Systems and methods for recirculating cells

Abstract

The present disclosure generally relates to systems and methods of recirculating cells such as immune cells. In certain aspects, target cells attached to a chamber are provided, where cells such as immune cells can attach and be activated by the target cells. In some embodiments, flushing fluid through the chamber can remove non-attached cells. In certain aspects, activated cells can be used for determining information about the subject. For example, an aging-related response to a disease (e.g., cancer) can be determined from the activated cells. Other aspects include exposing cells such as immune cells to malignant cells such as cancer cells, kits or devices for conducting various methods described herein, or the like.

Description

[0001] SYSTEMS AND METHODS FOR RECIRCULATING CELLS

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 695,199, filed September 16, 2024, entitled “Systems and Methods for Recirculating Cells,” incorporated herein by reference in its entirety.

[0004] FIELD

[0005] The present disclosure generally relates to systems and methods of recirculating cells such as immune cells.

[0006] BACKGROUND

[0007] Immunosenescence refers to the gradual deterioration of the immune system that occurs with aging. It is characterized by a decline in the function of innate and adaptive immune responses. This includes a reduced number and altered phenotype in antigen-presenting cells (APC), impaired antigen processing and presentation and reduced production of naive T cells, leading to a narrow T cell repertoire diversity. Immunosenescence has significant clinical implications, particularly for immune activation in the context of vaccine response, where reduced vaccine efficacy and the need for higher vaccine doses or boosters are often observed in the elderly. Therefore, immunosenescence represents a multifaceted challenge in the context of vaccine response, and understanding the mechanistic underpinnings of this phenomenon is needed for developing effective vaccination strategies for geriatric populations.

[0008] SUMMARY

[0009] The present disclosure generally relates to systems and methods of recirculating cells such as immune cells. The subject matter of the present disclosure involves, in some cases, interrelated products, alternative solutions to a particular problem, and / or a plurality of different uses of one or more systems and / or articles.

[0010] In one aspect, a method is disclosed. In one set of embodiments, the method comprises exposing target cells attached to a surface of a chamber to a plurality of immune cells. According to some embodiments, at least some of the immune cells attach to the target cells. In another set of embodiments, the method comprises removing at least some of the non-attached immune cells from the chamber. In yet another set of embodiments, the method comprises culturing the target cells and the immune cells together in the chamber to activate at least some of the immune cells. In some embodiments, the method comprises removing at least some of the non-activated immune cells from the chamber.

[0011] In another set of embodiments, the method comprises exposing target cells attached to a surface of a chamber to a plurality of immune cells. According to certain embodiments, at least

[0012] 12795415.1 some of the immune cells attach to the target cells. According to some embodiments, the method comprises flushing at least some of the non-attached immune cells from the chamber at least two times. In certain embodiments, the method comprises activating the immune cells within the chamber via exposure to the target cells.

[0013] In yet another set of embodiments, the method comprises exposing antigen-presenting cells attached to a surface of a chamber to a plurality of T cells. In certain embodiments, at least some of the T cells attach to the antigen-presenting cells. The T cells, in certain embodiments, are from a human subject at least 40 years old. In some embodiments, the method comprises flushing at least some of the non-attached T cells from the chamber. In accordance with some embodiments, the method comprises activating the T cells within the chamber via exposure to the antigen-presenting cells. In certain embodiments, the method comprises culturing the activated T cells.

[0014] In certain aspects, the method comprises exposing antigen-presenting cells attached to a surface of a chamber to a plurality of immune cells. According to certain embodiments, at least some of the immune cells attach to the antigen-presenting cells. According to one set of embodiments, the method comprises removing at least some of the non-attached immune cells from the chamber. In some embodiments, the method comprises activating at least some of the immune cells within the chamber via exposure to the antigen-presenting cells.

[0015] Other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments of the disclosure when considered in conjunction with the accompanying figures.

[0016] BRIEF DESCRIPTION OF DRAWINGS

[0017] Non-limiting embodiments of the present disclosure will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the disclosure shown where illustration is not necessary to allow those of ordinary skill in the art to understand the disclosure. In the figures:

[0018] Fig. 1A and IB illustrate a system with a fluid containing target cells and immune cells and a methodology to activate at least some immune cells, in one embodiment.

