, is described in WO 2018 / 234445, which is incorporated herein by reference. In this oEWOD device, the microdroplets move 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 includes what are called "virtual" electrowetting electrode locations, which are generated by selectively illuminating areas of the semiconductor layer embedded within the walls. 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. Further suitable oEWOD devices are described in WO 2020 / 104769, which is also incorporated by reference, and may be of particular relevance to the present method as it is particularly relevant when the microdroplets contain cells. Other oEWOD devices are described in WO 2020 / 169965, which is also incorporated by reference.

[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 composite 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 wavelengths in the range of 400-1000 nm on said conductor layer, said photoactive layer having a thickness in the range of 300-1000 nm; a first dielectric layer on said conductor layer, said first dielectric layer having a thickness in the range of 120 to 160 nm; the first composite wall comprising: A second composite 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 following: 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 a microdroplet, and either of the dielectric layers is coated with a biocompatible coating or an anti-fouling coating.

[0067] Optionally, an intermediate layer of silica between the dielectric layer and the antifouling / biocompatible coating, the intermediate layer having a thickness of 1-10 nm; an A / C power source providing 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 band gap of the photoactive layer adapted to impinge on the photoactive layer to induce a corresponding virtual electrowetting electrode location on the surface of the first dielectric layer; There may also be means for manipulating the point of impingement of the electromagnetic radiation on the photoactive layer to change the configuration of virtual electrowetting electrode locations, thereby forming at least one optically mediated electrowetting path along which microdroplets can be moved.

[0068] Methods and apparatus for high throughput microdroplet manipulation are described in co-pending application PCT / GB2021 / 050148, and such apparatus and techniques would be suitable for carrying out 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 the oEWOD chip. Such a configuration is optimized for facilitating high throughput, flexible loading and processing of microdroplets. The first optical assembly can form a plurality of oEWOD traps on the surface of the chip to cause a plurality of microdroplets on the surface of the chip to form an array of microdroplets corresponding to the first array of oEWOD traps, and the second optical assembly can form a second array of oEWOD traps on the surface of the chip. The oEWOD traps of one or more of the second array are aligned with the oEWOD traps of 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, an apparatus for manipulating microdroplets using optically mediated electrowetting is provided, the apparatus comprising: A first composite wall comprising: First board a first conductor layer on the substrate; an optically active layer of said conductor layer; a 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 composite wall comprising: Second board a second conductor layer on the substrate; a 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 dielectric layer and the second dielectric layer 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 found that providing the first and / or second dielectric layers in continuous layers with a thickness of less than 20 nm makes the droplets more stable, and therefore the droplets 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 of more than 20 nm results in less controlled droplet movement on the substrate, and therefore a greater likelihood of the droplets exhibiting uncontrolled movement that deviates from the illumination area. As a result, uncontrolled droplets may make accurate and efficient oEWOD operations, such as droplet merging and splitting, more difficult.

[0072] In some embodiments, the first and / or second dielectric layers 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 source of electromagnetic radiation 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 a 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 comprise an intervening layer of silicon oxide. The advantage of the intervening layer is that it can be used as a binding layer for the antifouling layer or the non-antifouling layer. The intervening layer is provided 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 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 a microdroplet. 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 may include one or more spacers to hold the first and second walls a predetermined distance apart, defining a microfluidic space suitable for containing the microdroplets. The physical shape of the spacer may be used to aid in the splitting, merging and elongation of the microdroplets within the device. In some embodiments, the microdroplets may contain one or more cells. The microdroplets may also include a medium, such as cell culture medium and / or a buffer.

[0078] The A / C source may be configured to apply a voltage between 0V and 100V 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 90V, or less than 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5V.

[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 constitute a pixelated array of light reflected from or transmitted through such array. The electrowetting location may be crescent-shaped toward the direction of movement of the microdroplets. The device may further include a photodetector for detecting an optical signal in the microdroplets located midstream or downstream of the device. The optical signal may be a fluorescent signal. The device may further include an upstream inlet for generating a medium consisting of 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 consisting of 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 a microfluidic space therebetween may form a periphery of a cartridge or chip. The device may further include a plurality of first electrowetting paths running simultaneously 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 location.

[0081] The device may further include an upstream inlet for introducing microdroplets into the microfluidic space, where 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 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 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 may have a thickness in the range of 70-250 nm. The photoactive layer is activated by electromagnetic radiation in the wavelength range of 400-1000 nm on the conductor layer, and the conductor layer may have a thickness in the range of 300-1000 nm. In some embodiments, the photoactive layer may 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 the conductive droplets, but also substantially eliminates the adverse effects on the droplets from dielectric pinhole defects, improving performance beyond what was anticipated despite the reduced electrowetting force from the use of two dielectric layers. As a result, optically mediated electrowetting can achieve powers of, for example, 0.01 W / cm for the simultaneous manipulation of thousands of droplets. 2 This can be achieved using low power illumination sources such as LEDs that generate low powers of 10,000 or more. For embodiments consisting of large area microfluidic devices with areas greater than 1 cm x 1 cm, the devices are suitable for simultaneously manipulating 10,000 or more droplets, and for ultra-large area devices, 50,000 or more droplets, 100,000 or more droplets, or 1,000,000 or more droplets.

[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 the droplets could not be moved. Through experimentation and testing, the inventors discovered that pinhole defects become a significant limitation in device performance, especially as the devices become larger.

[0087] The dielectric layer always has sparse pinhole defects that make it conductive in small isolated areas. In the optimized known process, 2 This results in a pinhole density of approximately 38 per square inch. Pinhole defects trap the droplets and prevent them from moving. The effect becomes 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 can provide a novel effect of nearly eliminating the effect of pinhole defects. Because the dielectric is located both above and below the droplet, a conductor path is only formed if a pinhole defect in the first dielectric layer is directly lined up 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 in enabling the manipulation of thousands of droplets simultaneously over a relatively large area.

