Method for selecting cells

Abstract

To provide a method for selecting cells using an EWOD or oEWOD device.SOLUTION: I) providing a panel of microdroplets comprising cells and medium; ii) providing a reporter panel of microdroplets comprising reporter entities; iii) merging microdroplets of the panel comprising cells with microdroplets of the reporter panel to form a panel of microdroplets; iv) monitoring the merged panel of microdroplets using a system that detects changes in the reporter entities based on one or more properties of the cells, and selecting a subset of microdroplets based on the changes in the reporter entities; A method of selecting cells using a microdroplet manipulation apparatus, comprising the steps of: forming a panel of selected microdroplets containing cells; v) obtaining a panel of selected microdroplets containing cells; and vi) selecting cells based on the final panel of microdroplets selected in step v).SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method of using a device to select cells based on desirable properties. These selected cells can be expanded for therapeutic or manufacturing uses. This method is useful for adoptive cell therapy (ACT), especially cellular immunotherapy. However, other applications, particularly the characterization of cells from a population, and generally when cells are genetically engineered, are also being considered. Furthermore, this method is also useful in cases where immunoglobulins or immunotherapeutic agents are used. Thus, it can also be used to select cells based on their ability to produce a desired molecule. Synthetic biology is a rapidly developing field that harnesses cells to develop medicines, fuels, food, and produce food and drink, or produce enzymes that break down plastics and other pollutants. The device of the present invention is in the field of microfluidics, and can produce microdroplets. The present invention relates to a microfluidic device capable of manipulating [Background technology]

[0002] Rapid, high-throughput processing of cells is a key challenge in research, development, and manufacturing. This is due to the increasing number of biologic therapeutics, such as cell therapy and cell-derived therapeutics (biologics). This is especially true for the generation of α- and β-actin-containing cells, which require cell identification and selection while further propagation is not possible. or need to preserve cells for testing. Sorting, assaying, and selecting cells Existing methods for this remain slow, despite the development of microfluidic devices. Difficult to handle.

[0003] Cell-based therapies are increasingly being approved, with the cells infused into patients having desirable properties and Furthermore, it is essential to ensure that the drug does not have any characteristics that could lead to any risk to the patient. Many diseases, including genetic disorders of the immune system, hemoglobin disorders, metabolic disorders, and cancer, are also important. For these diseases, cells extracted from the patient are genetically manipulated (gene therapy) and then introduced into the patient's body. Other applications of cell-based gene therapy include the treatment of eye diseases and disorders. is known.

[0004] In some cases, T cells are genetically engineered to contain a gene known as a chimeric antigen receptor (CAR). These CAR-T cells express cell surface markers that The important advantage is that they can bind to cancer cells via ATP and induce specific anti-tumor responses. Currently approved treatments involve autologous reinfusion of T cells, In the future, allogeneic T cells may be used. However, they must have a stable phenotype. , using CAR-T cells expressing the correct CAR on their surface without modification, and This CAR is "on target / off-tumour" and even Does not exhibit "off-target" binding, such as targeting of healthy cells It is very important that CAR-T cells are effective against the target before infusion. The two currently approved CAR T-cell therapies There are many known deficiencies regarding dosage consistency, patient safety, and the impact of the manufacturing process. These deficiencies appear to be shared across the fields of adoptive cell and gene therapy. It is understood.

[0005] Important underlying factors include: Extreme variability in yields across treatments and manufactured doses may lead to poor patient outcomes. Efficacy is unpredictable. Known off-target effects (e.g., CNS off-target and cardiac off-target) It can cause serious side effects in some patients. Due to the variability in yields from leukocyte depletion, it is important to maximize cell viability. and the input cell subpopulation must be carefully selected. · Contaminants (e.g., B cells) can ruin the culture. The lack of a simple, automated method to measure T cell activity against tumor targets has led to a lack of patient response. It is difficult or impossible to predict the response.

[0006] Additionally, cells may be used in a variety of applications, including therapeutic indications, assays, research, food and beverage, agriculture, and as a source of raw materials. As a material, it can be employed to make or manufacture products for many uses. They can be extracted from cells as secreted products, like immunoglobulins, or as drug molecules. The product is then produced by lysing the cells to recover the desired intracellular product. This effectively uses cells for this purpose.

[0007] Of particular interest is the production of immunoglobulins from hybridomas and lymphocytes. Identifying cells that produce immunoglobulins with specific antigen-binding capabilities is often difficult. Preferably, if specific antigen-binding properties are determined, the cells can be expanded or Such immunoglobulins are usually monoclonal antibodies. Which antibodies are used therapeutically or in assays and research? It may be necessary to screen thousands of cells to select those with the desired The intermediate step between harvesting and identifying the cells, which produces the product, is loss and / or It is recommended to load cells directly onto the device to reduce handling steps, as this may result in reduced performance. Without prior FAC classification, it is difficult to distinguish between desirable and undesirable cells. The ratio of the droplets to the total droplets is significantly reduced. The ability to

[0008] Selecting cells with desirable product-producing properties is crucial to ensure that the cells are viable in the test. Cells that can be damaged or destroyed and produce the desired product are selected. This is not desirable in some cases.

[0009] Thus, streamlining the labor-intensive process of screening cells for desirable properties. to allow for the selection and subsequent propagation or further testing of such cells, preferably A robust method for rejecting specific cells with undesirable characteristics, reducing overall time and cost Therefore, it is important that cells can be not only tested but also recovered. Microfluidic platforms have been proposed to address this need, but clinical applications remain limited. Practical and reliable methods have yet to be demonstrated in the field, especially for multiplexing multiple cells. The ability to perform assays has proven elusive, and furthermore, the large number of cells The same is true for the ability to process multiple samples simultaneously at high throughput. Much of the research in this field has been dominated by the use of fixed structures such as isolation pens, which have limited the ability to detect cells. Some platforms lack the flexibility to run multiple assays on individual Although it is not possible to investigate at the cellular level, evaluation is performed using multiple cells. In less flexible systems, there is a mechanism to allow for sequential evaluation, or to stop evaluation once it has finished. Once the cells are isolated, there is no mechanism to maintain them for further proliferation, so only a single assay can be performed on the cells. This means that assay miniaturization reduces the reagent consumption during the assay process. This reduces the amount of waste and reduces the cost of consumables.

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

[0011] The use of such chip-based devices advantageously allows for a wide range of sizes This allows for the manipulation of microdroplets, which can be dynamically reprogrammed under digital control. This device structure provides programmable operation steps, such as independent control of microdroplets. Compared to previous approaches, this allows for a more sophisticated and integrated workflow and also allows for microfluidic This allows for the control of a higher density of microdroplets over the entire area of ​​the chip surface. Summary of the Invention [Problem to be solved by the invention]

[0012] The present disclosure utilizes the flexibility of EWOD or oEWOD microfluidic chips to Allows high throughput processing of small droplets and therefore multiplexing in screening applications The present invention provides a method for selecting cells that solves the need for cell processing. includes the cells, parts of cells and / or cell-derived species disclosed herein.

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

[0014] Provided herein are (bio)chemical / (biological)molecular entities, or other cells (cells - a method for selecting cells to study their interactions with other entities, such as cell-cell interactions For example, this method may be used to detect targeted cell proliferation when two or more selected cells are brought into close proximity. This may include cell-cell interactions.

[0015] Provided herein are methods for examining secretions from single cells or cell-cell interactions. For example, this method may be used to select cells for the purpose of determining whether two or more selected cells are in close proximity. When attached, this can include targeted cell-cell interactions. [Means for solving the problem]

[0016] According to one aspect of the present disclosure, a method for selecting cells in an EWOD or oEWOD device The method comprises the steps of: i. of microdroplets containing at least medium or medium and at least one cell. providing a test panel; ii. At least one reporter of the microdroplet containing one or more reporter entities. providing a power panel; iii. Mixing the test panel microdroplets with at least one reporter panel microdroplet. merging to generate a panel of merged assay microdroplets; and iv. detecting a change in said reporter entity based on the presence of at least one characteristic. A panel of assay microdroplets is monitored using a detection system that can detect the presence of and v. Select a subset of microdroplets from the panel based on changes in reporter entities wherein the subset contains the selected cells. and Equipped with.

[0017] wherein the test panel of microdroplets containing at least a medium contains at least one cell and A test panel of microdroplets containing a medium is prepared by dividing the test panel into at least one A reference panel of cell-containing microdroplets is prepared.

[0018] and / or further comprising: a test panel of microdroplets containing at least one cell; Before or after any of (i) to (v), the device is divided into at least two panels, and the two panels are The panel contains at least one medium or medium suitable for merging with the reporter panel microdroplets. a test panel of microdroplets containing a medium and at least one cell, and A reference panel of microdroplets containing one cell.

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

[0020] The methods described herein can include at least one splitting operation, e.g., splitting Before step (i), a panel of microdroplets is first divided into microdroplets containing at least the medium. Prepare a test panel of droplets and a reference panel of microdroplets containing at least one cell It's something like that.

[0021] If the microdroplet panel of step (i) contains one or more cells, There is no need for a prior split operation.

[0022] Alternatively or additionally, one or more further division operations may be performed at any suitable step of the procedure. The division can include a test panel of microdroplets containing at least a medium, and / or or generating a test panel of microdroplets containing at least a medium and at least one cell. It is possible.

[0023] The splitting procedure allows the cells to produce secretions without any secretions being present in the medium. The dividing step can be carried out so that the secretion is contained in a plurality of samples having different concentrations of the secretion. Alternatively or additionally, the splitting operation can be performed such that child droplets of The daughter microdroplets can be run at different times so that they contain different concentrations of secretion.

[0024] The splitting operation comprises splitting one or more reference panels of microdroplets containing at least one cell, and Forming one or more test panels of microdroplets containing or not containing at least one cell This could lead to:

[0025] The method may include selecting at least one cell according to a property of interest. and a step of including both a test panel of microdroplets comprising at least a medium. Includes.

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

[0027] Alternatively, a method for selecting cells in an EWOD or oEWOD device is provided, The method includes the following steps: i. providing a panel of microdroplets comprising one or more cells and medium; ii. dividing the panel of microdroplets into at least two panels of microdroplets; at least one reference panel of microdroplets containing at least one or more cells; and forming at least one microdroplet test panel comprising a ground; iii. Reporting of at least one microdroplet containing one or more reporter entities. providing a power panel; iv. Mixing the test panel microdroplets with at least one reporter panel microdroplet. merging to form a panel of merged assay microdroplets; and v. detecting an alteration in said reporter entity based on the presence of at least one property. 1. Monitor a panel of assay microdroplets using a detection system capable of Steps, and vi. Based on the change in reporter entity in the corresponding assay microdroplet panel a subset of microdroplets from the reference panel, said subset containing the selected cells; Steps including, Equipped with.

[0028] The methods of the present disclosure involve detecting or determining one or more properties of a cell. In this case, the characteristics of a cell can be determined by the presence of one or more cell surface molecules, the presence of one or more cellular activities, or the presence of a cell surface molecule. the presence of one or more cell secretions, and / or the presence of one or more intracellular products, It can be any suitable characteristic, such as any one or more.

[0029] The characteristics of the cells may be determined by any suitable means according to the methods described herein. The method may include one or more levels capable of determining or detecting the presence of a property. Such reporter entities can be provided within the microdroplets. This is because it merges with the microdroplet being tested for said property.

[0030] The methods described herein may involve the selection of cells, in which case at least The panel of microdroplets containing at least one cell is divided into two groups, and the microdroplets containing at least one cell are separated into two groups. A panel of droplets is generated, which is defined here as the reference panel. The splitting or division operation may be performed at any suitable time or step in the procedure, i.e. A reference panel is constructed at the appropriate time. This reference panel is used in the following steps depending on the cells being selected: Therefore, the reference panel can be constructed at different time points / steps. It may contain a subset of cells.

[0031] The dividing step also results in the production of at least one test panel of microdroplets. The droplet test panel may include the medium from the precursor microdroplets. The medium may also contain secretions from the cells. The discrimination results of the test panel and the selection of the desired characteristics are determined by the presence of at least one cell. This results in the selection of the corresponding reference microdroplets. Thus, the identification result of the test panel is , associated with progenitor cells and the same are selected.

[0032] Prior to the division step, a panel of microdroplets containing at least one cell is subjected to cell division, The cells can be cultured under conditions that allow for clonal growth. and merging a microdroplet panel containing at least one cell. Other conditions may include providing the cells with additional medium by adding supplemental medium. Merging a microdroplet panel containing a base with a microdroplet panel containing at least one cell. This allows cells to receive supplements such as growth factors, nutrients, cell signaling molecules, and chemicals. This includes supplying

[0033] If cell division within the microdroplet panel is allowed, the microdroplet reference panel and test panel The panel can be split and a reference panel or microdroplet is included in the microdroplet test panel. The test panel contains at least one clonal copy of a cell corresponding to the cell being tested. The identification results of the cells are associated with progenitor cells and the same are selected.

[0034] According to one embodiment, a test panel of microdroplets containing at least one cell is prepared by lysing said cell. The cells can be merged with a panel of microdroplets containing drugs to treat the cells. A trip is required when the cell property to be determined is the presence of an intracellular product; or Intracellular products can be directly detected using reporter entities that can enter cells. can be done.

[0035] Thus, cells in the microdroplets forming the panel are exposed to specific reporter entities. Once the cells divide, the cells in the panel Each microdroplet can split to form at least two daughter microdroplet panels, Each microdroplet contains at least one cell. See panel At least one microdroplet panel and at least one other panel can be used in the method of the present invention. ("Test Panel"). Thus, the Reference Panel and the Test Panel are corresponding or comparable The test panel contains cells related to the corresponding cells in the reference panel, and the results of the test panel are compared to the corresponding cells in the reference panel. The microdroplet test panel and the microdroplet reference panel are They contain cells derived from original progenitor cells and are therefore genetically identical or clonal. This can be explained as follows.

[0036] According to any embodiment of the present disclosure, within any panel of microdroplets containing at least one cell The cells are examined for their morphological characteristics, and the morphological characteristics identify a subset of cells. This test may be performed prior to step (ii) of the method. Alternatively, it may be performed before or after any step of the method. Size, shape, adhesion, membrane features such as blebs, and / or vacuoles It may include the presence of intracellular features.

[0037] Selection of cells by any aspect defines a subset of cells and discards or isolates the remaining cells. means to exclude. Selection can be either positive (selection of cells based on the presence of a characteristic) or negative (Selection of cells based on the absence of negative characteristics). The discarded / rejected droplets are then removed from the device. It may be removed from the panel or left in the panel and removed from consideration.

[0038] According to some embodiments, prior to step (i), a microdroplet comprising at least one cell is may be evaluated in at least one assay to determine a characteristic of the cell; The assay comprises the panel of microdroplets containing at least one cell and at least one and merging at least one panel of microdroplets containing a reporter entity of The method is carried out by determining a change in at least one reporter entity based on the presence of a characteristic. Thus, an initial inspection or identification can be performed before any division step. .