[0019] Figs. 2A-2D illustrate a microfluidic device and characterization methods of the same, in another embodiment.

[0020] 12795415.1 Figs. 3A-3E illustrate a flow cytometry analysis of mouse bone marrow cells differentiation into macrophages and dendritic cells, OVA peptide presentation, and cytokine secretion, in yet another embodiment.

[0021] Figs. 4A-4E illustrate activation and proliferation of OVA-I- specific CD8+ T cells on- chip and off-chip, in still another embodiment.

[0022] Figs. 5A-5I illustrate antitumoral efficacy of CD8+ T cells stimulated by OVA- presenting MQs and DCs, in accordance with another embodiment.

[0023] Figs. 6A-6E illustrate an in vivo characterization to evaluate the immunogenicity and antitumoral efficacy of OVA peptides, according to still another embodiment.

[0024] Fig. 7A and 7B illustrate a chamber and simulated fluidic flows inside the chamber, in yet another embodiment.

[0025] Fig. 8 illustrates confocal images of the on-chip presentation of the OVA peptide, in one set of embodiments.

[0026] Fig. 9 illustrates confocal images of on-chip activation of OVA-specific CD8+ T cells, in another set of embodiments.

[0027] Fig. 10 illustrates confocal images of antitumoral efficacy of OVA-I-specific CD8+ T cells, in yet another set of embodiments.

[0028] DETAILED DESCRIPTION

[0029] The present disclosure generally relates to systems and methods of recirculating cells, such as immune cells. In certain aspects, target cells attached to a chamber are provided, where cells such as immune cells can attach and be activated by the target cells. In some embodiments, flushing fluid through the chamber can remove non-attached cells. In certain aspects, activated cells can be used for determining information about the subject. For example, an aging-related response to a disease (e.g., cancer) can be determined from the activated cells. Other aspects include exposing cells such as immune cells to malignant cells such as cancer cells, kits or devices for conducting various methods described herein, or the like.

[0030] For example, certain embodiments generally relate to systems and methods of recirculating, attaching, and activating immune cells in a chamber. In some embodiments, immune cells are introduced to a chamber with a plurality of target cells. These may be added in any suitable order. The immune cells may attach to the chamber and / or become activated, e.g., by interacting with the target cells. For example, some embodiments are directed to target cells (e.g., antigen presenting cells) attached to a surface of the chamber, where T-cells can attach and thereby become activated T-cells, e.g., upon interaction with the target cells. In some cases, nonattached immune cells can be replaced from the chamber through various methods (e.g., flushing with a solution, optionally containing new cells). In some cases, this may activate more immune

[0031] 12795415.1 cells, e.g., upon interaction with the target cells. Without wishing to be bound to any particular theory, it is believed that replacing non-activated immune cells may lead to a higher activation of immune cells compared to conventional methods.

[0032] Some aspects described herein include culturing target cells and immune cells together in a chamber, for example, in a microfluidic chamber. In some cases, target cells and / or immune cells may be cultured together with an agent suspected of altering an immune response, which may have numerous applications (e.g., drug screening, etc.), such as is explained in more detail below. Certain systems and methods disclosed herein may help determine the characteristics of a subject. For example, in some cases, age-related responses of the immune system (e.g., increased immunity or decreased immunity) may be studied, used to treat a subject (e.g., a subject having a tumor), or the like.

[0033] Some non-limiting embodiments of the present disclosure relate to systems and methods of activating immune cells and administering these cells to a subject. The activated immune cells (e.g., activated T cells) may be capable of fighting a disease (e.g., cancer) that the subject has or is at risk for. Such activated immune cells may be used, for example, to enhance an immune response in the subject. For example, such activated immune cells may be used to treat or prevent cancer, treat certain symptoms of aging, or the like. As another non-limiting example, activated immune cells (e.g., activated B cells) may secrete a product (e.g., an antibody) that can fight an infection.