[0089] For large-area or ultra-large-area devices suitable for simultaneously manipulating more than 100,000 droplets, or even more than 1,000,000 droplets, the number of pinhole defects becomes a significant limitation in the performance of the device, since 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 in the device, thereby impairing or disrupting the operation of the system. Thus, the advantage of the present invention in countering the effects of pinhole defects is of particular importance in the operation of ultra-large-area devices containing many small droplets.

[0090] FIG. 18 is a microfluidic device, in particular an oEWOD device 100. The oEWOD device as 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 said substrate 104, said first transparent conductor layer 106 having a thickness in the range of 70-250 nm, a photoactive layer 108 activated by electromagnetic radiation with a wavelength in the range of 400-850 nm on said conductor layer 106, said photoactive layer 108 having a thickness in the range of 300-1500 nm, and a first dielectric layer 110 on said 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 of providing such a thin layer, which must be continuous. However, in theory it can have a thickness of 0.1 nm to 20 nm.

[0091] The device 100 also comprises a second composite wall 112 including a second substrate 114, which may be made of glass, and a second transparent conductor layer 116 on said substrate 114. The second conductor layer 116 may have a thickness in the range of 70-250 nm. A second dielectric layer 118 may be on the transparent second conductor layer 116, 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 the lower practical thickness limit 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 20-180 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 intermediate bonding layer 124 is provided on the first dielectric layer 110 and can also be provided on the second dielectric layer 118. The thickness of the intermediate bonding layer can be between 0.1 nm and 5 nm. The thickness of the intermediate layer can 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 can 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 intermediate layer is that it can be used as a bonding layer for anti-soiling or non-anti-soiling layers and can be hydrophobic.

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

[0094] 18, an incident light 130 can be used to impart a light sprite pattern 131 that illuminates a portion of the photoactivator 110 and holds the microdroplet 122 in a stationary position within the microfluidic space 121. An oil-based carrier phase 134 can be delivered to the microdroplet 122 through holes 136 in the device to replenish important nutrients and components to keep the contents within the microdroplet 122, such as one or more cells, alive and healthy. In some cases, the oil phase 134 can provide important nutrients, medium, and contents 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 made of glass, metal, or engineering plastics. 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 in the range of 70-250 nm, preferably 70-150 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. The 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, and electromagnetic radiation is applied into 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 layer of hydrogenated amorphous silicon 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 anti-fouling layer to help establish the desired microdroplet / carrier fluid / surface contact angle at the various virtual electrowetting electrode locations. The anti-fouling layer additionally aims to prevent the contents of the microdroplet from adhering to the surface and being reduced as the microdroplet moves through the chip. For optimal performance, the anti-fouling layer should help establish the microdroplet / carrier fluid / surface contact angle, which should be in the range of 50°C to 180° when measured as an 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 may comprise polymers of acrylic esters, such as methyl methacrylate, or their dielectrics substituted with hydrophilic groups (e.g., alkoxysilyl). Either or both of the anti-fouling layers are hydrophobic to ensure optimal performance. In some embodiments, an intervening layer of silica less than 20 nm thick may be inserted between the anti-fouling 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, and define a microfluidic space in which the microdroplets are contained. Preferably, before being encapsulated, 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 deformation of the spherical microdroplets that leads to improved electrowetting performance (e.g. better microdroplet merging force). In some examples, 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 apart at a predetermined distance. Spacer options include beads or pillars, ridges formed from an intermediate resist layer created by photopatterning, etc. Alternatively, the spacers can be formed by the use of evaporated materials such as silicon oxide or silicon nitride. Alternatively, a layer of film, including flexible plastic film, with or without an adhesive coating, can be used to form the spacer layer. Various spacer shapes can be used to form thin 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 the microdroplets, followed by the division of the microdroplets 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 can promote the correct direction of droplet flow when loading the chip 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 a source of electromagnetic radiation having a wavelength in the range of 400-850 nm, e.g. 550 nm, 620 nm, 660 nm, and having an energy above the band gap of the photoactive layer. Suitably, the photoactive layer is adapted to receive radiation having an incident intensity of 0.005-0.1 Wcm. -2 The electromagnetic radiation source is activated with a virtual electrowetting electrode position in the range of 0.005–0.1 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 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 by a reflective screen such as a digital micromirror device (DMD) illuminated with light from an LED or other lamp. This allows very complex patterns of virtual electrowetting electrode positions to be rapidly created and destroyed on the first dielectric layer, thereby allowing microdroplets to be precisely moved along essentially any virtual path with tightly controlled electrowetting forces. Such electrowetting paths can be viewed as being 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 may be a composite of two or more dielectric materials. The dielectric layers may be made of, but are 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 mixtures or composites 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 top and bottom composite walls, creating a sealed microfluidic device and defining channels and regions within the device. The structure may occupy the gap between the walls of 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 invention require the manipulation of at least one microdroplet in 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 allows the separation of biological species or reactions. They allow the possibility of working with single cells and are suitable for high throughput experiments. Microdroplets can encapsulate cells and / or reporter entities and keep the same separated until merging is desired.

[0106] The microdroplets may be of any shape or size, but are 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 more. 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 appropriately to form smaller sized microdroplets (child microdroplets).

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

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

[0109] In a microfluidic device, the 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 preferably further include surfactants and other additives to maintain the stability of the microdroplets. The surfactants localize at the microdroplet / carrier interface, thereby stabilizing the microdroplets by lowering the surface tension. When the weight ratio of aqueous microdroplets to carrier is low, the microdroplets tend to shrink over time, a phenomenon that leads to a loss of reactivity within them. One way to counteract this effect is to use a hydrated carrier phase. In general, the above-mentioned carriers do not have a high ability to dissolve water, so 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 as 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 layer 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, for example gels or coated microspheres. They may further contain cell culture media in formulations including nutrients, energy sources such as carbohydrates, buffers, etc.