[0039] In one embodiment, once selected, the cells are then cultured for expansion or further testing. removed from its position.

[0040] According to another aspect of the present disclosure, cells are selected by use of an EWOD or oEWOD device. A method is provided for detecting a smeared image, the method comprising the steps of: i. preparing a panel of microdroplets containing at least one cell and a medium; Pu and, ii. at least one reporter microdroplet containing one or more reporter entities; providing a panel of: iii. Microdroplets of the panel containing at least one cell are transferred to at least one reporter. merging the microdroplets of the first and second panels with each other to form a panel of combined microdroplets. and, iv. detecting a change in said reporter entity based on one or more properties of the cell; A subset of microdroplets is selected based on the change in the reporter entity to contain the cells. Using a detection system capable of forming a selected panel of small droplets that are merged monitoring the panel of microdroplets; v. Harvesting a panel of selected microdroplets containing cells, and optionally, (ii)-(iv) are repeated one or more times, with one or more different reporter entities used in step (ii). and vi. Select cells based on the final panel of microdroplets selected in step (v) Steps and Equipped with Prior to step (iii) and / or step (v), a microorganism containing at least one cell is The panel of small droplets is divided into at least two panels of microdroplets, said panels being As stated above, that is, a. Contains at least medium and enough to merge with the reporter entity panel microdroplets A suitable test panel, and b. A reference panel of microdroplets containing at least one cell is.

[0041] In one embodiment, the cells are cultured prior to dividing the panel of microdroplets containing at least one cell. The cells are cultured to allow cell division, and the test panel of microdroplets contains cells, optionally cells from a reference panel. The cells may be adapted to contain clonal cell copies of the cells.

[0042] Alternatively, according to one aspect, the present invention provides a method for the preparation of cells by the use of an EWOD or oEWOD device. The present invention provides a method for selecting a metric, the method comprising the steps of: i. providing a first panel of microdroplets containing at least one cell; , ii. A panel of reporter microdroplets containing one or more reporter entities is generated. At least one preparation step, iii. a microdroplet of the first panel and a microdroplet of at least one reporter panel; and merging the first and second panels of the assay microdroplets to form a first panel of merged assay microdroplets; iv. detecting a change in said reporter entity based on one or more cellular properties; selecting a first subset of microdroplets based on said change in reporter entity; Assay microdroplets using a detection system capable of forming a first selection panel of monitoring the panel; v. A reporter panel of microdroplets containing one or more reporter entities is generated. At least one preparation step, vi. Microdroplets from a selected panel of microdroplets and further microdroplet reports and merging the microdroplets from the first panel to form a second panel of merged assay microdroplets. forming a panel; vii. detecting a change in said reporter entity based on one or more cellular properties; selecting a second subset of microdroplets based on said change in reporter entity; and A second selected panel of assay microparticles can be formed using a detection system. monitoring a second panel of droplets, said subset being selected; the monitoring step containing cells; Equipped with Prior to step (iii) and / or step (v), the panel of cell-containing microdroplets may be and the two panels are divided into at least two panels of microdroplets, the two panels being: a. A microdroplet containing at least medium and suitable for merging with the reporter panel microdroplet. a microdroplet panel, and b. A reference panel of microdroplets containing at least one cell is.

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

[0044] The division of the microdroplets can be equal or unequal, with one microdroplet splitting into two or more microdroplets. The two or more microdroplets may be divided into the same size or different sizes.

[0045] In one embodiment, the cells are cultured prior to dividing the panel of microdroplets containing at least one cell. The cells are cultured to allow cell division, and the test panel of microdroplets contains cells, optionally cells from a reference panel. The cells may be adapted to contain clonal cell copies of the cells.

[0046] According to one embodiment of the method disclosed herein, the particles of the microdroplets in step (i) are The cell may contain at least one cell, or the original cell may have undergone cell division. In some cases, the cells may contain more than one cell. The goal is to characterize cell types across the homogenous cell population being assayed. The characteristics of cells can vary. This method allows for the generation of microdroplets containing multiple different cells of the same type. The step of discarding or disposing of the cells may be included, and the cells are not produced by cell division. do not have.

[0047] According to one embodiment of the method disclosed herein, the microdroplets in step (i) The panel may contain at least a culture medium. The microdroplets containing at least a culture medium may include: The cells have previously been split or divided from the microdroplets containing the cells in the medium. Previously, a panel of microdroplets containing at least one cell was separated from a panel of microdroplets containing medium. The medium can optionally contain signaling molecules or cells. This allows the cells to produce secretions. Therefore, the medium in the microdroplet test panel is The secreted product may include a secreted product of the cell, which is exposed to a reporter entity to define a property of the cell. The method further comprises discarding or disposing of the microdroplets that do not contain the secretion of interest. The droplets containing the medium may have a different volume than the droplets containing the cells. .

[0048] Further optionally, the reporter panel used to select cells may be a reporter panel that includes, for example, specific cell surface Desired properties, such as cell surface properties, such as expression of a marker or secretion of a desired molecule The reporter entity in such a round allows the identification of cells that have It can be an entity that binds to or interacts with a surface molecule or a secreted molecule.

[0049] Optionally, a selection step may be performed to identify cells with desirable cell interaction properties, such as cell binding. Cells that have undergone cell migration, activation, cell fusion, cell uptake, cell death, etc. can be identified. The reporter in such a round of selection can be a cell, such as a tumor cell. do.

[0050] Optionally, the selection step looks at viability, activation and / or proliferation characteristics or potential. The assay may include an assay for:

[0051] Optionally, if cells are present in the microdroplet test panel, a microdroplet is inserted to release the cellular contents. Such a step is necessary to ensure that the cells have already undergone cell division events and Additionally, if a reference panel of microdroplets each containing at least one cell is created will be taken into consideration.

[0052] If it is determined that no further analysis is necessary, the microdroplets may be removed from the panel at any point during the analysis. For example, the microdroplets can be used to identify cells that do not contain the desired properties. The reporter cells do not undergo division, but rather induce cell death. The reporter cells are then transfected with antibodies that bind to the antigen. If it does not have the desired properties, such as emissions, it can be discarded.

[0053] Although it may be desirable to analyze any type of cell using the methods of the present invention, The cells may be of the same type, e.g., B cells or T cells (lymphocytes), and may be of the same type as described herein. The method can be used to select cells that have a particular characteristic, which characteristic is present in the cells. The cells may be naturally occurring or genetically engineered into the cells. The cells in the droplet may be a combination of reporter cells and primary cells, or Different cell types, such as epithelial cell cultures that combine different phenotypes to form tissue-like structures, It may be desirable to analyze different co-cultures. The cells may be natural. The cells may be artificial. The cells may be microcells. The method of the present invention may also be acellular. Parts of the cell, such as the nucleus and / or mitochondria, may also be used. The method may use liposomes.

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

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

[0056] The methods of the present invention are suitable for use in the manufacture of desired products such as molecules / compounds / chemicals. These can be used to select cells with desired properties. In the present invention, the cells may be genetically engineered bacterial cells, etc. [Brief explanation of the drawings]

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

[0058] The inventors believe that the deficiencies of prior art methods for cell selection are overcome by the methods disclosed herein. We have found that this problem can be overcome by using the method disclosed herein. This results in better product quality, predictability, and more favorable cell selection. is brought about.

[0059] The disclosed method is particularly unique because it proposes to test cells for multiple properties. The cells with the desired properties and best profile are selected and used for cell therapy. This has the best safety profile. The disclosed method does not allow further expansion after selection. The desired cells can be selected in a format that allows for their selection or testing. Cellular characteristics include the presence or absence of specific cell surface molecules, the presence or absence of secreted products, the presence or absence of intracellular products, and the presence or absence of cellular These include morphology, viability, proliferation ability, and / or the presence of desirable cell-cell interaction activity. Examples include, but are not limited to:

[0060] The method of the present disclosure is carried out on a microfluidic device capable of manipulating microdroplets, and There are two cells or parts of cells. The cells may be natural. The cells may be artificial. The cells may be microcells. At least one liposome There may be a

[0061] [Device] The present invention relates to a method that can be carried out in a microdroplet manipulation device such as an EWOD or oEWOD device. Any suitable method for manipulating microdroplets according to the methods described herein. The device can be employed.

[0062] Devices for manipulating droplets or magnetic beads have been previously described in the art. See, for example, U.S. Patent No. 6,565,727 and U.S. Patent Publication No. 2013-0233425 See U.S. Patent Publication No. 2015-0027889. For droplets, this is Typically, droplets are transferred to a cartridge or microfluidic device, e.g., in the presence of an immiscible carrier fluid. By passing the liquid through a microfluidic channel defined by two opposing walls of the tube, Embedded in the wall of the cartridge or tube is a dielectric Each electrode is connected to an A / C bias circuit and is rapidly charged at intervals. can be switched on and off automatically to change the electrowetting properties of the layer. This creates localized, directional capillary forces that can be used to propel the droplet along a predetermined path. occurs.

[0063] Electrowetting on Dielectric (EWOD) is a method for wetting a liquid with a substrate. It is well known that when an electric field is applied between a liquid and a surface, the liquid becomes more likely to wet the surface than in its natural state. The effect of electrowetting is achieved by applying a series of spatially varying electric fields to a substrate. By applying a field and following a series of spatial variations to increase the wettability of the surface, the fluid It can be used to manipulate (e.g., move, split, or change shape). Droplets manipulated in trowetting-based devices are typically deposited between two parallel plates. It is sandwiched between two electrodes and is activated by digital electrodes.

[0064] Variations of this approach based on optically mediated electrowetting have been proposed, e.g. For example, U.S. Patent No. 6,958,132, U.S. Patent Publication No. 2015-0298125, and No. 9,815,056. Generally in the size range below 10 μm. An improved version of this approach, which allows for the simultaneous manipulation of thousands of microdroplets, has been developed in the United States. No. 2018 / 234445, which is incorporated herein by reference. In this oEWOD device, the microdroplets are deposited in a microfluidic chamber defined by a containing wall. It moves through a space, for example, a pair of parallel plates with a microfluidic space between them. At least one of the containing walls may be a "virtual" electrowetting electrode. The semiconductor layer includes a so-called "position," which selectively illuminates an area of ​​the semiconductor layer buried within the wall. By selectively illuminating the layers with light from separate light sources, virtual A virtual path of the electrowetting electrode position can be transiently generated, Furthermore, suitable oEWOD devices are available from the International and US Patent Publication No. 2020 / 104769, which is also incorporated by reference. This may be particularly relevant when the microdroplets contain cells, and therefore may be particularly relevant to the present method. Other oEWOD devices are described in WO 2020 / 169965. and is incorporated herein by reference.

[0065] Another disclosure of oEWOD is in the non-patent literature (Park, Sung-Yong, Michael A. Teitell, and Eric PY Chiou, “Single-sided continuous optoelectrowetting (SCOEW) for drop let manipulation with light patterns "Lab on a Chip 10.13 (2010): 1655-1661) 1 is a single-sided open configuration platform as described.

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

[0067] Optionally, a 1-10 nm thick shielding layer is provided between the dielectric layer and the antifouling / biocompatible coating. The intervening layer of Rica, across first and second composite walls connecting the first and second conductor layers; an A / C power supply that supplies voltage; Induce corresponding virtual electrowetting electrode positions on the surface of the first dielectric layer an energy higher than the band gap of the photoactive layer adapted to impinge on the photoactive layer for at least one second electromagnetic radiation source having energy; Manipulating the point of impact of electromagnetic radiation on the photoactive layer to create a virtual electrowetting electrode position At least one device capable of changing the arrangement of the microdroplets and thereby moving the microdroplets. means for forming an optically mediated electrowetting path; good.

[0068] Methods and apparatus for high-throughput microdroplet manipulation are disclosed in co-pending application PCT / G B2021 / 050148, and such devices and techniques are The device disclosed therein would be suitable for carrying out the method. It includes a switchable optical assembly, both fixed and switchable on the surface of the oEWOD chip. Such a configuration can generate an array of reproducible light spots. Optimized for throughput, flexible loading and ease of processing. The assembly forms multiple oEWOD traps on the surface of the chip to , an array of microdroplets corresponding to the first array of EWOD traps. A second optical assembly can be formed on the surface of the chip to form an oEWOD trap. A second array of one or more EWOD traps can be formed. are aligned with the oEWOD traps of the first array. The contents of the array are examined and one or more of the microdroplets are detected by a second array of oEWOD traps. adjusting the first optical assembly while the first optical assembly is held in place by the PCT / GB2021 / 050148 is incorporated herein by reference.

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

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

[0071] Surprisingly, the first and / or second dielectric layer can be formed into a continuous layer with a thickness of less than 20 nm. By providing more, the droplet becomes more stable, and therefore the droplet comes to rest on the substrate. In contrast, the present inventors have found that the first and / or second Increasing the thickness of the dielectric layer makes the droplet motion on the substrate less controlled, Therefore, the droplets are more likely to exhibit uncontrolled movement away from the illuminated area. As a result, uncontrolled droplets can cause problems such as droplet merging and This can make accurate and efficient oEWOD operations, such as segmentation, more difficult.

[0072] In some embodiments, the first and / or second dielectric layer has a thickness of between 1 nm and 20 nm. The thickness may be 2 nm to 20 nm, 3 nm to 20 nm, 4 nm to 20 nm m, 5nm~20nm, 6nm~20nm, 7nm~20nm, 8nm~20nm, 9n m~20nm, 10nm~20nm, 12m~20nm, 14nm~20nm, 15nm It may be 1 to 15 nm, 1 to 10 nm, or 18 nm to 20 nm. m, 1 to 5 nm, 5 to 10 nm, 5 to 15 nm, or 10 to 15 nm.

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

[0074] The apparatus further includes first and second composite walls across which the first and second conductor layers are connected. and a corresponding transient element on the surface of the first dielectric layer. a photo-excitation layer adapted to impinge on the photoactive layer to induce a photowetting location; at least one source of electromagnetic radiation having an energy higher than the band gap of the layer; The impact point of electromagnetic radiation on the photoactive layer can be manipulated to induce transient electrowetting. At least one electrowetting device capable of changing its configuration and moving the microdroplets. and a microprocessor for forming a routing path.

[0075] The device may further comprise an intervening layer of silicon oxide. The advantage of the intervening layer is that it The intervening layer can be used as a binder layer for either the antifouling layer or the non-antifouling layer. The thickness of the intervening layer can be between 0.1 nm and 5 nm. The thickness of the layer is 0.1, 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3 0.5, 4 or 4.5nm or more, 5nm, 4.5, 4, 3.5, 3, 2 It may be 0.5, 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 are microfluidic channels adapted to contain microdroplets. They can be spaced less than 200 μm apart to define a body space. The width of the space may be 2 to 50 μm. Between: 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 more. In some embodiments, the microfluidic space is 50, 48, 46, 44, 42, 40, 38, 36, 34, 32, 30, 28, 26, 24, 22, 20, 18, 16, 14, 1 It may be less than 2, 10, 8, 6 or 4 μm.