[0034] In some embodiments, target cells are used to activate at least some of the immune cells. Target cells may have different features that may allow them to interact with and be recognized by immune cells, e.g., target cells may have surface receptors or may release antigens. The immune cells activated via target cells may perform different functions within the immune system, in various embodiments.

[0035] In some embodiments, target cells may be one of any type of cells capable of presenting a biological marker (e.g., antigen-presenting cells). Some antigen-presenting cells may undergo a cellular response that can trigger a biological marker (e.g., an antigen). The biological marker may be any marker that may induce an immune response in immune cells. Some biological markers may be entities such as carbohydrates, metabolites, allergens, nucleic acids, proteins, peptides, lipids, small molecules, etc. Biological markers may also be a combination of more than one entity type (e.g., lipopolysaccharides). The markers released by antigen-presenting cells may be an entity on the surface of antigen-presenting cells (e.g., epitopes), which may allow an immune cell to bind to the surface of the antigen-presenting cell. Thus, target cells may offer numerous pathways for to be recognized my immune cells. According to some embodiments, as

[0036] 12795415.1 schematically illustrated in Fig. 1A, target cells 30 can release biological marker 50, which can be recognized by immune cells 40.

[0037] Biological markers may be presented by biological entities other than antigen presenting cells, for example, viruses and bacteria. In some cases, viruses and / or bacteria can present a biological marker that may originate an immune response in which immune cells may participate. For example, bacteria may have surface receptors that may be identified by immune cells as a threat, thus immune cells may elicit a response to destroy bacteria. In some embodiments, target cells are bacteria or viruses. In some cases, these may be contained in other cells.

[0038] In some embodiments, target cells comprise monocytes. Monocytes are a type of white blood cells that are part of the immune system of many organisms. Some non-limiting examples of monocytes are lymphocytes, basophils, neutrophils, and / or eosinophils. In some embodiments, monocytes can undergo cell differentiation and become another type of cell (e.g., dendritic cells). Therefore, in some embodiments, target cells may originate from another type of cell.

[0039] In some embodiments, target cells may include one or more types of target cells. Certain embodiments are related to activating immune cells by exposing them to one or more types of target cells. Some immune cells may benefit from being exposed to one or more types of target cells for any of many reasons (e.g., be activated more efficiently). In certain embodiments, two types of target cells can be used to activate immune cells. In some embodiments, one or more target cell types originate from target cells. For instance, one or more types of target cells (e.g., macrophages and dendritic cells) may have originated as a result of cell differentiation from another target cell (e.g., monocytes).

[0040] In some embodiments, at least some of the target cells are attached to the surface of a chamber. Using a chamber with attached target cells may provide some advantages, as will be discussed later. Target cells attached to the chamber's surface may have different surface coverage. In some cases, the target cells attached to the surface of the chamber may cover at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In some cases, the target cells attached to the surface of the chamber may cover no more than 100%, no more than 90%, no more than 80%, no more than 70%, no more than 60%, no more than 50%, no more than 40%, or no more than 30%. Combinations of any of these ranges are possible. For example, the target cells may cover at least 30% but no more than 90% of the surface of the chamber.

[0041] In some embodiments, immune cells can be activated through the systems and methods disclosed here. Immune cells are generally activated to prevent and / or fight a threat to an

[0042] 12795415.1 organism, which may happen in the presence of an external agent (e.g., a pathogen) or an internal agent (e.g., cancer cells). In some cases, immune cells are also activated in the presence of other immune cells (e.g., T-cells can activate B cells). Activated immune cells are important for various roles within the immune system, as will be discussed later. In some embodiments, immune cells comprise T-cells. T-cells are a type of immune cell capable of contributing to the eradication of other biological entities (e.g., other cells). For example, activated immune cells may help coordinate an immune response (e.g., helper T cells) and / or destroy malignant cells (e.g., cytotoxic T cells). T-cells may be activated through many of the methods known in the art and may be used as activated immune cells in any of the methods disclosed here.