[0111] The disclosed method includes using a microdroplet containing at least a single cell. The cell is the cell that is assayed or tested for selection, optionally based on the characteristics of the cell. Given that the object to be selected is the characteristics of the cell, the initial microdroplet generally contains a single cell. Thus, the disclosed method involves a step of 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 said 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, for example, contain between 50 and 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 in 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 required components (cells, reporter entities, etc.) 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 method is 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 method of the invention may be cell-free and may use parts of cells, such as the nucleus and / or mitochondria. The method of the invention may use liposomes. The cells may be taken 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, an insect cell, a fungal cell, a bacterial cell, or an amoeba cell. It may also be a cell fusion such as a hybridoma.

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

[0121] If the cells are derived from a sample / biological sample, it may be any 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 or pre-ejaculatory fluid, female ejaculate, sweat, feces, hair, tears, bladder fluid, pleural and peritoneal fluid, pericardial fluid, lymphatic fluid, fibrinogen, bile, interstitial fluid, menstrual, pus, sebum, vomit, vaginal fluid, mammary gland fluid, mucous secretion, stool, pancreatic juice, sinus lavage, bronchopulmonary aspirate, blastocyst fluid, and umbilical cord blood. Alternatively, it may be taken from a tissue specimen.

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

[0123] In some embodiments, the panel of microdroplets containing at least one cell contains the same type of cell, for example lymphocytes such as T cells. Thus, the cells may be preselected before being included in the microdroplets. However, some contamination of any biological cell may occur, and different types of cells may also be included in the microdroplets, for example, B cells when T cells are the desired type. The method of the present disclosure allows such microdroplets to be rapidly identified and not 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 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 Zn finger nucleases, TALEN, CRISPR / Cas9 base and prime editing), non-viral gene transfer (such as nanoparticle transfer), gene knockdown, gene knock-in and gene manipulation using RNA, such as gene silencing or activation, or optogenetics. Genetic engineering generally involves the introduction of genetic elements into cells by any suitable means.

[0130] Genetic engineering may involve simply introducing a mutating entity into the genome of a cell to see if the random mutation results in a desired cellular characteristic. The disclosed method allows for screening of a large number of cells with random or deliberate mutations, and selecting cells with desired characteristics after mutation. This allows for optimization of cell selection, particularly cell-based manufacturing. This may 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 may be a viral vector containing a sequence of interest that one wishes to express or include in the cell. The nucleic acid may be a non-viral "naked" vector, such as a minicircle, for expression in the cell. The nucleic acid may contain gene editing components to edit genes in the cell. The genetic element may contain a desired sequence to allow any desired genetic engineering of the cell, for example to express 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 method of the present disclosure may thus comprise the further step of merging a microdroplet of the panel containing at least one cell with a microdroplet of 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 the 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] During the preparation of a panel of microdroplets containing cells, some microdroplets may be empty because only medium is encapsulated during the encapsulation step, or because cells die between the encapsulation step and the method step, or the microdroplets may be missing cells as a result of the division step. 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 provides a panel of microdroplets containing single cells. Encapsulation of a single cell 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 that contain the desired number of cells, and thus the effective rate at which single cells can be encapsulated. Thus, the encapsulation process selected by the user of the device may result in a minority of droplets that contain one or more cells. However, those skilled in the art are tolerant of such situations, and these empty microdroplets are subjected to the same split and merge operations and ignored for the purposes of selection. 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, it will be understood by those skilled in the art that a proportion may be devoid of one or more cells. The disclosed method can rapidly identify such empty microdroplets where cells are required 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 include intentionally empty microdroplets as a control or position marker.

[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, it will be understood by those skilled in the art that a proportion may lack one or more reporter entities. The disclosed method identifies such empty microdroplets and removes all that do not have the required reporter entities, but droplets that do not have 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 may include the presence 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) 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. Subsequent rounds of selection 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.) that serve to distinguish cell types. A particular combination of cell surface molecules or markers can be used to identify a particular cell.

[0145] In one embodiment, the cell surface marker is a CD molecule. CD 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, but are not limited to, specific immune functions. 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 molecules or markers are cell surface receptors, cell surface transporters, cell adhesion proteins, cell surface signaling molecules, and cell surface molecules 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 have been 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 is responsible for interacting with potential target molecules and targeting the modified cell to any cell expressing the matching molecule. The antigen recognition domain is usually 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 normally 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 a further subset 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 the 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, receptors, etc. Cells secrete proteins, including glycoproteins or lipoproteins, chemicals, nucleic acids, polymers, or molecules. Secretion is the step of moving a substance from the inside of the cell to the outside, and uses different pathways depending on the cell type. For example, extracellular vesicles may be involved in any of the embodiments of the invention. Bacterial cells may rely on secretion capabilities to adapt and survive.

[0152] The secretion may be a natural secretion of the cell, e.g., an immunoglobulin such as an antibody from a B cell, or the secretion may be the result of genetic engineering of the cell by introducing a gene or coding sequence for the secretion, hi one embodiment, the cell is a B cell or a hybridoma and the secretion 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 cells. Thus, the reporter entity interacts with the cell secretions. The cell secretions can be separated from the cells by dividing the microdroplet 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 cells. 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. Engineered cells are 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. The reporter entity may therefore 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, an immunoglobulin, etc. 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 medicine, a vaccine, an antiviral agent, etc.

[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 (increase in the number of cells in the microdroplet) or based on proliferation markers. Proliferation markers can be detected using reporter entities specific to them.