[0077] The exposed surfaces of the first and second dielectric layers maintain the first and second walls a predetermined distance apart. and a microphone suitable for containing microdroplets, which may include one or more spacers for holding the microdroplets. The physical shape of the spacer determines the separation and merging of microdroplets within the device. In some embodiments, the microdroplets may be used to aid in the elongation and propagation of one or more droplets. The microdroplets may also contain media such as cell culture media and / or buffer solutions. may include:

[0078] The A / C source is connected to the first and second composite walls by a 0.0 V power supply. In some embodiments, the A / C source is 0, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 8 It may be configured to apply a voltage of 0 or greater than 90V, or may be configured to apply a voltage of 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, 10 or less than 5V That's fine too.

[0079] The first and second composite walls are formed by first and second dielectric layers on the first and second dielectric layers, respectively. The anti-fouling layer on the second dielectric layer may further comprise a hydrophobic anti-fouling layer. The radiation source is a pixelated array of light reflected from or transmitted through such an array. The electrowetting positions are arranged in three directions along the direction of the droplet movement. The device may further include a microdroplet located midstream or downstream of the device. The optical signal may include a photodetector for detecting a fluorescent light signal. The apparatus further comprises generating a medium comprising an emulsion of aqueous microdroplets in an immiscible carrier fluid. The device may further include an upstream inlet for introducing the fluid into the microfluidic space. Emulsification of aqueous microdroplets in an immiscible carrier fluid through a microfluidic space via a port The fluid may include an upstream inlet for directing a flow of a medium comprising:

[0080] The first and second composite walls defining a microfluidic space therebetween are disposed on a cartridge or chip. This device can form the periphery of the loop. The device may further include a trowetting path. adapted to intersect with the first electrowetting path to form a coalescence site. The electrowetting path may further include a plurality of second electrowetting paths.

[0081] The device may further include an upstream inlet for introducing microdroplets into the microfluidic space. In this case, the diameter of the microdroplet is often 20% or more larger 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 include a second Transient electrowetting can also impinge on and change the photoexcitable layer of A second pattern of locations may be formed.

[0083] The electromagnetic radiation source may be an LED light source, with a power of 0.005-0.1 Wcm -2 Level voltage In some embodiments, the electromagnetic radiation source can provide 0.00 5~0.1Wcm -2 or 0.005, 0.0075, 0.01, 0 0.025, 0.05 or 0.075Wcm -2 More than one implementation is possible. In form, the electromagnetic radiation source is 0.1, 0.075, 0.05, 0.025, 0.01, 0. 0.0075, 0.005 or 0.0025Wcm -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 to 250 nm. The layer is activated by electromagnetic radiation in the wavelength range of 400-1000 nm on the conductor layer, and the conductor The body layer can have a thickness in the range of 300 to 1000 nm. Alternatively, the photoactive layer may be made of amorphous silicon.

[0085] In some embodiments, the microdroplets are microdroplets defined by two opposing walls. The fluid can pass through the space, and each wall is below the breakdown voltage of the dielectric layer. The dielectric layer includes a sufficiently low voltage applied across the dielectric layer. The use of two dielectric layers with a sufficiently low voltage across them reduces the destructive potential of the conductive droplets. Not only does it prevent unwanted ionization, it also virtually eliminates the adverse effects of dielectric pinhole defects on droplets. The reduction in electrowetting force due to the use of two dielectric layers As a result, the performance of optically mediated electrowetting For example, the power requirement is 0.01 W / cm for simultaneous manipulation of thousands of droplets. 2 That low power This can be achieved using a low power illumination source such as an LED that generates a large amount of light. For embodiments comprising large area microfluidic devices greater than 10 cm x 1 cm, the device It is suitable for simultaneous manipulation of more than 50,000 droplets, and for ultra-large area devices, it is suitable for 50,000 droplets. 00 or more droplets, 100,000 or more droplets, or 1,000,000 or more droplets Suitable for simultaneous manipulation of droplets.

[0086] In some embodiments, large area devices can be utilized to handle thousands of droplets. The inventors have previously demonstrated that a single dielectric layer can be used to simultaneously manipulate droplets. Attempts were made to create larger devices, but the inventors encountered defective areas where the droplets could not move. Through experimentation and testing, the inventors discovered that the We found that through-hole defects are a significant limitation of device performance.

[0087] The dielectric layer always has sparse pinhole defects, which cause the dielectric layer to have small arcs. In a known optimized process, 2 Approximately 3 per A pinhole density of 8 is obtained. The pinhole defects trap droplets and prevent them from moving. The effect is even more profound when using droplets of conductive media such as buffer solutions. It's time.

[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 is virtually immune to pinhole defects. This creates a new effect: the dielectric is both above and below the droplet. , the conductor path is such that pinhole defects in the first dielectric layer penetrate the second dielectric They can only form if they are directly aligned with a pinhole defect in the layer. The probability of this happening is extremely small. This pinhole reduction property achieved by the presence of the second dielectric layer is relatively large. This is key to enabling the simultaneous manipulation of thousands of droplets over a large area.

[0089] Manipulating over 100,000 droplets or even over 1,000,000 droplets simultaneously For large-area or ultra-large-area devices suitable for this purpose, a single droplet may contact a pinhole defect. The probability of contact is so high that the number of pinhole defects becomes a significant limitation on the device performance. A single droplet trapped in a pinhole defect can block the movement of other droplets within the device. This may impair or interrupt the operation of the system. The advantage of the present invention, which counteracts the effect of pinhole defects, is that it can be used to measure an ultra-large area containing a large number of microdroplets. This is of particular importance in the operation of the device.

[0090] 18 is a microfluidic device, specifically an oEWOD device 100. Such an oEWOD device comprises a first substrate 104, which can be made of glass, and a a first transparent conductor layer 106 on the substrate 104, the first transparent conductor layer 106 having a thickness in the range of 70 to 250 nm; The first transparent conductor layer 106 and a light having a wavelength in the range of 400 to 850 nm on the conductor layer 106. A photoactive layer 108 activated by electromagnetic radiation in the range of 300 to 1500 nm. the photoactive layer 108 having a thickness of 110.degree. C.; and a first dielectric layer 11 on the photoactive layer 108. 0. The first dielectric layer 110 has a thickness of less than 20 nm. The lower limit of the thickness of the layer is that it is at least partially continuous. However, the methodology for providing such a thin layer is crucial. Typically, the thickness can be from 0.1 nm to 20 nm.

[0091] The device 100 also includes a second substrate 114, which may be made from glass. and a second composite wall 112 including a transparent conductive layer 116 on the substrate 114. The second conductor layer 116 may have a thickness in the range of 70 to 250 nm. The dielectric layer 118 may be on a transparent second conductor layer 116, and the second dielectric layer 118 may be on a transparent second conductor layer 116. Like the first dielectric layer, the second dielectric layer must be continuous. The practical lower limit of thickness is determined by manufacturing constraints, but it is between 1 nm and 20 nm. The exposed surfaces of the first dielectric layer 110 and the second dielectric layer 118 may be Microfluidic spaces spaced 20-180 μm apart and adapted to contain microdroplets 122 The photoactive layer 108 is made of amorphous silicon. The second conductive layer is made of ITO.

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

[0093] A hydrophobic layer 126 is disposed on the intermediate bonding layer 124. Examples of hydrophobic layers include full The intervening tie layer 124 is optional, and the channel walls 120 are made of SU 8 or may be part of the glass structure. It is provided between the dielectric layers 110 , 118 and the hydrophobic layer 126 .

[0094] As shown in FIG. 18, incident light 130 imparts a light sprite pattern 131. The incident light 130 can be used to illuminate a portion of the photoactive material 110 and generate droplets. 122 is held in a stationary position within the microfluidic space 121. The oil-based carrier phase 134 is The droplets 122 are supplied with one or more cells or other liquids through the holes 136 in the droplets 122. It replenishes the vital nutrients and components that keep the contents of your body alive and healthy. In some cases, the oil phase 134 may be used to enhance cell growth, viability, and / or productivity. It can provide nutrients, medium, and ingredients important for the growth of the plant.

[0095] The first and second substrates 104, 114 are made of a material having mechanical strength. For example, the first and second plates are made of glass, metal, or engineering plastic. In some embodiments, the substrate 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 formed on one of the first and second substrates 104, 114. Located on the surface, it usually has a thickness in the range of 70 to 250 nm, preferably 70 to 150 nm. At least one of these layers is made of a transparent conductive material such as indium tin oxide (ITO). thin films of conductive metals such as silver, conductive polymers such as PEDOT, or similar These layers can be a continuous sheet or a series of discrete structures such as wires. Alternatively, the conductor layer may be a mesh of conductive material, which may be used to reduce electromagnetic radiation. The rays are projected into the gaps in the mesh.

[0097] The photoactive phase 108 is capable of generating localized regions of charge in response to stimulation by a source of electromagnetic radiation. It is made of a semiconductor material that can be used for the semiconductor device. For example, it has a thickness in the range of 300 to 1500 nm. In some embodiments, the photoactive layer may be a visible light emitting layer. The dielectric properties of this layer are preferably 10 7 V / m In some embodiments, the dielectric constant is greater than 3. The dielectric layer is selected from alumina, silica, hafnia, or a thin non-conductive polymer film. can be.

[0098] Alternatively, at least the first dielectric layer, and preferably both, may be coated with an antifouling layer, Desired microdroplets / carrier fluid / The antifouling layer may also help establish a contact angle with the surface. The purpose is to prevent the droplets from adhering to the surface and decreasing as they move through the chip. For optimal performance, the anti-fouling layer helps establish the contact angle between the microdroplet / carrier fluid / surface. The contact angle is measured at 25°C as an air-liquid-surface three-point interface. In some embodiments, these layers are These layers have a thickness of less than 100 nm and are typically formed as monolayers. are polymers of acrylic esters such as methyl methacrylate, or hydrophilic groups (e.g. The antifouling layer may contain those dielectrics substituted with a group such as alkoxysilyl. One or both are hydrophobic to ensure optimal performance. , a thick layer is added between the antifouling coating and the dielectric layer to provide a chemically compatible bridge. An intervening layer of silica less than 20 nm thick may be inserted.

[0099] the first and second dielectric layers, and thus the first and second walls, are at least 10 μm; The microfluidic space preferably has a width in the range of 20 to 180 μm and contains microdroplets. Preferably, before being enclosed, the microdroplets themselves are at least 10% wider than the width of the microdroplet spaces. 20% larger characteristic diameter. Therefore, upon entering the tip, the microdroplets are compressed and spheres leading to improved electrowetting performance (e.g., better microdroplet merging force) In some instances, the first and second dielectric layers are It can be coated with a hydrophobic coating such as orosilan.

[0100] In some embodiments, the microfluidic space has first and second walls separated by a predetermined distance. The spacer may include one or more spacers to hold the particles together. Spacer options include beads or or pillars, and ribs formed from an intermediate resist layer produced by photopatterning. Alternatively, by using evaporated materials such as silicon oxide or silicon nitride, Alternatively, a spacer can be formed with or without an adhesive coating. The layer of film, including the flexible plastic film, is used to form the spacer layer. Various spacer shapes can be used to create narrow channels, tapered forming a channel, or a partially enclosed channel defined by the line of the pillars With careful design, these spacers can be used to separate the droplets. Assisting deformation, followed by microdroplet splitting and effective manipulation of the deformed microdroplets Similarly, these spacers prevent cross-contamination between droplet populations. It can be used to physically separate zones of the chip to separate them under fluid pressure. This can encourage the flow of droplets in the correct direction when the drop is injected.

[0101] The first and second walls are biased using an A / C power supply attached to the conductor layer. A potential difference is applied between the two electrodes, and the potential difference is suitably 0 to 50 volts. The D structure is typically used for wavelengths in the range of 400 to 850 nm, e.g., 550 nm, 620 nm, 6 60 nm and associated with an electromagnetic radiation source with energy exceeding the band gap of the photoactive layer Preferably, the photoactive layer is used in a range of 0.005 to 0. 0.1Wcm -2 The virtual electrowetting electrode position is activated in the range of The magnetic radiation source is 0.005 to 0.1 Wcm -2 or 0.005, 0.0 0.75, 0.01, 0.025, 0.05 or 0.075Wcm -2 That's all In some embodiments, the electromagnetic radiation source can be 0.1, 0.075, 0.05, 0 0.025, 0.01, 0.0075, 0.005 or 0.0025 Wcm ―2 Less than It may also be a bell.

[0102] If the source of electromagnetic radiation is pixelated, it may be illuminated with light from an LED or other lamp. A reflective screen such as a digital micromirror device (DMD) is used to project light directly onto the screen. This allows for virtual electrowetting. Highly complex patterns of electrode locations can be quickly created and destroyed on the first dielectric layer. This allows for the microdroplets to be moved using precisely controlled electrowetting forces. This allows for precise movement along essentially any virtual path. The electrowetting path is a virtual electrowetting path on the first dielectric layer. The stimulating electrode position can be considered to be constructed from a continuum of stimulating electrode positions.

[0103] The first and second dielectric layers may be composed of a single dielectric material or may be composed of two or more It may be a composite of dielectric materials. The dielectric layer is made from Al2O3 and SiO2. It may be, but is not limited to this.

[0104] A structure may be provided between the first and second dielectric layers. The structure between the two may be epoxy, polymer, silicon or glass, or a mixture of these. The can be made of composite materials, including but not limited to straight, angled, The structure between the first and second dielectric layers is The upper and lower composite walls can be connected to create a sealed microfluidic device, Defines channels and regions within the device. The structure occupies the gap between two composite walls. Alternatively or additionally, the conductor and dielectric may already have a walled structure. The film can be deposited on the substrate.

[0105] [Microdroplet] The method of the present invention requires the manipulation of at least one microdroplet in the device. Microdroplets generally contain, for example, cells / cells, reporter entities, or a combination of the two. Microdroplets refer to droplets of liquid, such as aqueous liquids, containing combinations of biological species or reactions. They provide detachable partitions, which allow the possibility of handling single cells and have high throughput. Microdroplets encapsulate cells and / or reporter entities. and keep the same separate until a merge 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 20 to 600 μm, but may be 20, 30, 40, 50, 60, 80, 100, 120, 140, 150, 160, 180, 200, 220, 24 0, 250, 260, 280, 300, 320, 340, 360, 380, 400, 42 0, 440, 460, 480, 500, 520, 540, 550, 560 or 580μ In some embodiments, the size of the microdroplets may be 600, 580, or more. , 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 less than 30 μm. Multiple microdroplets can be merged to form larger droplets. Alternatively, large droplets can be divided into smaller droplets. It is also possible to form droplets (child microdroplets).

[0107] If the droplet is too small for the height of the microfluidic device, the droplet will not fit into the microfluidic chamber. Since the droplet does not come into contact with either side wall of the nozzle, it cannot be moved by the oEWOD. In contrast, droplets that are large relative to the geometry of the device move within the microfluidic device. They are difficult to move and / or move slowly, simply blocking other droplets of the correct size, They interfere with merging with droplets of the correct size, often disrupting other operations.