[0043] The plurality of immune cells may be a suspension of cells. According to some embodiments, the suspension of cells are immune cells suspended in a fluid. The suspension of cells may have a different concentration of cells in a certain volume. The suspension of cells may be at least IxlO4cells / mL, at least IxlO5cells / mL, at least IxlO6cells / mL, at least 2xl06cells / mL, at least 3xl06cells / mL, at least 4xl06cells / mL, at least 5xl06cells / mL, at least IxlO7cells / mL, or at least IxlO8cells / mL, and / or no more than IxlO8cells / mL, no more than IxlO7cells / mL, no more than 5xl06cells / mL, no more than 4xl06cells / mL, no more than 3xl06cells / mL, no more than 2xl06cells / mL, no more than IxlO6cells / mL, no more than IxlO5cells / mL, or no more than IxlO4cells / mL.

[0044] Immune cells may have different applications after being activated. According to some embodiments, activated immune cells can target cancer cells. For example, in a subject with a healthy and strong immune system, activated immune cells can often identify and kill cancer cells, and thereby may reduce the risk of a tumor growing in the subject. However, in some cases, when the subject has an unhealthy and weaker immune system, the immune cells within the subject may not be able to kill some cancer cells. In such cases, it may be advantageous to activate immune cells using the systems and methods disclosed and administer the activated immune cells to the subject. The subject may or may not have a risk of developing cancer or may already have cancer. In some cases, the immune cells being activated arise from the subject, although in some cases, the activated cells may be given to a different subject. In an example embodiment, as shown in Fig. IB, activated T cells can identify and kill at least some B16-OVA melanoma cancer cells.

[0045] In some embodiments, the systems and methods disclosed in which immune cells are activated (e.g., for killing cancer cells) are similar or the same as immune cells activated inside a subject (e.g., immune cells in lymph nodes of the subject). In some embodiments, activated immune cells can determine cancer (e.g., help diagnose cancer in a subject) based on the immune response.

[0046] 12795415.1 In some embodiments, immune cells target pathogens and / or other immune cells. The pathogens may be bacteria, viruses, fungi, or the like. Some immune cells may be activated and exposed to different types of bacteria (e.g., cocci, bacilli, spirilla, etc.). Some immune cells exposed to bacteria may be activated and can be used for different purposes when administered to a subject (e.g., fight certain bacterial infections, improve the immune system, etc.). In some cases, some immune cells may be exposed to a virus and may also become activated. The virus may be contained in another cell. In some cases, the systems disclosed may determine infectious diseases.

[0047] A plurality of immune cells may include activated and / or non-activated immune cells. To perform certain functions, immune cells may need to be activated. Activated immune cells may participate in roles during an immune response such as eliminating malignant cells, signaling other immune cells, or the like. Although activated immune cells may be useful during an immune response, among other functions, non-activated immune cells may also have some relevance. For example, non-activated immune cells may be able to interact with other cells and determine whether they pose a threat or not, which may be important to keep an immune system regulated.

[0048] After exposing a plurality of immune cells to target cells, immune cells can be attached to immune cells or non-attached immune cells in relation to the target cells. In some embodiments, at least some immune cells are attached to the target cells, where some attached immune cells are generally considered activated immune cells. However, in some embodiments, non-attached immune cells may also be activated immune cells. For example, non-attached immune cells may be activated by an antigen suspended in a fluid. Non-attached immune cells may be activated in the presence of other non-attached immune cells through different mechanisms. For example, non-attached immune cells (e.g., T-cells) may secrete a biological marker (e.g., cytokines), which may activate other immune cells.

[0049] The systems and methods disclosed here may include a fluid in a chamber. In some cases, the fluid may be an aqueous solution (e.g., a buffer, e.g., phosphate buffered saline, cell culture media, etc.), a biological extract (e.g., blood), a solvent, etc. The fluid may contain other substances such as living cells, dead cells, lysed cells, nutrients, biological markers, proteins, fatty acids, or the like.