[0161] In one embodiment, the cells are selected based on morphology. Such selection can be determined, for example, by visual inspection of the cells using suitable optical detection means. Cell morphology includes 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 cell is selected based on the result of interaction with the reporter entity, and 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 cell can be any type of cell, such as a tumor cell, a cell line, a tumor-like cell, a bacterial cell, or an immune system presenting cell (such as an antigen presenting cell). In response to the reporter entity cell, the selected cell can be activated, induced to secrete a molecule, and / or promote the cell activation or death of the reporter entity cell, or the secreted product can bind to it.

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

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

[0165] [Reporter] The methods described herein involve the use of at least one panel of microdroplets comprising 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 comprising one or more reporter entities and each different.

[0166] Any panel of microdroplets comprising 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 capable of interacting 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 their derivatives: 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. Proteins It may 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 the reporter entity cell. The reporter entity is associated / conjugated to a structure such as a bead or a label such as a visible, chromogenic, luminescent, fluorescent, phosphorescent label, a protein complementary ligand such as split fluorescent, split luminescent, or fluorogenic.

[0169] Alternatively or additionally, the change in the reporter entity may be detected and labelled (visible, luminescent, fluorescent or phosphorescent labelled) by a second entity. The second entity may for example detect the binding of the reporter entity to the cell and be a second antibody or a 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, colour or fluorescent signal. The skilled artisan 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 "multiple element reporter The effects of multiple reporters on the expression of a marker can be assessed in a "multi-element reporter assay" or "multi-reporter cell-surface marker assay," which is an assay in which a multiplicity of features are examined by a system of reporter entities, each of which is capable of producing an individual detectable change.

[0172] In one embodiment, at least one of the reporter entities comprises at least one reporter entity 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 undesirable. A change in the reporter entity cell is detected. Such a change may be cell death or destruction, cell activation, or cell aggregation. These changes may be observed visually (at 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 presence of an increased cell surface molecule. Alternatively or additionally, the reporter entity 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, which generates a measurable signal (e.g., a luciferase gene generates light when the necessary 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 a fluorescent dye-conjugated secondary antibody; ii. secondary antibody-bound beads and fluorescent dye-bound antigen or ligand, or iii. Antigen or ligand bound to a carrier surface and a secondary antibody bead with a fluorescent dye attached It includes at least one of the following:

[0174] Such reporter entities are useful for the detection of molecules that have binding capacities such as antibodies, receptors, enzymes, etc. A person 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 the simple observation of a visible event, albeit under magnification (microscope). Thus, the detection event can be the visual detection of a change.

[0177] Detection in some embodiments 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 signal 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 an assay can be used to determine the characteristics (phenotype) of cells, and individual cells can be screened by the markers they express, the activities they have, or the products they may produce or secrete.

[0179] The detection system allows for high-quality imaging over a wide area, for example using a sensitive camera. In some embodiments, the detection system supports multi-channel fluorescence detection, allowing 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. The signals of different reporter entities are detected and the intensities are spatially quantified for individual microdroplets, facilitating high-throughput screening of cells.

[0180] [choice] The methods disclosed herein allow for the selection of a subset of cells depending on the characteristics of the cells. The cells in the microdroplet panel are selected based on a particular characteristic, such as a cell surface marker / molecule, an internal cell product, or a secretion from the cell. The characteristic may involve the specificity / activity of the secretion or intracellular product. The selected or sorted cells form a subset of the microdroplet panel. The remaining microdroplets containing at least one cell are discarded or ignored. The subset of microdroplets thus selected may be subjected to a further step in which the characteristics of the cells are further investigated or assayed, so that a further subset may be selected. When the final step of the method described herein is performed, the 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 both originate from the same precursor microdroplet. In one embodiment, the division of the precursor microdroplets produces a reference panel microdroplet containing at least one cell and a test panel microdroplet containing at least culture medium. The culture medium may contain secretions from the cell, which is a property of said cell. In an alternative embodiment, the precursor microdroplets produce a reference panel microdroplet containing at least one cell and a test panel microdroplet 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 that is being evaluated in the assay to determine the cell's properties. The selection step may involve lysing the cells in the test panel, which is undesirable when selecting cells for further growth.

[0183] In this way, whatever the 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 them 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 the reporter entities are detected using different means, e.g., fluorescent, luminescent, phosphorescent labels or events. Detection means may also include protein complementation events, such as split fluorescent, split luminescent, or fluorogenic ligands.

[0185] The selected cells may be further dispensed from the device and cultured to undergo cell division prior to further use. In one embodiment, the cells are for injection 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. A medium is simply a substance for culturing cells. This medium may be a minimal medium that contains enough substances to support the cells, and the content of such a minimal medium depends on the type of cells to be encapsulated. The components required vary depending on the type of cells to be studied, and a person skilled in the art will know how to identify a medium suitable for the task.

[0188] In general, cell culture media may include a carbon source (e.g., a sugar such as glucose), water, one or more salts, a pH buffer, a source of amino acids, 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 (such as 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. It therefore requires supplementation to be a "complete" medium. It is commonly 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. As EMEM is a non-complex medium, it is generally enriched with additional supplements or higher concentrations of serum to make it compatible with a wide range of mammalian cells. RPMI is a versatile medium with broad applicability to mammalian cells, particularly hematopoietic cells. RPMI is a modified version of McCoy's 5A, developed for long-term culture of peripheral blood lymphocytes and supports 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 growth of hybrid cells such as mouse hybridoma cells for antibody preparation. It is also often used for storage / suspension of peripheral blood mononuclear cells. Many types of cell culture media are available and should be chosen to optimize 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 test panel microdroplets may also contain cells, especially 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 generate a reference panel of cells containing at least one microdroplet. This preserves clonal copies of the cells, and allows the selection of cells from the reference panel or microdroplet even if any subsequent step stimulates the cells of the test panel, causing damage to the cells. In particular, this damage may occur during the process of lysing the cells to access genetic material or intracellular products.