[0108] Generally, the microdroplets usually contain the contents of the microdroplets (cell(s), nutrients, reporters, etc.). It consists of an aqueous phase containing target entities, assay reagents, hydrogel beads, polymers, etc. In some cases, the microdroplets may further comprise an internal phase of an immiscible fluid surrounding the aqueous material. This allows the formation of multiple emulsions, such as double emulsions and droplet-in-droplet emulsions. do.

[0109] In microfluidic devices, tiny droplets are separated from each other by an immiscible carrier phase or fluid. Suitable carrier phases include silicone oil, mineral oil, or fluorocarbon oil. Exemplary fluorocarbon oils include H FE7500, HFE7700 or FC-40. The carrier phase is preferably Surfactants and other additives may also be included to maintain the stability of the microdroplets. The surfactant localizes at the droplet / carrier interface, thereby reducing the surface tension. When the weight ratio of aqueous droplets to carrier is low, The microdroplets tend to shrink over time, which means that the reactivity within them is lost. One way to counteract this effect is to use a hydrated carrier phase. Generally, the above-mentioned carriers do not have a high water dissolving ability, so the hydration reaction is the formation of micelles or secondary microdroplets of water or aqueous buffer within the carrier phase. This buffer solution may be the same as or different from the composition of the microdroplets themselves. In some embodiments, these micelles or secondary microdroplets may be It can contain up to five times the salt content of the droplet itself and can optionally contain glycerol. Typically, these micelles and secondary microdroplets are an order of magnitude smaller. The dispersion layer may contain lipophilic compounds that can diffuse in and out of the droplets.

[0110] The microdroplets used in the disclosed method vary depending on which panel they are on. The microdroplets may be aqueous in nature, e.g., gel or coated microdroplets. Additional components may be included, such as components to provide a support structure such as a sphere. Furthermore, a formulated cell culture medium containing nutrients, an energy source such as carbohydrates, a buffer, etc. may include:

[0111] The methods of the present disclosure involve using microdroplets containing at least a single cell. The cells are optionally assayed or tested to select for a specific characteristic of the cells. Considering that the target to be selected is the characteristics of a cell, the initial droplet is Generally, they contain a single cell. Therefore, the methods of the present disclosure are suitable for panels containing a single cell. This involves selecting only the microdroplets containing the cells and discarding the empty or multi-occupied cells. After this step, the cells are allowed to divide and the microdroplets contain at least one cell. It becomes like this.

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

[0113] A panel of microdroplets is formed by a plurality of microdroplets. A collection, group, or array of droplets, each of which has a similar content. For example, in a panel of microdroplets containing cells, each of said microdroplets contains at least one cell. The panel of microdroplets may alternatively be a cell culture It may include nutrient medium, reporter entities, selected cells, expanded cells, nucleic acids, etc. A panel of microdroplets contains, for example, 50 to 700,000 microdroplets.

[0114] In the disclosed methods, a panel of microdroplets containing at least one reporter entity is provided. Such microdroplets may contain reporter entities as further defined herein. It may contain at least one.

[0115] The disclosed method involves selecting a subset of a panel of microdroplets. The kit then produces a new panel of microdroplets for further use in the disclosed methods. It can be formed.

[0116] Microdroplets are manipulated within the device using real or virtual electrowetting electrodes. The droplets are arrayed to facilitate the arrangement of merging, splitting, and detection events. Merge and split events may be manipulated to create a real or virtual element. This can be done using electrowetting electrodes. Manipulating the microdroplets to achieve the desired results Any suitable layout may be used to separate the droplets. It can also be operated using other methods such as

[0117] Microdroplets typically encapsulate the necessary components (e.g., cells, reporter entities) in aqueous formulations. The microdroplets are prepared by emulsification techniques. In another example, the emulsification is a step emulsification. It may be generated on or adjacent to the fluidic device.

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

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

[0120] Cells may be obtained from cell cultures, e.g., stem cells, pluripotent cells, or cultures of genetically engineered cells. It may be taken.

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

[0122] The cells may be isolated from a patient or individual. The methods described herein may be used to obtain such cells. The cells can be screened and then used to give back to the patient (autologous cell transplant). , may be isolated from an individual and selected to be administered to a patient (allogeneic cell transplantation).

[0123] In some embodiments, the panel of microdroplets containing at least one cell is The droplets contain a variety of cells, such as lymphocytes, including T cells. However, any biological cells may be preselected before inclusion. However, some contamination may occur, e.g., when T cells are of the desired type, Different types of cells may also be included in the microdroplets, including B cells. The method rapidly identifies such microdroplets and prevents them from being selected into a subset of microdroplets. To do so.

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

[0125] The cells may be human or mammalian cells. The cells may be derived from any living organism, such as an organ or tissue. It may be of any suitable type and of any tissue origin.

[0126] The cell may be an immune system cell. Such cells include monocytes, macrophages, Osteoclasts, neutrophils (polymorphonuclear leukocytes), dendritic cells, microglial cells, mast cells, T cells ( Helper T cells, regulatory T cells, cytotoxic T cells, and natural killer T cells These include B cells, natural killer cells, and hematopoietic stem cells.

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

[0128] The cells may be genetically engineered before being encapsulated in the microdroplets. The cells may be genetically engineered after encapsulation.

[0129] Genetic engineering of cells can be achieved through transduction (viral gene transfer), gene editing (Zn finger transcription), Nucleases, TALENs, CRISPR / Cas9 base and prime editing, etc. non-viral gene transfer (e.g., nanoparticle transfer), e.g., gene silencing RNA-based gene knockdown, gene knock-in, and gene targeting, including stimulating or activating and by any suitable method, including genetic engineering or optogenetics. Engineering generally involves introducing genetic elements into cells by any suitable means.

[0130] Genetic engineering simply involves introducing a mutation into the genome of a cell, causing random abrupt changes. It may be determined whether the mutation results in a desirable cellular characteristic. This allows for the screening of large numbers of cells with random or deliberate mutations, After the mutation, cells with desired properties can be selected. This allows for optimization, especially in cell-based manufacturing. This can be called "directed evolution."

[0131] The genetic element optionally comprises a nucleic acid operably linked to a promoter, The nucleic acid sequence encodes the desired product, such as CAR.

[0132] Genetic elements are viral vectors that contain sequences of interest that you want to express or include in cells. The nucleic acid may be a non-coding vector, such as a minicircle, for expression in a cell. It may also be a viral "naked" vector. The nucleic acid is used to edit genes in cells. The genetic elements may include gene editing components for, for example, creating new cell surface molecules. In order to be able to carry out any desired genetic engineering in the cells, such as expressing It may contain a new sequence.

[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 at least one genetic element is The method of the present disclosure thus provides a panel of microdroplets comprising at least one The panel microdroplets containing the cells are mixed with the panel microdroplets containing at least one genetic element. This may comprise the further step of merging the droplets to form panels of merged microdroplets. Such microdroplets contain a small number of genetically engineered cells according to the selection methods described herein. The successful genetic manipulation is then determined by assay, and the unmodified cells are The cells are discarded.

[0134] Genetic engineering involves providing a gene sequence under an inducible promoter. The genetically engineered cells are then adapted to produce the product encoded by the gene sequence. It's stimulating.

[0135] When preparing a panel of cell-containing microdroplets, only the medium was present during the encapsulation step. The cells may be encapsulated or die between the encapsulation and method steps. Therefore, some microdroplets may be empty. Alternatively, the splitting step may result in a small amount of liquid in the microdroplets. In this process, cells are encapsulated, The resulting population of droplets has an occupancy rate described by Poisson statistics.

[0136] The disclosed method utilizes actively controlled fluid dynamics to improve the efficiency of single cell encapsulation. Optionally, the encapsulation process can be used in conjunction with a droplet generation method. The system delivers a panel of microdroplets containing single cells for clonal expansion during the process. This makes it possible.

[0137] However, some of the available encapsulation processes are inefficient and these systems is the desired number of cells encapsulated per droplet, as described by Poisson statistics. The percentage of droplets containing a number of cells decreases, resulting in an effective rate of encapsulating a single cell. Therefore, depending on the encapsulation process chosen by the device user, However, it is possible that the cells may be a minority of cells containing one or more of these cells. These empty droplets are tolerant to such conditions and are subject to the same splitting and merging operations. , are ignored for the purposes of selection. Thus, the selection method described here is flexible and , can be used in conjunction with a variety of cell encapsulation methods.

[0138] Thus, a "panel of microdroplets containing at least one cell" is If the term "octopets containing at least one cell" is used, the percentage It will be understood by those skilled in the art that the method of the present disclosure may be devoid of one or more cells. To rapidly identify such empty microdroplets where cells are needed, we removed them from further consideration. Therefore, the microdroplets containing at least one cell can be discarded. In the panel, virtually every microdroplet can contain at least one cell However, some or all of the droplets may be empty. Empty microdroplets may be intentionally included as markers.

[0139] The panel of microdroplets contains cells with known properties or known products / molecules such as antibodies. Control microdroplets may also be included, such as microdroplets containing a reporter entity, which A negative control may also be included, which does not produce a positive result with the reporter entity. They may be cells or products / molecules that are not expected to produce a result, or they may be empty droplets. Alternatively, it may be a droplet of culture medium.

[0140] Similarly, "a panel of microdroplets containing one or more reporter entities" may be used. The term "droplets containing one or more reporter entities" is used It will be understood by those skilled in the art that, in some cases, the proportion may lack one or more reporter entities. The disclosed method identifies such empty microdroplets and allows the necessary reporter entities to be inserted. Remove all droplets that do not have the required reporter entity, but do not drop during the assay. In some cases, droplets without reporter entities may be included in a subset of the panel. to act as a negative control. may include:

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

[0142] Such characteristics include the presence or absence of one or more of the following: That is, Cell surface molecules, activation profile, proliferation ability, secretion ability, affinity of secreted products, secretory production functional behavior of the cell, its ability to make intracellular products, viability, morphology, and / or cell-cell interactions. The functions are cell killing, cell activation, cell aggregation, etc.

[0143] Cells can be selected based on any combination of desired properties and can be isolated substantially simultaneously ( Thus, one or more reporter entities can be tested in a single step (multiple times) or sequentially. One or more reporter molecules may be required, or may contain at least one reporter entity. A microdroplet panel may be required. Successive selection rounds will result in at least one A subset of microdroplets containing cells can be selected.

[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., proteins, lipids, and glycans). These cells express surface molecules that help distinguish between cell types. Certain combinations of markers can be used to identify specific cells.

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

[0146] In one embodiment, the cell surface molecule or marker is a cell surface receptor, a cell surface trans transporters, cell adhesion proteins, cell surface signaling molecules, and cell-cell interactions. It is a cell surface molecule that carries this responsibility.

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

[0148] Optionally, the artificial cell surface marker is a chimeric antigen receptor (CAR). Prototype recognition domain, extracellular hinge domain, transmembrane domain, intracellular signaling domain The antigen recognition domain is exposed to the outside of the cell and is potentially The modified cells interact with a target molecule and target the modified cells to any cells that express the compatible molecule. The antigen recognition domain is usually expressed as a single-chain variable fragment (scFv). Derived from the variable regions of monoclonal antibodies linked together, but engineered single They can also be derived from domains, typically using ligand / receptor pairs that bind to each other. Antibody-free approaches have also been used to develop CARs, such as

[0149] Cell surface markers (native or modified) are detected on a first subset of cells, or on cells During selection to define a second or further subset of cells, the cells are selected. It may also be used to select

[0150] In one embodiment, cell selection may be based on cell surface markers or molecules. The reporter entity may be one that interacts with a cell surface marker or molecule.

[0151] In one embodiment, the property to be determined is the presence or absence of one or more secretions from the cell. Many cells are capable of secreting proteins, e.g., mammalian cells constantly release proteins into the extracellular matrix. Different specialized cells have specialized secretions, for example, immune cells They express secreted proteins such as cytokines, immunoglobulins, ligands, and receptors. The vesicles contain proteins, chemicals, nucleic acids, and polymers, including glycoproteins or lipoproteins. Secretion is the process of moving substances from the inside of the cell to the outside. For example, extracellular vesicles are an embodiment of the present invention. Bacterial cells rely on secretory capabilities to adapt and survive. It may be.

[0152] The secretions were the natural secretions of the cells, such as immunoglobulins like antibodies from B cells. Alternatively, the secreted product may be a genetically modified product of a cell into which the gene or coding sequence of the secreted product has been introduced. In one embodiment, the cells are B cells or hybridomas. and the secreted product is a monoclonal antibody.

[0153] The secretions may or may not have desired properties, such as binding capacity. This can be measured by one or more reporter entities.

[0154] In one embodiment, cell selection may be based on secretion from the cells. The entity interacts with the cell secretions. The cell secretions form microdroplets, at least The reference microdroplet contained one more cell and the test microdroplet contained the medium in which the secreted product was present. By separating them, they can be separated from the cells.

[0155] In one embodiment, the property to be determined is one or more intracellular products produced by the cell. Most, if not all, cells contain glycoproteins and lipoproteins. Although it can be used to produce proteins, including proteins, some cells, especially Bacteria can make chemicals, nucleic acids, and other polymers. The cells are modified to produce the desired product, which is not secreted but is retained intracellularly. It may be possible to release it, but the cells must be lysed to release it.

[0156] Intracellular products may or may not have desirable properties, such as catalytic activity. This can be examined by one or more reporter entities.

[0157] The product may be a native product of the cell, or may be a gene or code for said product. It may be the product of genetic engineering of the cells into which the sequence is introduced.

[0158] In one embodiment, cell selection is based on the detection of an intracellular product. The target entity may interact with the product. The product may be detected in situ by a control entity or the product may be detected by a cell in the test microdroplet. They may also be separated from cells by lysis.

[0159] The intracellular product may be any suitable biological or chemical molecule. are hormones, enzymes, cell signaling molecules, signal transduction molecules, immunoglobulins, etc. The intracellular product may be a protein. The intracellular product may be RNA, DNA, or a hybrid thereof. The intracellular product may be a pesticide, a toxin, an antibiotic, a fuel, a medicine, or a nucleic acid. The therapeutic agent may be a chemical substance such as a vaccine or an antiviral agent.

[0160] In one embodiment, cells are selected based on their proliferation potential. Such selection can be achieved by cell division induction. by detecting the presence of vents (increased cell numbers in the droplets) or proliferation markers Proliferation markers can be determined based on specific reporter entities. can be detected using

[0161] In one embodiment, cells are selected based on morphology. Such selection can be carried out, for example, by using suitable This can be determined by visual inspection of the cells using optical detection means. , size, shape, presence or absence of cell adhesion, presence or absence of bleb-like cell membrane protrusions, vacuole-like One or more of the following may be mentioned: presence or absence of intracellular entities.