[0050] Some embodiments are related to flushing fluid through the chamber. Flushing fluid through the chamber may be advantageous for certain reasons. Some reasons may intend to add an entity or remove an entity. In some cases, flushing fluid may help incorporate immune cells and / or target cells in the chamber, add nutrients beneficial for cell growth (e.g., cell media), remove other immune cells, remove dead cells, etc. In some cases, the fluid flushed through the

[0051] 12795415.1 chamber may not contain new cells, although in some cases, new cells may be added during a fluid flush. In some cases, flushing fluid may still be beneficial for various reasons (e.g., flushing out dead cells, unreactive cells, spent media, debris, etc.).

[0052] The fluid flushed through the chamber may have different volumes. The volume of the fluid may depend on the size of the chamber, the viscosity of the fluid, the contents of the fluid, and / or other factors that may affect the fluid being flushed. If more than one flush is used, the flushes may use the same or different fluid volumes. In some cases, the volume of the fluid may be chosen to take advantage of fluidic properties unique to certain volumes (e.g., microfluidic volumes). The volume of the fluid flushed through the chamber may be, for example, greater than 500 nL greater than 1 microliter, greater than 2 microliters, greater than 5 microliters, greater than 10 microliters, greater than 20 microliters, greater than 50 microliters, greater than 100 microliters, greater than 500 microliters, or greater than 1,000 microliters. The volume of the fluid may be no more than 2,000 microliters, no more than 1,000 microliters, no more than 500 microliters, no more than 100 microliters, no more than 50 microliters, no more than 20 microliters, no more than 10 microliters, no more than 5 microliters, no more than 2 microliters, no more than 1 microliter, or no more than 500 nL. Combinations of any of these ranges are possible.

[0053] The flow rate of the fluid being flushed may be different. The flow rate of the fluid being flushed may depend on factors such as the size of the chamber, the viscosity of the fluid, the contents of the fluid, and / or other factors. The flow rate of the fluid being flushed may be, for example, greater than 100 nL / min, greater than 500 nL / min, greater than 1 microliter / min, greater than 30 microliters / min, greater than 60 microliters / min, greater than 300 microliters / min, greater than 600 microliters / min, or greater than 1,000 microliters / min. The flow rate of the fluid being flushed may be no more than 1,000 microliters / min, no more than 600 microliters / min, no more than 300 microliters / min, no more than 60 microliters / min, no more 30 microliters / min, no more than 1 microliter / min, no more than 500 nL / min, or no more than 100 nL / min. According to certain embodiments, the flow rate of the fluid being flushed may be between 100 nL / min and 1,000 microliters / min, 500 nL / min and 600 microliters / min, etc.

[0054] In some embodiments, flushing fluid through the chamber may help remove at least some of the non-attached immune cells. In some cases, removing non-attached immune cells may allow other immune cells to interact with and be activated by target cells. In some cases, non-attached immune cells may be dead cells, which may no longer be useful during an immune response. In certain embodiments, removing non-attached immune cells is directed to removing immune cells that may no longer be capable of becoming activated immune cells to be removed

[0055] 12795415.1 from the chamber. For example, after cultivating immune cells and target cells together, some non-attached immune cells may not be activated by the target cells and therefore may need to be removed. Without being attached to any particular theory, it is believed that removing some non-attached immune cells may help activate at least some T-cells, or at least reduce the interactions of T-cells with cells that do not activate the T-cells.

[0056] In some embodiments, flushing fluid through the chamber may be performed a plurality of times. Flushing fluid through the chamber may be performed at least one time, at least two times, at least three times, at least four times, at least five times, at least ten times, or at least twenty times, etc., and / or no more than twenty times, no more than ten times, no more than five times, no more than four times, no more than three times, no more than two times, or no more than one time. In some embodiments, flushing fluid through the chamber may be performed a plurality of times to remove at least some of the non-attached immune cells from the chamber. In some cases, flushing fluid through the chamber may allow the system to benefit from the effects of flushing fluid (e.g., more activation of T-cells). If more than one flush is used, the volume of fluid flushed may independently be the same or different.

[0057] The immune cells and target cells may be cultured together in the chamber in some embodiments. It may be advantageous, for example, to culture the target cells and / or immune cells together in the chamber for various reasons (e.g., grow more immune cells). In some embodiments, culturing the immune cells in the chamber may allow the number of immune cells to increase (e.g., due to cell division), for example, doubling their numbers, or more. In some embodiments, culturing immune cells and target cells together may activate at least some of the immune cells. The cultured immune cells may be activated and increase the number of activated immune cells compared to conventional methods.