[0192] When such a division is employed to separate cells and medium, precursor microdroplets containing at least one cell are cultured under conditions that allow the secretion to accumulate in the medium. Such conditions include providing a medium to promote secretion, such as a stimulatory or signaling molecule (e.g., a cytokine), or including a cell, such as an antigen-presenting cell, that interacts with the cell to induce the formation of the secretion. Alternatively, if the secretion is genetically produced by the cell, manipulations can be applied to condition the expression of the secretion, such that such 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 of the microdroplet panel is split to form at least two panels of child droplets. These child droplets may be approximately the same size, or the splitting may be asymmetric, with the dimensions of the microdroplets in one panel being larger. Once the microdroplets are split, one child droplet may receive at least one cell, and such microdroplet forms a reference panel.

[0194] The presence of the 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, and 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 at optimal conditions for cell maintenance, with controlled conditions such as environment and temperature, 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 WO2020 / 104769.

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

[0197] [Cell division] The disclosed method may include a step in which a panel of microdroplets containing at least one cell is allowed 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 cells have undergone mitosis). Prokaryotes (bacteria and archaea) may undergo binary division, where the genetic material is evenly separated into two daughter cells. These cells may be described as clones, since the genetic material should be identical.

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

[0199] In the disclosed method, cell division or clonal expansion is permitted or encouraged to occur within the panel of microdroplets. The 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 type of cell 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 may be bound using antibodies directed against these receptors. Alternatively, activation may be achieved using suitable 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 the cell does not become activated or undergo cell division, the lack of production of further copies of that cell (clone) may result in the microdroplet not being selected. 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 of the microdroplet panel is split to form at least two panels of child droplets. These child droplets can be approximately the same size, or the splitting can be asymmetric, with the dimensions of the microdroplets in one panel being larger. Once the microdroplets are split, each child droplet receives at least one cell. Microdroplets that do not have 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 cells of the reference panel of microdroplets to be selected based on assays and / or tests for properties of cells in the panel of microdroplets containing at least one cell, since both contain genetically identical cells.

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

[0207] [Identification and tracking of microdroplets] The microdroplets of the present disclosure can be suitably manipulated on a device as described herein. Control of the manipulation of the microdroplets can be implemented by a computer. The location of the microdroplets on the device allows for identification of the microdroplets, such as those derived from a fission event of a precursor microdroplet, so that the correlated or corresponding microdroplets can be identified. This allows for the selection of a microdroplet from the reference panel when a reporter entity shows a result in a test panel. In addition, if one of the microdroplets generated from a fission event is retrieved 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 result 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 panel.

[0209] From the position of the microdroplet within the panel, it is possible to determine the identity of the microdroplet. The position of the microdroplet and its identity can be controlled by software. Once a property of a cell is determined within a microdroplet that is merged with a reporter entity microdroplet, the result can be correlated with a 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 the reporter panel droplets to merge can be adjusted. Software algorithms can be implemented to make the assay steps responsive to time-dependent events occurring on the chip. For cell division, image recognition software can be used to count the number of cells in the 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 fluid-holding pen in a miniature valve structure or a closed chamber structure in a microfluidic chip. In some embodiments, the process of splitting the droplet is performed by application of a mechanical structure or deformation of the microstructure. In some embodiments, the droplet is split using acoustic manipulation.

[0211] In some embodiments, the process of merging 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, in particular cell selection methods that allow for further use of the cells after selection is completed.

[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 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 containing the selected cells; Equipped with a test panel of microdroplets containing at least one 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, 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.

[0215] Thus, the cells may be selected on the basis of 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 said 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 said medium; iii. providing at least one reporter panel of microdroplets 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 the reporter entities in the corresponding assay microdroplet panel, said subset comprising the selected cells; and Equipped with.

[0217] 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 medium; ii. providing a panel of at least one reporter microdroplet comprising one or more reporter entities; iii. merging a microdroplet of at least one cell-containing panel with a microdroplet 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 a cell, and selecting a subset of the 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 comprising 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 adapted to merge with the microdroplets of the reporter entity panel; and b. A reference panel of microdroplets containing at least one cell It is.

[0218] The following features may be applied to any of the methods described herein.

[0219] The microdroplets of any panel may be subjected to multiple merging and splitting operations for the purpose of diluting the contents of the microdroplets or for the purpose of "washing" the contents of the microdroplets. Such operations include merging a 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 successively 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 are merged to form a new microdroplet. This pairing step may be performed one or more times, 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. A set of microdroplets for this merging 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 a microdroplet 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 a reporter entity that determines a cell surface property of the cells, such as the presence or absence of a particular cell marker or molecule. The test panel of microdroplets can then be merged with a further reporter panel of microdroplets, involving the use of a reporter entity that determines a property of a cell-cell interaction, such as the ability to activate or kill another cell type.

[0224] Alternatively, in another embodiment, these steps are reversed, and cells are 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 secondly with a reporter entity that determines a cell surface property of the cell, such as 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 suitable point in the method, which may thus 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, and a reference panel of microdroplets is kept containing 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 constructed panel of microdroplets (test panel) may contain culture medium or culture medium and at least one cell. Before the splitting step, the cells may be cultured under conditions that allow either cell division or cell secretion.

[0227] In certain embodiments, the method comprises:

[0228] 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 an alteration in said reporter entity based on the presence of one or more cell surface markers and selecting a subset of the microdroplets based on said alteration in reporter entity to form a selected panel of microdroplets comprising at least one cell; v. harvesting a panel of selected microdroplets containing at least one cell and optionally repeating steps (ii)-(iv) one or more times, where 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 split into at least two panels of microdroplets containing at least one cell, said two panels being: a. a test panel of microdroplets containing at least one cell; and b. A reference panel of microdroplets containing at least one cell It is.