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

[0163] Based on the results of direct or indirect (through secreted products) interactions with reporter cells Cell selection based on the target gene can determine properties suitable for therapeutic guidance.

[0164] The cells may be selected based on the presence or absence of any number of desirable and / or undesirable characteristics. These may be selected based on any combination of the characteristics described herein. good.

[0165] [Reporter] The methods described herein comprise the step of generating at least one microorganism comprising at least one reporter entity. Regarding the use of panels of droplets: 2, 3, 4, 5, 6, 7, 8, 9, 10 or more Any suitable number of panels may be employed, such as a panel of one or more reporter entities. Well, each one is different.

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

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

[0168] The reporter entity may be a cell or a part thereof or a derivative thereof (such as a product or secretion). (e.g., a reporter molecule) and this interaction produces a detectable change in the reporter entity. These changes include binding, annealing, aggregation, separation, amplification, conformational The reporter entity may be a reporter entity cell. The reporter entity is suitable for directly monitoring a detectable change, such as in the case of Structures, such as beads or labels, such as visible, color-emitting, luminescent, fluorescent, or phosphorescent labels, Ligands for protein complementation, such as split-fluorescence, split-emission, or fluorogenic To relate / connect.

[0169] Alternatively or additionally, the change in the reporter entity is detected by a second entity, resulting in a label. The second entity may be labeled (visible, luminescent, fluorescent, or phosphorescent). The antibody detects the binding of the target entity to the cells and can be a secondary antibody or its derivative. Additionally, a reporter entity may be conjugated or linked to an enzyme, and the substrate of the enzyme may be linked to a second entity. The specific antigens may be added to elicit a visual, chromogenic, or fluorescent signal. 1. The reader will be aware of numerous assay procedures for determining the presence of an analyte.

[0170] Generally, the reporter entity is an antibody or a fragment or derivative thereof (antigen binding fragment (Fab), single chain variable fragment (scFv), miniaturized antibody, or antibody (e.g., phibody).

[0171] In one embodiment, the test panel of microdroplets comprises microdroplets containing multiple reporter entities. Thus, the cells are exposed to a panel of "multi-element reporters." "multi-element reporter assay" or "multiple reporter cell surface markers" This can be assessed by a multi-reporter cell-surface marker assay. Many of the properties are assays tested by a system of reporter entities, and each reporter The entity is capable of producing a distinct detectable change.

[0172] In one embodiment, at least one of the reporter entities is Cells include somatic cells. Cells include tumor cells, cancer cells, cell lines, tumor-like cells, presentation cells, and attached cells. Any suitable cells, such as genera or "off-target" cells with which interaction is undesirable. The reporter entity may be of the type: These changes can include cell death or destruction, cell activation, and cell aggregation. They can be observed visually (with appropriate magnification) or by measuring the intracellular components, cytokines, The presence of cell signaling molecules, such as ATP, or increased cell surface molecules in the microdroplets It may also be observed through a second entity that detects the presence of a suitable analyte. Additionally, the reporter cells may contain at least one reporter gene associated with an event such as receptor activation. The cells may be genetically engineered to contain two reporter genes. Utilizing reporter genes, intracellular signaling cascades generate measurable signals. activates (e.g., luciferase gene produces light when the necessary substrate is provided) ).

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

[0174] Such reporter entities are useful for detecting molecules that can bind to them, such as antibodies, receptors, and enzymes. Those skilled in the art of assay design will be able to identify all possible combinations of reporter entities. and how characteristics can be detected and transmitted. .

[0175] For CAR-T cells, the following entities are required: CD4, CD8, and CD19. , scFv domain marker (for specificity and / or affinity screening), B Cell markers (or markers of other unwanted contaminating cells), and / or differentiation markers ( (to select the relevant sub-panel) is a preferred feature.

[0176] [detection] Changes in the reporter entity can be detected. As mentioned above, detection can be achieved by amplification (detection). It may also be a simple observation of visible events, albeit through a microscope. The detection event may be the visual detection of a change.

[0177] In some embodiments, detection is optical, e.g., the label employed is optical. They may be visually, chromogenically, luminescently, fluorescently or phosphorescent. All may employ suitable detection techniques. It is possible.

[0178] The detection system screens the signals of each individual reporter entity in each microdroplet. In one example, a panel of merged microdroplets can be used to image The results of such an assay are: It can be used to determine the characteristics (phenotype) of cells and identify individual cells according to the characteristics they develop. markers they express, the activities they possess, or the substances they may produce or secrete Screening can be done by product.

[0179] The detection system allows for wide-area, high-quality imaging using, for example, a highly sensitive camera. In some embodiments, the detection system supports multi-channel fluorescence detection and dark field. High-quality imaging, brightfield imaging, and cell morphology detection are possible. The lens is supported by multiple objectives, including a high NA (60x) lens. Different droplets are generated for each microdroplet, facilitating high-throughput screening of cells. The signal of the reporter entity is detected and the intensity is spatially quantified.

[0180] [choice] The methods disclosed herein allow for the selection of subsets of cells depending on cell characteristics. The cells in the microdroplet panel are labeled with cell surface markers / molecules, internal cellular products, or They are selected based on certain characteristics, such as secretions from cells. The specificity / activity of the intracellular product may be involved. The selected or sorted cells may be subjected to microdroplet panel analysis. The remaining microdroplets containing at least one cell are discarded. The selected subset of microdroplets is then either selected or ignored. Further steps are taken to further investigate or assay the resulting further subsets. When the final step of the method described herein is performed, at least one cell The final subset of microdroplets containing the selected cells.

[0181] The selected cells are then dispensed from the device. Once selected and dispensed from the device The cells may then be subjected to further assays or to other methods to promote cell maintenance and / or proliferation. After dispensing, the cells may optionally be cultured for infusion or reinfusion into the patient. After dispensing, the cells may be used to produce therapeutics such as immunoglobulins or pharmaceuticals. It may be propagated for further use.

[0182] In one embodiment, the selected cells are selected from a reference microdroplet containing at least one cell. The reference panel contains microdroplets that correlate with the microdroplets in the test panel. , since both are derived from the same precursor microdroplet. The droplets are split into a reference panel microdroplet containing at least one cell, and a reference panel microdroplet containing at least one cell. In both cases, test panel microdroplets containing the culture medium are generated. The culture medium contains secretions from the cells. In an alternative embodiment, the precursor microdroplets may contain at least A reference panel microdroplet containing one cell each and a test panel microdroplet containing one cell each. Generate microdroplets, and thus a reference panel of said microdroplets determines the characteristics of the cells. Cloning a panel of microdroplets containing at least one cell to be evaluated in an assay for The selection step involves selecting cells in the test panel that are clones or clone copies of the cells. This may involve lysing the cells, which may be necessary if selecting the cells for further growth. Not desirable.

[0183] Thus, in either embodiment, the results obtained from assaying the microdroplet test panel The results can be directly correlated with the cells in the reference panel, and the selected cells were The advantage of such a step is that it does not harm the cells or cause tumor cells. There are several factors that are undesirable in the preparation of cells to be used for therapeutic purposes, such as exposure to that is, that the cells have not been exposed to the reporter entity.

[0184] Cells may be selected based on the presence of a trait, the absence of a trait, or a mixture of both. Thus, cells, extracts or secretions therefrom may be purified using the methods described herein. In one embodiment, the cells can be tested or assayed for a number of properties. Multiple properties are tested simultaneously in one step. Changes in the body can be detected using different means, such as fluorescent, luminescent, or phosphorescent labels or events. The detection means may also be split fluorescence, split emission or or a protein complementation event such as a fluorogenic ligand.

[0185] The selected cells may be further dispensed from the device and cultured to produce cells prior to further use. In one embodiment, the cells are intended for injection into a patient. In some embodiments, after dispensing a panel of selected microdroplets, the microdroplets contained therein are The cells are treated to expand the population and / or cultivate the clones.

[0186] After dispensing from the device, a subset of the microdroplets contains secretions, metabolites, and other cellular and and / or the acellular body, which 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, including but not limited to any one or more of may be imposed.

[0187] [Culture medium] For the methods of the present disclosure, the cells are encapsulated in microdroplets. The cells are generally provided in a medium, usually an aqueous medium. Medium simply refers to the liquid used to culture the cells. This medium is a minimal medium containing enough material to support the cells. The contents of such a minimal medium will depend on the type of cells to be encapsulated. The components required will vary depending on the cell type being studied and will be readily apparent to those skilled in the art. 2. The reader will understand how to identify the appropriate medium for

[0188] Generally, cell culture media contains a carbon source (e.g., a sugar such as glucose), water, and one or more salts. The medium may contain, if necessary, a pH buffer, an amino acid source, and nitrogen. It may be supplemented with molecules or agents that support or promote division or cell survival. It requires the right nutrients, growth factors, signaling molecules (such as cytokines), vitamins, and salts. These include: proteins, 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; It does not contain proteins or growth promoters. Therefore, it is not considered a "complete" supplement. The medium should be supplemented with 5-10% fetal bovine serum (FBS). DMEM has been found to be widely applicable to mammalian cell culture and is It can also be used to culture bryozoans. An alternative medium is Eagle's Minimum Essential Medium (EMEM). EMEM contains a balanced salt solution, non-essential amino acids, and sodium pyruvate. EMEM is a non-complex medium and is therefore generally fortified with additional supplements or higher concentrations of serum. RPMI is a medium containing 20% ​​soluble cellulose, making it compatible with a wide range of mammalian cells, especially hematopoietic cells. RPMI is a general-purpose medium that can be widely applied to cells. It was developed for the 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 is suitable for the culture of fresh human lymphocytes, Fusion protocols and hybrid cells such as mouse hybridoma cells for antibody preparation. It has a wide range of applications for mammalian cells, including cell proliferation. For preserving / suspending peripheral blood mononuclear cells. Many types of cell culture media are available and can be tailored to suit the cell species of choice. They should be selected to optimize the survival of the species.

[0190] The disclosed method comprises: distributing a panel of microdroplets containing at least one cell to at least one A reference panel of microdroplets containing one cell and a reference panel of microdroplets containing at least one medium Those skilled in the art will appreciate that the microscopic analysis of several test panels may involve dividing the sample into test panels. The droplets may also contain cells, particularly precursor droplets containing at least one cell. It will be understood that this is the case if cell division occurs in the droplets.

[0191] In one embodiment, the microdroplet containing at least one cell has already undergone a splitting operation. A reference panel of cells containing at least one microdroplet has been generated. As a result, clonal copies of the cells are preserved and can be used to stimulate cells in the test panel at any subsequent step. Even if the cells are damaged by the stimulation, they can still be selected from the reference panel or microdroplets. In particular, this damage can cause cells to lose their ability to access genetic material or intracellular products. This may occur during the dissolution process.

[0192] When such division is employed to separate cells and medium, at least one cell The precursor microdroplets containing the secreted product are cultured under conditions that allow the secreted product to accumulate in the medium. Suitable conditions include the secretion of stimulatory or signaling molecules (e.g., cytokines). or to induce the formation of secretions. These include cells such as antigen-presenting cells that interact with the target cells. If a secretion is genetically designed to be produced by the cell, the manipulation can alter the production of the secretion. It can also be applied to condition expression, and such expression can be regulated by chemicals such as lactose. Such appropriate signals must be provided to initiate expression. can be.

[0193] Once the cells are cultured to promote secretion, the panel of microdroplets can be partitioned. The splitting operation may be performed using real or virtual electrowetting electrodes. In an embodiment, each microdroplet in the microdroplet panel is split to form at least two panels of daughter droplets. These child droplets may be roughly the same size, or the division may be asymmetric, with one The droplets on the panel can be large in size. When the droplets split, they form a single droplet. The droplets may receive at least one cell, and such microdroplets may be used as a reference panel. Form.

[0194] The presence of secretions in the medium can be confirmed as described herein. A series of markings on the microdroplets of the test panel, such as merging one or more panels of the microdroplets, It is useful to employ a splitting event, and the secretions in the microdroplet test panel are It may be diluted or washed.

[0195] Microdroplets containing cells and media can be used to control conditions such as the environment and temperature to maintain the cells. To maintain cells in optimal conditions, As described in 104769, saturating levels of nutrients, buffers, nitrogen, oxygen and and / or carbon dioxide. Accompany.

[0196] Those skilled in the art will appreciate that microdroplets containing at least one cell may be dispersed in a solution containing at least one cell, even if the microdroplets are aqueous in nature. It will be understood that the term "medium" essentially includes the medium.

[0197] [Cell division] The disclosed method involves allowing a panel of microdroplets containing at least one cell to undergo cell division. Cell division may include a step of allowing a parent cell to divide into two or more daughter cells. In this context, each daughter cell is genetically identical to the parent cell (eukaryotic In the case of organisms, the cells undergo mitosis. They may undergo division, separating the genetic material equally into two daughter cells. Since the genetic material should be identical, they are sometimes referred to as clones.

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

[0199] In the disclosed methods, cell division or clonal expansion can occur within a panel of microdroplets. Cells are provided with appropriate culture medium, nutrients, and cytoplasm in the microdroplets. Addition of related auxiliary molecules such as steroids, vitamins, activators, hormones, or growth factors Suitable conditions for allowing cell division can be provided, such as the conditions for cell division. It will be appreciated that the conditions required will vary depending on the cell type in question.

[0200] In one embodiment, the cells present in the panel of microdroplets containing at least one cell are These immune cells include lymphocytes (NK cells, B cells, or T cells). Cells may require activation to induce clonal expansion. Activation involves, among other things, Activation may also be accompanied by signaling molecules such as allergic cytokines. Additionally, this may involve binding to specific cell surface receptors on immune cells. These receptors can be bound using antibodies directed against them. Alternatively, appropriate cells ( They can also be activated using antigen-presenting cells (APCs, etc.).

[0201] In one embodiment, the panel of microdroplets containing at least one T cell comprises an anti-CD3 antibody. A panel of microdroplets containing at least one T cell activator, such as a CD4+ antibody or an anti-CD28 antibody, is prepared. It can contain or merge with rules.

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

[0203] If the cells are not activated or cell division does not occur, the cells (clones) The lack of further replica generation may result in the microdroplet not being selected. Thus, activation and / or division capacity are properties selected for in the methods of the present disclosure. could be.

[0204] When cells are cultured to promote cell division, the panels or microdroplets can divide. The splitting operation can be performed using real or virtual electrowetting electrodes. In one embodiment, each microdroplet in the microdroplet panel is split into a small number of daughter droplets. These child droplets are roughly the same size, or they form at least two panels. There may be asymmetry, with the droplets on one panel being larger in size. When the droplets are separated, each daughter droplet receives at least one cell. The droplets can be discarded or excluded from selection. Cells in the divided microdroplets are substantially intact. genetically identical or cloned.

[0205] In one embodiment, after cell division, the panel of microdroplets containing at least one cell is A reference panel of microdroplets and test microdroplets that can be further used in the methods described herein. The disclosed method is divided into two panels, one containing genetically identical cells. To achieve this, an assay is performed to assess the properties of cells in a panel of microdroplets containing at least one cell. Based on the results and / or the test, cells for the reference panel of microdroplets can be selected. To perform Noh.