[0058] In some embodiments, target cells and / or immune cells are obtained from a subject. According to some embodiments, target cells obtained from the subject may be used to activate immune cells, e.g., as discussed herein. Target cells obtained from a subject may include any kind of target cell included in the present disclosure. For example, target cells obtained from the subject may be target cells (e.g., macrophages) that can be exposed to immune cells. Using target cells obtained from a subject may be advantageous for many reasons, e.g., train immune cells to be activated by human target cells, where the immune cells can then be used to fight cancer cells.

[0059] In some embodiments, the subject is human. The human may have target cells that may be used to activate immune cells. The target cells may be human target cells that are related to the immune system (e.g., dendritic cells). However, in other embodiments, the subject may be

[0060] 12795415.1 non-human. For example, the subject may be a non-human mammal, such as a monkey, cow, sheep, goat, horse, rabbit, pig, mouse, rat, dog, cat, etc.

[0061] The subject from which the cells are obtained may be of at least a certain age. According to some embodiments, the subject is at least 10 years, at least 20 years, at least 30 years, at least 40 years, at least 50 years, at least 60 years, at least 70 years, or at least 80 years old. The age of the subject may impact how their immune cells respond in the presence of target cells. As a nonlimiting example, a subject of 20 years of age may have a stronger immune system than subject human of 70 years of age, which may be indicative of how their immune cells react in the presence of target cells. In such case, the activated immune cells of the subject of 20 years of age may be able to kill cancer cells more efficiently than the activated immune cells of the subject of 70 years of age.

[0062] Immune cells may be cultured, activated, and / or administered to a subject in certain aspects. In some cases, culturing cells (i.e., isolating cells and growing them) may allow immune cells to grow in an environment outside the body of the subject, which may offer different advantages (e.g., support the growth of immune cells of the subject). The immune cells that are cultured and activated in the chamber may be administered to a subject, and the subject may be the same or a different subject than the subject from where the cells originated. According to some embodiments, immune cells (e.g., T-cells) are cultured and activated and administered to the subject in different ways (e.g., intravenously). Some non-limiting examples of situations where subjects may benefit from having cells cultured, activated, and administered into their body include a weakened immune system, autoimmune disorders, cancer, infections, etc.

[0063] In some embodiments, a secreted product from the immune cells may be obtained. In some cases, certain immune cells (e.g., B cells) may secrete a product (e.g., antibodies). The secreted product may be any type of antibody (e.g., IgE, IgG, IgM, etc.) that is secreted from immune cells. The secreted product may be part of the fluid that is flushed through a chamber and later collected. The secreted product may be any product that is released by the immune cells after being activated and that may have relevance to the immune system.

[0064] In some embodiments in which a secreted product is obtained, the secreted product may also be administered into a subject. The subject may benefit from having a secreted product administered for different purposes. For example, the secreted product may include monoclonal antibodies that may recognize an entity that may pose a threat to the subject (e.g., a virus), where the monoclonal antibody may bind to the virus and prevent its entry to healthy human cells.

[0065] In some embodiments, the activated immune cells may be exposed to dissociated cells. In some embodiments, the dissociated cells comprise tumor dissociated cells, e.g., micro- organospheres. According to one set of embodiments, micro-organospheres comprise tissue

[0066] 12795415.1 samples that may have similar properties to a tumor (e.g., human tumor). Activated immune cells may be exposed to dissociated cells (e.g., micro-organospheres) for different reasons, e.g., to replicate the tumor environment in a chamber, screening a potential tumor in a subject, etc. In some cases, such micro-organospheres may contain cells originating from a patient, for example, extracted from a small patient biopsy, (e.g., for quick diagnostics to guide therapy), from resected patient tissue, including resected primary tumor or part of a dysfunctional organ (e.g., for high-throughput screening), and / or from already established PDMCs, including patient- derived xenografts (PDX) and organoids. In some cases, these may be contained with gel droplets. The gel droplets may be formed with or in oil. Any of the gel droplets supporting biological tissue (e.g., dissociated cells) may contain cells originating from a patient and / or tissue culture. For example, the cells may be extracted from a small patient biopsy. These gel droplets may be formed from primary cells that are normal (e.g., normal organ tissue) or from tumor tissue. For example, in some variations, the gel droplets may be formed from cancerous tumor biopsy tissue. See, for example, U.S. Pat. Nos. 11,555,180 and 11,628,382.