[0229] The immune cell may be a T cell. 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 for other unwanted contaminating cells), and / or differentiation markers (to select the relevant subpanel), 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, a method is provided: 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 comprising at least one genetically modified hematopoietic stem cell; ii. providing a panel of at least one reporter microdroplet comprising 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 a cell and selecting a subset of the microdroplets based on said change in reporter entity to form a panel of selected microdroplets comprising at least one cell; v. harvesting a panel of selected microdroplets containing at least one cell and optionally repeating steps (ii)-(iv) one or more times, where 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 split 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 It is.

[0232] In a different embodiment, a method is provided: 1. A method for selecting cells based on their production of immunoglobulins 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 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; 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 the reporter entity, said subset containing the selected cells; Equipped with A test panel of microdroplets containing at least one medium is prepared by dividing a panel of microdroplets containing 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 before or after any of steps (i)-(v) into at least two panels of microdroplets, 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, a method is provided: 1. A method for selecting genetically engineered cells based on their production of pharmaceutical products in an EWOD or oEWOD device, the 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 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 agent; and v. selecting a subset of microdroplets from the panel based on a change in the reporter entity, said subset containing the selected cells; Equipped with A test panel of microdroplets comprising at least one 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 split 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 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 applicable to any of the methods described herein.

[0235] Manipulation of the 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 transgenes introduced by genetic engineering, and can also look for specific cell surface markers, cell products, or cell secretions.

[0237] In an alternative embodiment, the method involves the selection of particular genetically modified cells based on the expression of a genetically added transgene, which can be used to produce a product, such as a chemical or protein.

[0238] A method for selecting genetically engineered cells using an EWOD or oEWOD device, comprising the steps of: i. providing a panel of microdroplets comprising at least one genetically engineered cell; ii. providing a panel of at least one reporter microdroplet comprising 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 a cell and selecting a subset of the microdroplets based on said change in reporter entity to form a panel of selected microdroplets comprising at least one cell; v. harvesting 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 comprising at least one cell is cultured to allow cell division and then split into at least two panels of microdroplets comprising at least one cell, said two panels comprising: a. a test panel of microdroplets comprising at least one cell; and b. a reference panel of microdroplets containing at least one cell; It is.

[0239] Optionally, the method can further comprise the step of lysing the cells in the microdroplets of the panel prior to merging with the reporter panel of microdroplets, such a step is generally performed after a reference panel of microdroplets, each containing at least one cell, has been established.

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

[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. EXAMPLES

[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 - Typical CAR-T workflow This workflow targets specific areas of the CAR-T cell manufacturing process, starting with the genetic modification of T cells and concluding before mass expansion and re-infusion of selected T cells 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; 2) activation assessment; and these steps can be performed in either order.

[0246] The patient's blood is drawn and T cells (unmodified) from the patient are isolated. These T cells are emulsified in microdroplets in the usual way and introduced into a microfluidic platform. The T cells are then transduced, which includes merging said panel of microdroplets, each containing a T cell, with a panel of microdroplets, each containing a vector as a pair, to form a first merged panel of microdroplets. The vector comprises a nucleic acid operably linked to a promoter, the nucleic acid sequence encoding a chimeric antigen receptor. After transduction, the T cells proceed to a first screening step. Empty or multiply occupied microdroplets are discarded, if desired (Figure 2).

[0247] Multi-element reporter assays can use detection systems that analyze multiple markers on the transduced T cells, and microdroplets are selected on this basis. 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 for 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-labeled detection antibodies). Suboptimal cells are removed here, as early as possible before expansion. This gives the best possible input for the 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 splitting a panel of microdroplets containing at least one CAR-T cell after the cells have been activated and allowed to divide.

[0249] The cells of the selected microdroplets are spread on the chip and the viability, activation and exhaustion behavior are evaluated. Activation screening includes, for example, screening for antitumor activity and efficacy. Detailed evaluation methods for antitumor cell activity are described in Example 3. According to the results of these assays, unnecessary microdroplets are discarded and a final panel of microdroplets is selected, which can be a subset of the reference panel. In the on-chip activity assay, for example, microdroplets containing a dose of T cells are merged with microdroplets containing tumor cells or tumor-like material 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 be sequenced and single-cell genomic data can now be obtained (Figure 3).

[0250] The final selected microdroplet subpanel T cells can be dispensed from the device into well plates and re-injected into the patient after mass expansion (Figure 3). This method allows for the best possible consistency and viability of the selected cells, allowing for quantification of efficacy and safety profile information.

[0251] Example 2—Immuno-oncology workflow Chinese hamster ovary (CHO) cells are modified to produce an immunotherapeutic drug (e.g., TCR), emulsified into microdroplets, and then loaded into the microfluidic platform. Empty or multiply occupied microdroplets are discarded. The remaining microdroplets containing a single CHO cell are incubated on-chip to promote the production of the immunotherapeutic drug. 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 emulsified separately and loaded 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. A second merging operation is used to add doses of the immunotherapeutic drug and to track which CHO cell each dose came from. The resulting assay is 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) are then incubated for 2 h at 5% CO2 and 37 °C in droplet medium (RPMI 1640 (10% FCS, 1% glutamine, 1% penicillin / streptomycin)) supplemented with 1 μM caspase 3 / 7, 50 μM Hoechst 33342, 0.1 nM TCR drug at a density of 3e6 / mL. Meanwhile, Pan T cells from human donors are thawed, washed with PBS, and then incubated for 30 min at 5% CO2 and 37 °C in 1 μM Deep Red, before being incubated in droplet medium (RPMI 1640) at a density of 2e6 / mL. Both cell suspensions are 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 early apoptosis, the merged microdroplets at t=0 time are scanned for caspase 3 / 7 fluorescence.