[0206] The method described here involves "culturing" cells to allow cell division. This means that such a technique simply grows cells in a microdroplet environment. Use appropriate cell culture media, nutrients, and other conditions favorable for growth, as discussed in the white paper. Those skilled in the art are aware of cell culture techniques that promote cell growth and division. Deaf.

[0207] [Identification and tracking of microdroplets] The microdroplets of the present disclosure are suitably manipulable on devices such as those described herein. The control of the droplet manipulation can be performed by a computer. is a correlation or corresponding microdroplet, e.g., a microdroplet resulting from a fission event of a precursor microdroplet. The droplets can be identified so that the droplets can be identified. When the subject shows a result on the test panel, a microdroplet can be selected from the reference panel. Additionally, one of the microdroplets generated from the fission event is collected from the microfluidic chip, When dispensed into an external microwell plate, the original precursor microdroplets (see reference The DNA sequencing, P, is performed on the associated daughter droplets. The results can be matched to results from off-chip assays such as CR or mass spectrometry.

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

[0209] From the location of the microdroplets within the panel, it is possible to determine the identity of the microdroplets. The location of the droplet and its identity can be controlled by software. Once the properties of the cells in the droplets merged with the original droplets are determined, the results can be transferred to the same cells. Correlation can be made with the associated corresponding microdroplets of the reference panel.

[0210] The assay steps above can be controlled by software to detect specific cell types or levels. Adapt or modify the sequence of steps depending on the exact requirements of your transporter assay. For example, in the case of slow-secreting cells, the reporter panel droplets can be merged. The latency period can be adjusted. The software algorithms are generated on the chip. It can be implemented to provide assay steps that respond to time-dependent events. In the case of cell division, image recognition software is used to count the number of cells in the microdroplets. Once the cell number reaches a certain level, a process can be triggered to split the droplets. In some embodiments, the microdroplets are transported through miniature valve structures or closures within a microfluidic chip. A droplet of fluid contained within a microstructure trap, such as a fluid-retaining pen within a chamber structure. In some embodiments, the process of splitting the droplets involves the application of mechanical structures or microstructures. In some embodiments, the droplets are split using acoustic manipulation. will be done.

[0211] In some embodiments, the process of merging microdroplets can be performed using mechanical actuation, electrophoresis, or other methods. This is done by colliding microdroplets with each other using ultrasonic waves, acoustic waves, or centrifugal force. .

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

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

[0214] According to one aspect of the present disclosure, a method for selecting cells in an EWOD or oEWOD device is provided, the method comprising the steps of: i. Testing microdroplets containing at least medium or medium and at least one cell providing a panel; ii. At least one reporter of the microdroplet containing one or more reporter entities. providing a turn panel; iii. Mixing the test panel microdroplets with at least one reporter panel microdroplet. merging the microdroplets to form a panel of merged assay microdroplets; and iv. detecting a change in said reporter entity based on the presence of at least one property. A panel of assay microdroplets is monitored using a detection system that can detect the presence of and v. Select a subset of microdroplets from the panel based on changes in reporter entities a step of: said subset containing selected cells; selecting a Equipped with The test panel of microdroplets containing at least medium contains at least one cell and medium. and further dividing the panel of microdroplets containing at least one cell into a plurality of microdroplets. and Further, the test panel of microdroplets containing at least one cell is subjected to steps (i) to (v). before or after the droplet is divided into at least two panels of microdroplets, The panel consists of tests on microdroplets containing at least medium or medium and at least one cell. a panel, the microdroplets being suitable for merging with the microdroplets of the reporter entity panel; a test panel of droplets, and a reference panel of microdroplets containing at least one cell.

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

[0216] Alternatively, a method for selecting cells in an EWOD or oEWOD device is provided, The method comprises the following steps: 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 panel of at least one reference microdroplet panel containing one or more cells, and dividing the sample into at least one test panel of microdroplets containing at least said medium; iii. at least one microdroplet reporter comprising one or more reporter entities; providing a panel; iv. Mixing the test panel microdroplets with at least one reporter panel microdroplet. and merging the microdroplets to form a panel of merged assay microdroplets. v. detecting a change in said reporter entity based on the presence of at least one property. A detection system capable of monitoring a panel of assay microdroplets is used. Tep and vi. Based on the variation of reporter entities in the corresponding assay microdroplet panel selecting a subset of microdroplets from the reference panel, said subset comprising: Selecting the subset, which comprises the selected cells. Equipped with.

[0217] According to another aspect of the present disclosure, cells are selected by use of an EWOD or oEWOD device. The present invention provides a method for detecting a cellular signal, the method comprising the steps of: i. Providing a panel of microdroplets comprising at least one cell and medium. and, ii. at least one reporter microdroplet containing one or more reporter entities; providing a panel; iii. A microdroplet of the panel containing at least one cell is transferred to at least one reporter. and merging the microdroplets of the panel to form a panel of merged microdroplets. Pu and, iv. detecting a change in said reporter entity based on one or more properties of the cell; and A subset of microdroplets is selected based on said change in reporter entity to identify the microdroplets containing cells. Using a detection system capable of forming a selected panel of droplets, merged micro- monitoring the panel of droplets; v. Collecting a selected panel of cell-containing microdroplets and repeating steps (ii)-(iv). The step (ii) may be repeated one or more times, and one or more different reporter entities may be used in step (ii). Pu and, vi. A step for selecting cells based on the final panel of microdroplets selected in step (v). Tep and Equipped with wherein, prior to step (iii) and / or step (v), at least one cell a panel of microdroplets comprising: The panel is a. A microdroplet containing at least a medium and suitable for merging with the reporter entity panel microdroplet. a test panel of microdroplets containing b. A reference panel of microdroplets containing at least one cell is.

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

[0219] The microdroplets of any panel may be used for the purpose of diluting the contents of the microdroplets or In order to "wash" the container, it may be subjected to multiple merge and split operations. Such a procedure involves depositing a panel of microdroplets containing a medium, buffer, or suitable aqueous solution. Merging a panel of microdroplets and dividing the merged microdroplets into at least two subdroplets. This step further dilutes the contents. These steps may be carried out consecutively to achieve the above-mentioned results.

[0220] In one embodiment, the microdroplets in the various panels are merged in pairs, forming a pair (two) of microdroplets. The droplets merge to form new microdroplets. This pairing step may be performed more than once. In this case, multiple microdroplets merge to form merged microdroplets. The number of steps to be taken may ultimately be 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. Each of these may be merged sequentially or nearly simultaneously.

[0221] When a set of microdroplets merges, one microdroplet from each panel merges almost simultaneously. The microdroplets are then merged to form a new panel of merged microdroplets. The droplet sets consisted of 2, 3, 4, 5, 6, 7, 8, 9, and 10 droplets from different panels. or more microdroplets.

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

[0223] In one embodiment, step (ii) comprises analyzing the cellular characteristics, such as the presence or absence of a particular cellular marker or molecule. This involves the use of reporter entities to determine cell surface properties. The microdroplet test panel is then Microdroplets can be merged with additional reporter panels, where different cell types Reporter entities that determine the properties of cell-cell interactions, such as their ability to activate or kill a species Involves the use of.

[0224] Alternatively, in another embodiment, these steps can be reversed, e.g., to allow for the differentiation of other cell types. Using reporter entities that determine properties of cell-cell interactions, such as their ability to activate or kill First, the cells are characterized, and second, cell characteristics, such as the presence or absence of specific cell markers, are identified. The cell surface properties of the protein are characterized using a reporter entity that determines the cell surface properties of the protein.

[0225] Multiple steps are used to merge the test panel of microdroplets with the reporter panel of microdroplets. When used, a split step may 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. The reference panel may therefore represent a subset of the cells initially obtained.

[0226] In one embodiment, a panel of microdroplets containing at least one cell is Before merging with the panel of reporter entities containing the reporter entities, the microdroplets are Both panels are divided into two microdroplet panels, and the reference panel of microdroplets contains at least one cell. These cells are maintained to contain cytotoxic substances that may be harmful to the cells or may be used for treatment, etc. Do not contact the reporter entity, which may interfere with further use of the cells in the other The constructed microdroplet panel (test panel) contains a medium or medium and at least one Before the division step, the cells may be in a state of cell division or secretion. They can be cultured under conditions that allow either.

[0227] In certain embodiments, the method comprises:

[0228] Using the EWOD or oEWOD device, nucleic acid encoding a CAR is transfected. 1. A method for selecting immune cells, such as introduced T cells, comprising the steps of: That is, i. at least one immune cell transfected with a nucleic acid encoding a CAR providing a panel of microdroplets containing cells; ii. a reporter, comprising one or more reporter entities specific for cell surface markers; providing at least one panel of microdroplets; iii. Adding a panel microdroplet containing at least one cell to a reporter panel microdroplet. merging the droplets with the droplets to form merged panels of microdroplets; iv. detecting a change 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 change in reporter entity to perform a small A detection system capable of forming a selected panel of microdroplets containing at least one cell. monitoring the merged panel of microdroplets using a system; v. Collecting a panel of selected microdroplets containing at least one cell, and optionally Steps (ii) to (iv) are repeated one or more times, and step (ii) is performed using one or more different reporters. using a target entity; vi. Cells based on the final panel of microdroplets selected in step (iv) or (v). selecting; Equipped with Prior to step (iii) and / or step (v), a microorganism containing at least one cell is The panel of droplets is cultured to allow cell division and then incubated with at least one and dividing the two panels into at least two panels of microdroplets containing the cells, a. a test panel of microdroplets containing at least one cell; and b. A reference panel of microdroplets containing at least one cell is.

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

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

[0231] In a different embodiment, the method provides: A method for selecting genetically modified hematopoietic stem cells using an EWOD or oEWOD device. and comprising the steps of: i. Preparing a panel of microdroplets containing at least one genetically modified hematopoietic stem cell. and ii. Packaging at least one reporter microdroplet containing one or more reporter entities. preparing a panel; iii. Adding a panel microdroplet containing at least one cell to a reporter panel microdroplet. merging the droplets with the microdroplets to form panels of merged microdroplets; iv. detecting a change in said reporter entity based on one or more properties of the cell; and selecting a subset of the microdroplets based on said change in reporter entity; Using a detection system capable of forming a panel of selected microdroplets containing cells monitoring the panel of merged microdroplets; v. Collecting a panel of selected microdroplets containing at least one cell, and optionally Steps (ii) to (iv) are repeated one or more times, and step (ii) is performed using one or more different reporters. using a target entity; vi. Cells based on the final panel of microdroplets selected in step (iv) or (v). selecting; Equipped with Prior to step (iii) and / or step (v), a microorganism containing at least one cell is The panel of droplets is incubated to allow cell division, after which at least one cell is generated. The microdroplet is divided into at least two panels of microdroplets containing vesicles, the two panels being as follows: be. a. Contains at least one cell and is suitable for merging with the reporter panel microdroplet a panel of suitable microdroplets, and b. A reference panel of microdroplets containing at least one cell is.

[0232] In a different embodiment, the method provides: Selecting cells based on immunoglobulin production in EWOD or oEWOD devices said method comprising the steps of: i. Microdroplets containing at least medium or medium and at least one immune cell providing a test panel; ii. at least one microdroplet reporter containing one or more reporter entities; providing a turn panel; iii. Mixing the test panel microdroplets with at least one reporter panel microdroplet. and forming a panel of merged assay microdroplets; iv. detecting the presence of said reporter entity based on the presence of at least one immunoglobulin. A panel of assay microdroplets is monitored using a detection system capable of detecting changes. a step of filtering; and v. Selecting a subset of microdroplets from the panel based on changes in reporter entities; the subset containing selected cells; Equipped with The test panel of microdroplets containing at least medium contains at least one cell and medium. The cells are prepared by dividing a panel of microdroplets containing at least one cell. Fabricating a reference panel of microdroplets and / or further comprising: a test panel of microdroplets containing at least one cell; dividing the droplet into at least two panels of microdroplets before or after any of steps (i) to (v); The two panels contain at least a medium or a medium and at least one cell, a test panel of microdroplets suitable for merging with the microdroplets of the Porter substance panel; and and a reference panel of microdroplets containing at least one cell.

[0233] In a different embodiment, the method provides: Genetically engineered cells based on the production of pharmaceuticals in EWOD or oEWOD devices 1. A method for selecting a target object, said method comprising the steps of: i. a microorganism containing at least a medium or a medium and at least one genetically engineered cell; providing a test panel of droplets; ii. at least one microdroplet reporter containing one or more reporter entities; providing a turn panel; iii. Mixing the test panel microdroplets with at least one reporter panel microdroplet. merging the microdroplets to form a panel of merged assay microdroplets; and iv. detecting a change in said reporter entity based on the presence of at least one agent. A detection system capable of monitoring a panel of assay microdroplets is used. Tep and, v. Selecting a subset of microdroplets from the panel based on changes in reporter entities; the subset containing selected cells; Equipped with The test panel of microdroplets containing at least medium contains at least one cell and medium. The cells are prepared by dividing a panel of microdroplets containing at least one cell. Creating a reference panel of microdroplets and / or the test panel of microdroplets containing at least one cell is subjected to step (i) (v) dividing the droplet into at least two panels of microdroplets before or after the first step, The panel includes at least a medium or a medium and at least one cell, and a reporter A test panel of microdroplets suitable for merging with the microdroplets of the solid panel, and a reference panel of microdroplets containing at least one cell;

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

[0235] Manipulation of microdroplets within any panel is achieved using real or virtual electrowetting electrodes. When microdroplets merge, multiple droplets are mixed to form a single microdroplet. This involves the coalescence of individual microdroplets.

[0236] Optionally, the method may include a further step to allow differentiation on the device prior to any merging step. The reporter entity may be a gene introduced by genetic engineering. The activity of the transgene can be determined and specific cell surface markers, cell products, or Alternatively, cell secretions can be looked for.

[0237] In an alternative embodiment, the method comprises: It involves selecting specific genetically modified cells, and the use of these cells produces products (chemicals). They can produce nutrients (such as proteins and enzymes).

[0238] A method for selecting genetically engineered cells using an EWOD or oEWOD device. ,The following steps, namely: i. Preparing a panel of microdroplets containing at least one genetically engineered cell Steps and ii. at least one reporter microdroplet containing one or more reporter entities; providing a panel; iii. Adding a panel microdroplet containing at least one cell to a reporter panel microdroplet. merging the droplets with the microdroplets to form panels of merged microdroplets; iv. detecting a change in said reporter entity based on one or more properties of the cell; and selecting a subset of the microdroplets based on said change in reporter entity; Using a detection system capable of forming a panel of selected microdroplets containing cells monitoring the panel of merged microdroplets; v. Collecting a panel of selected microdroplets containing at least one cell and staging Steps (ii) through (iv) are repeated one or more times, and one or more different reporter entities are identified in step (ii). The steps used; vi. A step for selecting cells based on the final panel of microdroplets selected in step (v). Tep and Equipped with wherein, prior to step (iii) and / or step (v), at least one cell A panel of microdroplets containing The microdroplet is divided into at least two panels each containing a single cell, the 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; is.