[0067] In some embodiments, the activated immune cells may be exposed to cancer cells. Activated immune cells may be capable of recognizing and killing cancer cells. In some cases, cancer cells in a subject may not be recognized by immune cells for immune-related reasons. In such cases, administering activated immune cells may help kill at least some cancer cells and benefit the subject.

[0068] Immune cells that are cultured and activated may be removed from the chamber. In some embodiments, at least some of the activated immune cells can be removed from the chamber for any of various reasons (e.g., downstream processing). The activated immune cells may be removed from the chamber and collected, e.g., for downstream processing, to be applied to a subject, etc.

[0069] Some of the immune cells may be sorted and / or counted, for example, using techniques such as flow cytometry and / or cell sorter. Flow cytometry may allow to measure a plurality of immune cells and determine any of different characteristics about the same (e.g., ratio of living cells to dead cells, ratio of activated cells to non-activated cells). In some cases, flow cytometry may be used to analyze the immune cells removed from the chamber and determine the strength of an immune response.

[0070] Some of the activated immune cells may be sequenced after being removed from the chamber. The activated immune cells may undergo certain techniques capable of determining a nucleic acid sequence of cells (e.g., single-cell sequencing). The sequence obtained for at least some of the activated immune cells may provide cell characteristics and / or monitor abnormalities (e.g., mutations).

[0071] 12795415.1 In some embodiments, immune cells are used to determine a disease and / or disorder in a subject. Some diseases and / or disorders that can be determined are cancer, infectious diseases, inflammatory diseases, neurodegenerative disorders, or the like. For example, inflammatory diseases such as diabetes, allergies, joint diseases, etc. may be determined. Some neurodegenerative disorders can also be determined (e.g., Parkinson’s disease). In certain embodiments, the system and / or methods disclosed can determine when an organ transplantation rejection occurs in a subject.

[0072] Immune cells may be used to determine immunosenescence (i.e., aging-related changes to the immune system) of a subject in certain cases. In some embodiments, immune cells are activated in the presence of target cells, where some immune cells belong to a younger subject (e.g., 25 years old) and some belong to an older subject (e.g., 75 years old). In some cases, the difference in ages may be at least 5 years, at least 10 years, at least 15 years, at least 20 years, at least 25 years, at least 30 years, at least 35 years, at least 40 years, at least 45 years, at least 50 years, etc.

[0073] In some embodiments, at least some of the immune cells can help determine an immune response of a subject, where the age of the subject may impact the response. For example, the response may be an immunosenescent response, which can be related to the age of the subject. The response may also be an antitumoral response, where the response is associated with the presence of a tumor.

[0074] After being activated, at least some of the activated immune cells may remain inside the chamber. The activated immune cells may be analyzed inside the chamber through different methods (e.g., light microscopy) that may reveal cell characteristics (e.g., cell size, cell shape, etc.) and / or cell function. In addition, in some embodiments, at least some of the activated immune cells can be removed and analyzed outside of the chamber.

[0075] In some embodiments, target cells and / or immune cells are exposed to an agent suspected of altering a response of the immune cells to the target cells. The agent suspected of altering an immune response (e.g., a drug, a toxin, etc.) may be introduced in the chamber, where the suspected agent may affect an immune response on the immune cells to the target cells, where the extent of the response may be advantageous or disadvantageous for different applications (e.g., drug screening, toxicity determination, etc.). For example, a potential drug may be tested for its efficacy on improving the immune response of immune cells to the target cells.