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

[0255] Example 4—Antibody Discovery [Cell viability] Unrelated hybridoma cells were stained with Zombie stain, emulsified within microdroplets and placed into the microfluidic platform. The cells were then assessed for viability by imaging the microdroplets (10x lens, Zombie green / GFP (excitation: 457 / 50, emission: 520 / 28): exposure time 0.54 s, lamp power 100%, BF: exposure time 0.1 s). Heat-killed unrelated hybridoma cells were used as a positive control. Cell viability remained at 100% (33 cells) for up to 5.3 h in the droplets and 3.6 h in the device. Furthermore, cell viability remained above 90% for up to 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 beyond the duration of the assay region. 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-binding beads and secondary detection antibodies) were loaded onto the Lightcast platform and merged in pairs to generate 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 stain - no cell death was detected during the experiment. The assay was run up to 16 hours 20 minutes. In microdroplets containing hybridoma and reporter, fluorescent aggregates started to appear at t=3 hours 20 minutes.

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

[0258] Further microdroplets were generated containing 2 M / mL beads, 200 nM AF488-labeled secondary antibody, and 10-100 nM [free anti-target antibody (anti-mouse AF488)] incubated in hybridoma medium for 4.5 hours at room temperature. The fluorescence of the beads was imaged (BB1, 10x objective, 2 second exposure time) over the range of free anti-target antibody concentrations tested, and a curve was generated showing filter fluorescence (au) versus anti-target antibody concentration (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 the unrelated hybridomas before emulsification and used for their identification on the microfluidic device. In this case, unrelated hybridomas are defined as hybridoma cells that secrete proteins that should not be detected by the reporter molecule. Hybridoma cell-containing microdroplets and reporter-containing microdroplets were loaded 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 only the reporter were also prepared. The negative control, and beads incubated with 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 clear AF488 signals 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 placed into the microfluidic platform. The microdroplets, each containing either the target or unrelated hybridoma cells, were sorted into a first panel. Media containing a reporter (target antigen-bound beads and [AF488-conjugated anti-mouse secondary antibody] 200 nM) was also emulsified into microdroplets and sorted into a second panel. The first and second panels were pairwise merged to generate a panel of merged microdroplets. 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 signal from the irrelevant hybridoma cell line was indistinguishable from the negative control. 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 was also collected and single cell secretion curves were generated showing the time dependence of antibody secretion from single cells. Beads were loaded at 128 nM and bead fluorescence was measured over 7 hours counting from merging of beads and antibodies. This result was reproduced in multiple runs. To collect single cell data, multiply occupied hybridoma cell microdroplets were discarded before merging the first and second panels.

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

[0264] Color-coded beads (solR1, R3, R5) were conjugated with cyno-targeted, off-target, and human-targeted antigens, respectively. Cell culture medium containing antigen-conjugated beads and anti-mouse AF488 detection antibodies was then emulsified into microdroplets and loaded onto the Lightcast platform. Microdroplets, each containing a single target or an unrelated hybridoma cell, were also obtained and split to generate a panel of microdroplets, each containing a secreted product, and a reference panel of microdroplets, each containing a corresponding hybridoma cell, to track which hybridoma cell each volume of secreted antibody came from. Microdroplets containing reporters (beads and detection antibodies) were then paired and merged with microdroplets containing secreted antibodies. The properties 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 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 the 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 the volume and facilitate the subsequent splitting step. 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. The microdroplets containing B cells are sorted into a reference panel, while the microdroplets containing a dose of antibody are sorted into another 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 determine the positive hits of the reporter-mAb panel. The microdroplets of the reference panel 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 loaded into the Lightcast platform. Empty or multi-occupied microdroplets are discarded, while the remaining microdroplets containing single bacterial cells are paired and merged with microdroplets containing cell culture medium to increase the volume and facilitate the subsequent division step. The merged microdroplets grow and split into clonal colonies. A set of microdroplets containing clonal bacterial cells is sorted into a reference panel, while the other set of microdroplets containing clonal bacterial cells or a set of microdroplets containing secretions of said bacterial cells are sorted into an assay panel of microdroplets. The assay panel of microdroplets is paired and merged with a reporter panel of microdroplets. At any stage, the bacterial cells can lyse and release their contents if the assay components are inside the bacterial membrane. An optical detection system is used to determine the positive hits of the merged reporter panel. The reference panel microdroplets containing bacterial cells corresponding to said positive hits are dispensed into a well plate and grown (Figure 17).

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

[0268] 20A-C are bright field images of an exemplary splitting operation. A microdroplet is split 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 elongated in the middle of a splitting operation. FIG. 20C shows a microdroplet that has split into a pair of daughter droplets. The operation allows history information to be maintained and all information of the progeny droplets to be obtained.

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

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

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

[0272] Example 8 - Modular design of microdroplet manipulation FIG. 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 to deviate from the original order and / or apply operations only to selected microdroplets, based on navigation of decision tree logic. In this way, assays can be dynamically reconfigured. Exemplary modules are shown, but are not limited to: microdroplet input and / or classification, microdroplet merging, incubation, assay, splitting, and export. Examples of microdroplet classification include, but are not limited to, classification based on size, content, and / or occupancy. Droplet merging allows for the delivery of reagents and / or reporters and allows for the controlled introduction of cells to promote cell-cell interactions. Droplet splitting allows for the recovery of material for downstream analysis or, in the case of cell recovery, further growth.