[0239] Optionally, the method further comprises: prior to merging with the reporter panel of the microdroplets, Such a step may comprise lysing the cells in the microdroplets. Generally, after a reference panel of microdroplets each containing at least one cell has been established, It will be implemented.

[0240] Cells can be of any type suitable for genetic modification, including mammalian, insect, plant, bacterial, and fungal. The cells may be of the type described above.

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

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

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

[0244] Example 1—Representative CAR-T Workflow The workflow begins with genetic modification of T cells followed by mass expansion of selected T cells. and target specific areas of the CAR-T cell manufacturing process that are completed before reinfusion into patients. (as depicted in Figure 1).

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

[0246] The patient's blood is drawn and unmodified T cells from the patient are isolated. These T cells are , emulsified into microdroplets in the usual way and introduced into a microfluidic platform. The T cells are then transduced, a step that involves the passage of said microdroplets, each containing a T cell. First, the panels are merged into a panel of microdroplets, each containing a pair of vectors. The method further comprises forming a panel of microdroplets containing the vector operably linked to a promoter. The nucleic acid sequence comprises a linked nucleic acid, the nucleic acid sequence encoding a chimeric antigen receptor. The T cells then proceed to the first screening step. The drops are discarded as needed (Figure 2).

[0247] Multicomponent reporter assays analyze multiple markers on transduced T cells. A detection system that detects the presence of a reporter can be used, and the microdroplets are selected based on this. -The scheme can be implemented using, for example, the following markers: CD4 / CD8, CD19, s cFv domain marker (for specificity and / or affinity screening), B cells markers (or markers of other unwanted cells), and / or differentiation markers (associated To analyze multiple markers on T cells, Microdroplets containing transduced T cells were then transferred to a reporter (primary antibody and fluorescent dye-labeled probe) The method involves merging the cells into microdroplets containing the desired antibody or antibody. This is the earliest possible stage before growth, and will improve subsequent growth and development. This provides the best possible input to the filing step. The drops proceed to the dosimetry and profiling stage (as depicted in Figure 2).

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

[0249] The selected microdroplet cells are spread on the chip and their viability, activation, and consumption behavior are evaluated. Activation screening includes, for example, screening for antitumor activity and efficacy. Detailed methods for evaluating antitumor cell activity are described in Example 3. According to the results of these assays, unwanted microdroplets are discarded and a final panel of microdroplets is selected. In on-chip activity assays, for example, For example, microdroplets containing a dose of T cells may be combined with microdroplets containing tumor cells or tumor-like material. The tumor cell killing activity of T cells was measured at various T cell / tumor cell ratios. The efficacy and safety profile can be evaluated at this stage. The sequence can be determined and genomic data of single cells can be obtained here (Figure 3).

[0250] The final selected microdroplet subpanel of T cells is dispensed from the device into a well plate. The cells can be grown in large quantities and then reinfused into the patient (Figure 3). Enables the best possible consistency and viability of cells, ensuring efficacy and safety profiles This allows for the quantification of file information.

[0251] Example 2—Immuno-Oncology Workflow Chinese hamster ovary (CHO) cells produce immunotherapeutic drugs (e.g., TCRs) The molecules are then emulsified into microdroplets and injected into a microfluidic platform. The remaining microdroplets containing a single CHO cell are discarded. The droplets are then incubated on-chip to facilitate the production of immunotherapeutics. The droplets containing the CHO cells are then split to obtain multiple doses of the drug produced by each cell. Target tumor cells were emulsified separately and then arrayed onto a microfluidic platform. The cell occupancy of each microdroplet is adjusted as desired. The T cells and tumor cells then merge. A second merge operation is used to add the doses of immunotherapeutic agent, determining how much each dose is. The resulting assay was incubated and analyzed to determine whether the cells were apoptotic. The detection system detects caspase 3 / 7 fluorescence, a fluorescent marker for the enzyme. The killing behavior of T cells is monitored using CHO cells that produce an effective amount of the test drug. The device can dispense the solution into a well plate (Figure 4).

[0252] Example 3 - Pan T cell killing assay [Adjustment Protocol] in EMEM (10% FCS, 1% glutamine, 1% penicillin / streptomycin) Tumor model cell lines (passage 59+1) cultured to 80% confluence were then cultured for 1 50 μM caspase 3 / 7, 50 μM Hoechst 33342, and 0.1 nM TCR drug 3e6 / mL droplet medium (RPMI 1640 (10% FCS, 1% glutamine, 1% phenanthrene) Incubate in 10% CO2 at 37°C for 2 hours in 1% ethanol (IU / Strep). On the other hand, Pan T cells derived from human donors were cultured in droplet medium (RPMI) at a density of 2e6 / mL. 1640), thawed, washed with PBS, and then added to 1 μM Deep Incubate in Red at 5% CO2 and 37°C for 30 minutes. The cytosolic suspension is emulsified into microdroplets (Figure 5).

[0253] [Droplet Panel] Microdroplets containing Pan T cells were sorted into the first panel, and microdroplets containing tumor cells were sorted into the second panel. The two panels were then merged into pairs, revealing various tumor cell / Pa Generate microdroplets containing both T cells and tumor cells at a ratio of n T cells. Negative control As an example, microdroplets containing only tumor cells are also included (Figure 6). To confirm that the merged microdroplets at t = 0 time were not catalyzed by caspase 3 / 7. Fluorescence is scanned.

[0254] [result] The results showed that the 50% survival rate was observed for approximately 28 hours, confirming the clear death behavior of cancer cells. This time was sufficient for tumor cell apoptosis to occur (Figure 7). To determine the caspase activity, the caspase intensity is measured for each droplet over time. To image T cells interacting with and sequentially killing tumor cells, brightfield microscopy was used. A microscope is used (Figures 8 and 9).

[0255] Example 4—Antibody Discovery [Cell viability] Unrelated hybridoma cells were stained with Zombie stain and emulsified in microdroplets using microfluidics. The cells were then placed in a microdroplet platform. The efficiency was assessed (10x lens, Zombie Green / GFP (excitation: 457 / 50, emission: 520 / 28): Exposure time 0.54 seconds, Lamp power 100%, BF: Exposure time 0.1 seconds Heat-killed unrelated hybridoma cells were used as a positive control. Viability remained at 100% (33 cells) for 5.3 hours in droplets and 3.6 hours in the device. Furthermore, cell viability remained at 90% for 20.3 hours in the droplets and 18.6 hours in the device. More than 28 cells were maintained in one drop (5 cells in one drop were not maintained throughout the assay). Therefore, excellent survival rates were demonstrated during and after the assay period. The gas resupply mechanism used in this system is partly responsible for the observed sustained biomass This may contribute to the survival rate (Figure 10).

[0256] [Negative control] Microdroplets containing hybridoma cells and reporters (antigen-bound beads and secondary Microdroplets containing the detection antibody are introduced into the light-casting platform, and paired with the target The microfluidics were: 1 bead / 2 cells, 1 bead / 1 cell, beads only, and cells only. Microdroplets containing hybridoma cells or negative controls containing only the reporter were generated (Figure 11). No fluorescence was detected from the droplets. The cells were also stained with Zombie Green dye. No cell death was detected during the experiment. The assay was continued for up to 16 hours and 20 minutes. In microdroplets containing hybridomas and reporter, fluorescent aggregates were detected at t = 3 hours 20 minutes. began to appear in.

[0257] [Bulk assay] Hybridoma cells were emulsified into microdroplets and introduced into a microfluidic platform. Hybrid containing a reporter (e.g., antigen-conjugated beads and AF488-conjugated secondary antibody) Hybridoma culture medium was also emulsified into microdroplets and then introduced into the device. In one panel, the reporter-containing microdroplets were sorted into the second panel. The second panel was merged in pairs and contained 0.5 M / mL beads in hybridoma medium, Microdroplets containing 0.5 μg / mL cells and 200 nM AF488-labeled secondary antibody were The beads were incubated at 37°C for 2 hours. Fluorescence was imaged (BB1, 10x objective, 2 s exposure time).

[0258] 2M / mL beads, 2, incubated in hybridoma medium at room temperature for 4.5 hours. 0.00 nM AF488-labeled secondary antibody, and 10–100 nM free anti-target antibody (anti-macromycin). Further microdroplets containing β-lactam AF488 were generated. The fluorescence of the beads was examined. Free anti-target antibody was imaged over a range of concentrations (BB1, 10x objective, exposure time 2 sec), and a curve showing filter fluorescence (a.u.) versus anti-target antibody concentration was generated (Figure 1 2).

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

[0260] [Unrelated / target hybridoma] Cell Tracker Deep Red fluorescent staining was applied to unrelated hybridomas before emulsification and The unrelated hybridomas were then used to identify them on a chromatographic device. Hybridoma cells that secrete proteins that should not be detected by the reporter molecule The hybridoma cell-containing microdroplets and the reporter-containing microdroplets are defined as: Droplets are introduced into a microfluidic platform, paired and merged, and a range of different Generate microdroplets containing unrelated or target hybridoma cell / bead ratios. Negative control microdroplets containing only the ion transporter were also prepared. Beads incubated with cells identified as did not glow for the duration of the experiment (16 hours). Incubate with target hybridoma (hybridoma producing anti-target antigen primary antibody) The merging beads showed clear AF488 signals 2 hours and 20 minutes after merging (Figure 13). ).

[0261] [Multiplexed spiking assay] Multiplexed spiking assays can be performed using either unrelated or target hybridoma cells. This assay was used to separate a mixed population of microdroplets containing either The hybridization required microdroplets of 100 μm each, all of which were imaged with over 10 times the resolution. Two separate cultures of target and irrelevant cells were grown and the medium was harvested. The target and unrelated hybridoma cells were then separately emulsified into microdroplets and subjected to microfluidic Each of these cells was either the target or an unrelated hybridoma cell. The microdroplets containing either the reporter (target antigen-binding beads) were sorted into a first panel. The medium containing the enzyme and 200 nM of [AF488-conjugated anti-mouse secondary antibody] was also injected into the microdroplets. The first and second panels were merged in pairs and then grouped into a A panel of microdroplets containing the same dye was generated. Filter fluorescence on the beads (exc. ​​457 / 50 nm) / au) over time for each merged microdroplet in the spiking run The secretion curve of the anti-target antibody was plotted for each hybridoma cell line (Fig. 13). The AF488 signal from the relevant hybridoma cell lines was indistinguishable from the negative control. All target hybridomas showed positive signals. Aggregates formed in droplets after approximately 4 hours. After 9 hours, the analysis began to be affected.

[0262] Single-cell secretion data were also collected, and a single-cell secretion curve was developed to show the time dependence of antibody secretion from single cells. The beads were added at 128 nM, and the bead fluorescence was determined from the merger of the beads and antibody. The results were reproduced in multiple runs. To collect data for multiple occupancy groups, we analyzed the data for multiple occupancy groups before merging the first and second panels. The hybridoma cell microdroplets were discarded.

[0263] [Multiplexed beads] Imaging hybridoma cells in culture using a microfluidic platform The beads containing high concentrations of SolR1, R3, and R5 beads were tested. Microdroplets containing 10 ... All intensity bands (R1, R3, R5) were well separated and analyzed for intensity quantification. We determined the optimal imaging parameters while still allowing adequate resolution of the noise. Good resolution between all intensity bands was achieved at 10x, and fair resolution at 4x. Ta.

[0264] Color-coded beads (solR1, R3, R5) contain the cytochrome P450 target antigen, The off-target antigen and the human target antigen are bound to the antigen-binding beads and anti-mouse antibody. The cell culture medium containing the AF488 detection antibody was then emulsified into microdroplets and applied to the Lightcast platform. Each contained a single target or unrelated hybridoma cells. Microdroplets each containing a secreted product are also obtained, and a panel of microdroplets each containing a dividing the cells to generate a reference panel of microdroplets containing corresponding hybridoma cells; The hybridoma cells from which each secreted antibody was derived were then tracked. The microdroplets containing the beads and the detection antibody are paired with the microdroplets containing the secreted antibody. The merged microdroplets and the control microdroplets were characterized by the intensity bands of the beads. Lightcast detection allows both decoding of AF488 and simultaneous quantification of AF488 fluorescence The system was used to monitor the negative control microdroplets containing no cells (reporter The merged microdroplets containing the hybridoma cells (only) and unrelated hybridoma cells remained dark. The absence of primary antibody was confirmed by the presence of reporter and free target antibody [25 nM]. Positive control microdroplets containing free target antibody bind to both target antigens (R1 and R5). Microdroplets containing secreted antibodies also bound to both target antigens (Figure 15). ).

[0265] [Antibody discovery workflow] B cells are emulsified into microdroplets and introduced into a microfluidic platform. The multi-occupied microdroplets are discarded. The remaining microdroplets containing single B cells increase in volume. To achieve this, the droplets are paired with microdroplets containing cell culture medium and merged to facilitate the subsequent division step. The merged microdroplets are then injected into the oocyte to facilitate the production of monoclonal antibodies. The cells are incubated in a microchip and split into daughter droplets to obtain multiple doses of the antibodies produced by each cell. The microdroplets containing B cells are sorted into a reference panel, while the microdroplets containing the antibody dose are sorted into a reference panel. The droplets are sorted into separate panels, each containing a microdroplet containing antigen-binding beads and a detection antibody. The optical detection system is paired with the reporter-mAb panel and merged. The positive hits are then evaluated to determine whether the positive hits contain B cells. Microdroplets of the reference panel are dispensed into well plates and grown (Figure 16).

[0266] Example 5 - Bacterial workflow Bacterial cells are emulsified in microdroplets and introduced into the Lightcast platform. The multi-occupied microdroplets are discarded, and the remaining microdroplets containing single bacterial cells increase in volume. To facilitate this, the droplets are paired with microdroplets containing cell culture medium and merged, followed by a subsequent splitting step. The merged microdroplets grow and divide into clonal colonies. A set of microdroplets containing bacterial cells is sorted into a reference panel, while a set of clonal bacterial cells is sorted into a reference panel. Another set of microdroplets containing bacteria, or a set of microdroplets containing secretions of said bacterial cells. The assays are classified into microdroplet assay panels. If the assay components are located inside the bacterial membrane, Even at this stage, bacterial cells can lyse and release their contents. , used to determine the positive hits of the merged reporter panel. Microdroplets of the reference panel containing bacterial cells corresponding to the α-amino acid sequence were dispensed into well plates and allowed to grow. (Figure 17).

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

[0268] 20A-20C are bright field images of an exemplary splitting operation. A microdroplet splits into two pairs of daughter microdroplets. The droplets are split into parallel droplets. Figure 20A shows droplets in an array awaiting the splitting operation. Figure 20A shows a microdroplet that has been elongated during the splitting operation. Figure 20B shows a microdroplet that has split into a pair of daughter droplets. This shows a microdroplet that has been created. The history information can be maintained by the operation, and all information on the descendant droplets can be obtained. do.