[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, bright field detection, etc. Intracellular imaging can reveal signals within parts of a cell or 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 FIG. 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 product of interest are loaded onto the platform and contained in droplets, which can be sorted by occupancy to select single cells. 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. Retention of a population is particularly useful when performing an assay step on a daughter microdroplet that may result in cell disintegration while retaining live cells for further testing in a second daughter microdroplet. Merging allows control of droplet size and provides cell droplets with components, e.g., medium to extend cell lifespan and / or reporters. Reporters can facilitate titer readouts by various detection modes to assess cell productivity. Microdroplets can be dispensed and collected from the platform. Recovering the cells allows for further growth or downstream analysis. In just one example, product recovery allows for evaluation of the product quality. The order of operations can be freely changed. For example, microdroplets 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 possible points throughout the workflow where the microdroplets can be optionally classified. Classification can be based on many factors, such as droplet size, contents, occupancy, assay results, or a combination thereof. Brightfield, fluorescence, luminescence, FRET are some examples of detection modalities to classify where a signal can be localized to a reporter or cell within the microdroplet.

[0276] Example 11 - Brightfield 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 showing activated calcium metabolism. Figure 28C shows a fluorescent image of the nuclear stain of the cells, and Figure 28D shows dead cells shown with 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 deemed 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 deemed to be a specific disclosure of each of (i) A, (ii) B, and (iii) 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. A method for selecting cells and / or parts of cells on the basis of at least one property in an EWOD (Electrowetting on Dielectric) or oEWOD (Optically Mediated Electrowetting on Dielectric) device, comprising the steps of: - providing a test panel of microdroplets comprising at least a culture medium or a culture medium and at least one cell; - providing at least one reporter panel of microdroplets containing one or more reporter entities; - merging the test panel microdroplets with at least one reporter panel microdroplet to form a panel of merged assay microdroplets; and 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 - selecting a subset of microdroplets from the panel based on the 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 further wherein the test panel of microdroplets comprising at least one cell is divided, before or after any of steps (i) to (v), into at least two panels of microdroplets, 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 optionally the cell characteristics are viability, cell activation ability, and / or cell killing ability.

3. 3. The method of claim 1 or 2, wherein prior to dividing the panel of microdroplets containing at least one cell, the cell is cultured under conditions allowing cell division, and optionally the conditions include merging a panel of microdroplets containing at least one cell with a panel of microdroplets containing medium; the reference panel of microdroplets and the test panel are partitioned such that the reference panel of microdroplets contains at least one clonal copy of a cell that corresponds to a cell contained in the test panel of microdroplets; and / or - the test panel of microdroplets containing at least one cell is merged with a panel of microdroplets containing an agent for lysing the cell; method.

4. 4. The method of any one of claims 1 to 3, wherein cells in any panel of microdroplets containing at least one cell are examined for their morphological properties, and said morphological properties may be used to select a subset of cells, optionally comprising size, shape, adhesion status, cell membrane status, 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 for determining a property of the cell, said assay being performed by merging said 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 at least one reporter entity based on the presence of said 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 into a merged assay panel of microdroplets, and the merged panel of microdroplets is then split, and optionally, at any stage of the method, unwanted or empty microdroplets are discarded.

7. 7. The method according to any one of claims 1 to 6, wherein the cells of a selected panel of microdroplets are dispensed from the device, optionally 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, metabolic products, etc. contained therein, including, but not limited to, any one or more of the following: ・Mass spectrometry, Surface plasmon resonance, - High performance liquid chromatography, and / or 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 a test panel correspond to the microdroplets of a 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, the cells are genetically engineered, optionally the cells may be genetically engineered prior to step (i), said genetic engineering comprising the steps of: Providing a panel of microdroplets each containing at least one cell; - providing a panel of modified microdroplets, each of which contains one or more genetic elements capable of modifying a cell; and - merging a 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: - proteins, including glycoproteins or lipoproteins; ・Chemical substances, [0043] a polymer, ・ Nucleic acid, ・Compound, A method for producing one or more of the above.

11. In the method according to any one of claims 1 to 10, the at least one reporter entity is one of the following entities: ・Antibodies, ・Antigen, Receptors, ・Substrate, Enzymes, Ligands, ・ Nucleic acid, ・Cells, Part of a cell, Extracellular vesicles, liposomes, [0043] a polymer, ・Chemical substances, Drugs, ・FRET reporter Chemiluminescent substances, - tissue samples, - a virus or bacteriophage, cytokines, and / or · 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 labelled for direct detection of the change and / or also includes a second labelled entity capable of detecting and reporting the change in the reporter entity, the label being 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: ・Immune cells, Bacteria, ・Fungal cells, ・Insect cells, ・Pluripotent cells, Cancer cells, - hybridoma cell fusions, ・Cell lines, ・Plant cells, ・Artificial cells, ・Microcells, Part of a cell, Extracellular vesicles, or liposomes, and optionally, 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, and optionally further comprising: a) the cell is a B lymphocyte and is capable of secreting immunoglobulins; b) the reporter entity is a reporter cell, and optionally a desired immunoglobulin can bind to the reporter cell and cause a change; or c) The one or more reporter entities are: - antigen-conjugated beads and fluorescent dye-conjugated secondary antibodies; secondary antibody-conjugated beads and fluorochrome-conjugated antigen, or - antigen and fluorochrome-conjugated secondary antibody beads bound to a carrier surface; is selected from The method, wherein monitoring the immunoglobulin properties comprises assaying antigen binding activity.

15. 15. The method of any one of claims 1 to 14, optionally comprising: the cells are genetically engineered to express either a cell surface receptor or a T cell engaging molecule; an immune cell, and said cell surface receptor is a chimeric antigen receptor, and optionally the method is for selecting a CAR-T cell; and / or - 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, and optionally one or more reporter entities are present in each microdroplet of the reporter panel, and optionally step (v) is a detection multiplex assay.

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