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

[0270] Example 7—Reporter beads in droplets in an emulsion of cell culture medium in oil Encapsulated cells Referring to Figure 22, the cells were grown in microdroplets in an emulsion of cell culture medium in oil. Images of cytokine-secreting cells co-encapsulated with kine reporter beads are shown. In the presence of secreted cytokines, a sandwich ELISA method using fluorescently labeled antibodies was performed. As shown in Figure 22A, the bright-field image shows the fluorescence of the reporter beads. The fluorescence image shown in Figure 22B shows the distribution of cells and beads within the droplets. Figure 22C shows which reporter beads react positively in the presence of ATP. The fluorescent image shown in Figure 22D shows the location of all cells with intact membrane integrity. The location of the needles is shown, and Figure 22E shows dead cells as shown by propidium iodide staining.

[0271] Referring to Figure 24, reporters were found in microdroplets in an emulsion of cell culture medium in oil. Images of antibody-secreting cells co-encapsulated with Turbeads are shown. In the presence of secreted antibody In this study, a reporter antibody was detected by a sandwich ELISA mechanism using a fluorescently labeled antibody. The bright-field image in Figure 24A shows the distribution of cells and beads within the microdroplets. The fluorescence image shown in Figure 24B shows which reporter beads were positive in the presence of secreted antibodies. Figure 24C shows the location of all cells with intact membrane integrity. However, Figure 24D shows dead cells.

[0272] Example 8 - Modular design for microdroplet manipulation Figure 23 shows how you can combine all or any of the following in any order to build your own workflow. This shows an overview of the modular design of the operation, which allows flexible combination of programmable parts. Possible decisions can be made at any point throughout the workflow and are organized into decision trees. (Decision tree) Deviating from the original order based on logic navigation, and / or In this way, the assay can be performed in a dynamic manner. Exemplary modules are shown, but are not limited to these. It is not intended to be a microdroplet injection and / or sorting system, a microdroplet merging system, an incubation system, or a microdroplet sorting system. Examples of microdroplet classifications include injection, assay, splitting, and export. classifications include, but are not limited to, classifications based on size, contents, and / or occupancy. Merging droplets allows for the delivery of reagents and / or reporters. This allows for the controlled introduction of cells to promote cell-cell interactions. This allows material to be recovered for downstream analysis or, in the case of cell recovery, for further expansion. It is possible.

[0273] The workflow shown in Figure 25 can be used to monitor cell secretion or cell-cell interactions. The various detection modes that can be used are shown in the figure. Detection can be performed by fluorescence detection, luminescence detection, FRET detection, etc. In intracellular imaging, the cells are imaged using a variety of methods, including, but not limited to, bright field detection. It can reveal signals within parts of the vesicle or cell vessels, examples of which include the nucleus, Golgi apparatus and / or mitochondria.

[0274] Example 9—Synthetic Biology Workflow, Mammalian Cell Line Development and / or CRISPR screening Figure 26 shows the potential applications of synthetic biology, mammalian cell line development, and / or CRISPR screening. This is an outline of an exemplary workflow suitable for screening, but is not limited to these screening methods. Cells engineered to produce a desired product are not intended to be used on a platform. The cells are then injected and contained in droplets, which can be sorted by occupancy to select single cells. Cells can be incubated and allowed to grow on the platform for a period of time. By monitoring the growth, healthy clones can be selected. Optionally, it can be split to control droplet size or to separate products or Collect either the cultured or uncultured cells, while retaining the population for alternative manipulations. While performing assay steps that may result in cell lysis on the daughter microdroplets, This is particularly useful for preserving live cells for further testing in daughter microdroplets. By merging, we can control the droplet size and assign components, e.g., cell life span, to the cell droplets. A medium for extending the cell culture and / or a reporter can be provided. Facilitate titer readouts with various detection modes to assess cell productivity. The microdroplets can be dispensed and collected from the platform. By harvesting the product, further propagation or downstream analysis can be performed. Recovery allows for evaluation of the quality of the product. The order of operations can be freely changed. For example, productivity Based on the evaluations performed during screening, such as top clones with high activity and proliferation Microdroplets can be dispensed using the micro-dispensing device.

[0275] Example 10 - Microdroplet sorting workflow Referring to FIG. 27, an exemplary workflow following an example sequence of modular operations is shown. It provides the possibility to optionally sort the microdroplets throughout the workflow. The classification is based on droplet size, content, occupancy, assay results, and The imaging can be based on many factors, including brightfield, fluorescence, and combinations thereof. Luminescence and FRET are used to localize the signal to the reporter or cell within the microdroplet. Some examples of detection modalities for classifying whether a particular

[0276] Example 11 - Bright-field imaging of cells encapsulated in microdroplets Referring to Figures 28A-28D, the cells were incubated in the presence of an intracellular calcium dye. Figure 28A shows a sample of a cell encapsulated in a microdroplet. Brightfield images of the droplets show the location and number of cells within each droplet. Figure 28C shows a fluorescence image of the nuclear staining of the cells, Figure 2 8D shows dead cells revealed by propidium iodide staining.

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

[0278] As used herein, "and / or" refers to the combination of two specified features or structures. Each component element is considered to be specifically disclosed regardless of the presence or absence of the other. For example, "A and / or B" means (i) A, (ii) B, and (iii) A and B, respectively. and each of which is considered a specific disclosure of the same, as if each were individually set forth herein. It is interpreted as follows.

[0279] Unless the context dictates otherwise, the description and definitions of the features set forth above are in accordance with the present invention. It is not intended to be limited to any particular aspect or embodiment, but rather to encompass all aspects and embodiments described. This applies equally to the embodiments.

[0280] Although the present invention has been illustratively described with reference to several embodiments, the present invention is not limited to the disclosed implementations. The present invention is not limited to the specific embodiments described above, and does not depart from the spirit and scope of the present invention as defined in the appended claims. Those skilled in the art will further appreciate that alternative embodiments may be constructed without departing from the spirit and scope of the present invention. .

Claims

1. In an EWOD or oEWOD device, cells and and / or a method for selecting a portion of cells, comprising the steps of: i. a microorganism comprising at least a medium or a medium and at least one cell; providing a test panel of droplets; ii. at least one of the microdroplets containing one or more reporter entities; preparing a reporter panel of iii. Mixing the test panel microdroplets with at least one reporter panel microdroplet. merging the droplets to form a panel of merged assay microdroplets; and iv. detecting a mutation in said reporter entity based on the presence of at least one property. A panel of assay microdroplets is monitored using a detection system capable of detecting the activation of and v. A subset of microdroplets from the panel based on changes in reporter entities a step of selecting a subset of cells, the subset comprising the selected cells. selecting a set; Equipped with The test panel of microdroplets containing at least a medium contains at least one cell and a medium. The microdroplets are prepared by dividing a panel of microdroplets containing at least one cell. making a reference panel of microdroplets having and / or further comprising: a test panel of microdroplets containing at least one cell; Before or after any of steps (i) to (v), the droplet is divided into at least two panels of microdroplets. The two panels are then placed in a small area suitable for merging with the microdroplets of the reporter entity panel. a test panel of microdroplets comprising at least a medium or a medium and at least one cell; and A method, wherein the reference panel is a microdroplet containing at least one cell.

2. 10. The method of claim 1, wherein the characteristic of a cell is the presence of one or more cell surface molecules; the presence of one or more cellular activities, cell morphology, the presence of one or more cellular secretions, and / or the presence of one or more intracellular products.

3. The method of claim 1 or claim 2, wherein the microorganism contains at least one cell. The method further comprising culturing the cells under conditions that allow cell division prior to dividing the panel of droplets.

4. 4. The method of claim 3, wherein the condition is a microdroplet containing at least one cell. merging the panel with a panel of microdroplets containing culture medium.

5. The method of claim 3 or claim 4, wherein the microdroplet reference panel and the test panel are The reference panel of microdroplets contains cells corresponding to the cells contained in the test panel of microdroplets. The method of claim 1, wherein the clone is divided to contain at least one clonal copy.

6. The method according to any one of claims 3 to 5, comprising at least one cell. The test panel of microdroplets is a panel of microdroplets containing an agent for lysing the cells. The way it is merged with the rule.

7. The method according to any one of claims 1 to 6, comprising at least one cell. Cells in any panel of microdroplets are examined for their morphological characteristics and A method in which morphological properties can be used to select subsets of cells.

8. 8. The method of claim 7, wherein the morphological characteristics include size, shape, adhesion state, cell The method includes determining the membrane state and / or the presence of intracellular features.

9. The method according to any one of claims 1 to 8, wherein, before step (ii), at least A panel of microdroplets containing one or more cells is then subjected to at least one assay for determining a characteristic of the cells. The assay may be performed in a single assay, the assay comprising a microorganism containing at least one cell. The panel of droplets is divided into at least one microdroplet containing at least one reporter entity. and merging the panels together and identifying at least one reporter entity based on the presence of said characteristic. The method is carried out by determining a change in

10. The method according to any one of claims 1 to 9, wherein after step (iv), the aqueous microfluidic at least one panel of droplets is merged with a merged assay panel of microdroplets; The method also includes subsequently splitting the merged panel of microdroplets.

11. The method according to any one of claims 1 to 10, wherein at any stage of the method, Undesirable or empty microdroplets are discarded.

12. The method according to any one of claims 1 to 11, wherein the selected panel of microdroplets The method wherein the cells are dispensed from the device.

13. 13. The method of claim 12, wherein after dispensing a panel of selected microdroplets, treating the cells contained in the sample to expand the population and / or cultivate the clones; Law.

14. 14. The method of claim 12 or 13, wherein the selected panel of microdroplets is separated. After injecting the cells, the cells and / or non-cell bodies such as secretions and metabolic products contained therein are analyzed as follows: Further analysis, including but not limited to any one or more of the following: i. Mass spectrometry; ii. Surface plasmon resonance; iii. High performance fluid chromatography, and / or iv. genetic analysis, including nucleic acid sequencing and PCR amplification; A method of imposing.

15. The method according to any one of claims 1 to 14, wherein the microdroplets on the test panel are transferred to a reference panel. The cells retained on the reference panel correspond to the microdroplets in the panel, and the cells retained on the test panel correspond to the microdroplets in the test panel. The method may be selected based on a change in the reporter entity that results from the mutation.

16. The method according to any one of claims 1 to 15, wherein the cells are genetically engineered. How to do it.

17. 17. The method of any one of claims 1 to 16, wherein the cells are cultured prior to step (i). The genetic engineering may be carried out by the following steps: a. Preparing a panel of microdroplets each containing at least one cell Top and b. Modifications that include one or more genetic elements, each of which can modify a cell providing a panel of microdroplets; c. The cell-containing microdroplets are transferred to microdroplets containing one or more genetic elements. and merging the droplets, each containing a genetically engineered cell, into the droplets. said merging step forming panels of drops; A method comprising:

18. The method according to any one of claims 1 to 17, wherein the cell characteristics include viability, cell proliferation, and cell viability. The method of claim 1, wherein the compound has cell activation and / or cell killing ability.

19. 19. The method of claim 1, wherein the cell characteristic is a reporter. Intracellular products, cell surface molecules and / or secreted substances that can be detected using the entities include: The products below, viz. i. proteins, including glycoproteins or lipoproteins; ii. Chemicals; iii. polymers, iv. Nucleic acids, v. compound; A method that can produce one or more of the following:

20. The method according to any one of claims 1 to 19, wherein at least one reporter element is The body is the following entity: i. Antibodies, ii. Antigen, iii. receptors, iv. Substrate; v. enzymes, vi. Ligands, vii. Nucleic acids, viii. cells, ix. Part of a cell; x. extracellular vesicles, xi. liposomes, xii. polymers, xiii. Chemicals; xiv. Drugs, xv. FRET reporter xvi. Chemiluminescent substances, xvii. tissue samples; xviii. Virus or bacteriophage, xix. cytokines, and / or xx. Protein, The method is any one or more of the following:

21. 21. The method of any one of claims 1 to 20, wherein the reporter entity detects the change. Labeled for direct detection and / or detection of changes in reporter entities The method also includes a second labeled entity that can be detected and reported.

22. 22. The method of claim 21, wherein the label is visible, luminescent, fluorescent, phosphorescent, or Protein complementation ligation, such as split-fluorescence, split-luminescence, or fluorogenic A method that is

23. The method according to any one of claims 1 to 22, wherein the detecting step comprises an optical detection scan. Step, method.

24. The method according to any one of claims 1 to 23, wherein the cells are of the following type: Wow, i. immune cells, ii. bacteria, iii. fungal cells, iv. Insect cells, v. pluripotent cells, vi. cancer cells, vii. hybridoma cell fusions; viii. Cell lines, ix. plant cells, x. Artificial cells, xi. Microcells, xii. Part of a cell, xiii. extracellular vesicles, or xiv. Liposomes, The method is selected from any one of the following:

25. 25. The method according to any one of claims 1 to 24, wherein the cells are lymphocytes. Law.

26. 26. The method of claim 25, wherein the cells are B lymphocytes and A method capable of secretion.

27. 26. The method of claim 24 or claim 25, wherein the reporter entity is a reporter cells, and optionally, a desired immunoglobulin binds to the reporter cells and causes a change. How can you rub it?

28. 26. The method of claim 25, wherein the one or more reporter entities are: Chi, i. antigen-conjugated beads and fluorescent dye-conjugated secondary antibodies; ii. secondary antibody-conjugated beads and fluorescent dye-conjugated antigen, or iii. antigen and fluorescent dye-conjugated secondary antibody beads bound to a carrier surface; is selected from The step of monitoring the immunoglobulin properties may include assaying antigen binding activity. A method including steps.

29. 26. The method of claim 25, wherein the cells are fused to a cell surface receptor or a T cell engaging molecule. The method of claim 1, wherein the gene is genetically engineered to express either

30. 30. The method of claim 29, wherein the cell is an immune cell and the cell surface receptor is The method is a chimeric antigen receptor.

31. 31. The method of claim 30, wherein the method is for selecting CAR-T cells. 。

32. 32. The method of any one of claims 1 to 31, wherein the cells are isolated from a patient. It is something, a method.

33. The method according to any one of claims 1 to 24, wherein the cells are adapted to produce a drug. The method of claim 1, wherein the bacterial cell is genetically engineered to

34. 25. The method of any one of claims 1 to 24, wherein the cells produce an immunotherapeutic agent. How it can be done.

35. The method according to any one of claims 1 to 34, wherein the microdroplets are formed by an immiscible liquid. How it is surrounded.

36. 36. The method of any one of claims 1 to 35, wherein one or more reporter entities are reporters. and optionally, step (v) is a multiplex assay detection. There is a way.

37. 25. The method of claim 24, wherein the cells are hybrids formed by a cell fusion process. The method is a hybridoma cell and is capable of secreting immunoglobulins.

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