[0076] The plurality of immune cells can be recirculated within the chamber in accordance with certain embodiments. For example, when being recirculated, non-attached immune cells that are not activated may find more target cells to attach and activate. As another example,

[0077] 12795415.1 recirculating immune cells may allow such cells to stay alive by being exposed to cell media. Referring back to Fig. 1A, as a non-limiting example, system 10 with fluid 20 can have immune cells 40 that are recirculated over target cells 30, according to some embodiments.

[0078] In some embodiments, target cells and immune cells are cultured together for a period of time. According to some embodiments, target cells and immune cells may be cultured together for at least 1 hour, at least 12 hours, at least 1 day, at least 2 days, at least 5 days, at least 10 days, at least 20 days, and / or, no more than 40 days, no more than 20 days, no more than 10 days, no more than 5 days, no more than 2 days, no more than 1 day, or no more than 12 hours.

[0079] Target cells and / or immune cells may be incubated inside the chamber in some embodiments. In some cases, target cells and / or immune cells may be incubated (i.e., cultured under controlled conditions), which may promote their population growth. In some cases, the incubation inside the chamber may increase the activation of immune cells in the presence of target cells. The incubation may have incubation parameters such as temperature, carbon dioxide level, etc., which may benefit culturing the cells. For example, the incubation parameters may be a temperature of 37 °C and a carbon dioxide level of 5%.

[0080] In some embodiments, target cells may be attached to a surface of the chamber, for example, a bottom surface. The surface may aid the recirculation of immune cells within the chamber.

[0081] In some embodiments, the chamber is made of a polymer. The chamber may be fabricated through different methods (e.g., spin-coating, laser cutting, drop casting, molding, etc.). The polymer may be of one or more different polymers (e.g., poly(methyl methacrylate), polyethylene, polypropylene, polyvinyl chloride, polyimide, etc.). In some embodiments a surface of the chamber may be coated with a glycoprotein or other suitable cell surface. The glycoprotein may be any glycoprotein (e.g., fibronectin, laminin, etc.) known in the art that facilitates cell attachment.

[0082] In some embodiments, the chamber has a dimension of a particular size. The chamber may have a largest dimension smaller than 1,000 microliters, smaller than 500 micrometers, smaller than 100 micrometers, smaller than 50 micrometers, smaller than 10 micrometers, or smaller than 1 micrometer, and / or larger than 1 micrometer, larger than 10 micrometers, larger than 50 micrometers, larger than 100 micrometers, larger than 500 micrometers, or larger than 1,000 microliters.

[0083] U.S. Pat. Apl. Ser. No. 63 / 695,199, filed September 16, 2024, entitled “Systems and Methods for Recirculating Cells,” is incorporated herein by reference in its entirety.

[0084] The following examples are intended to illustrate certain embodiments of the present disclosure, but do not exemplify the full scope of the disclosure.

[0085] 12795415.1 EXAMPLE 1

[0086] A microfluidic device was fabricated using poly(methylmethacrylate) (PMMA) in this example. For three layers of the device, the dimensions are 22 mm width, 28 mm length, and 0.5 mm thickness (Fig. 2A). One inlet and outlet were present on the top layer. For the middle layer, the dimensions of the cut-out area were 5 mm width, 18 mm length, and 3 mm diameter of the reservoirs. After treating the surfaces of the layers with 70% isopropyl alcohol (IPA), the layers were attached to develop the final form of the device (Fig. 2B). The optical image of the device is shown in Fig. 2C. After fabricating the device, it was coated with fibronectin to enhance the cell adhesion inside the device. The cells were successfully seeded with media inside the device (Fig. 2D).

[0087] EXAMPEE 2

[0088] In this example, bone marrow cells were isolated from the tibia and femur of young and old C57BE / 6 mice and the absence of macrophages (MQs) (CD1 lb+ / F4 / 80+) and dendritic cells (DCs) (CDl lc+ / F4 / 80-) before the off-chip differentiation procedure (Fig. 3A) was confirmed. After 7 days of off-chip differentiation, both young (Fig. 3B) and old (Fig. 3C) mice had bone marrow cells that were differentiated into MQs (CDl lb+ / F4 / 80+) and DCs (CDl lc+ / F4 / 80-). Ovalbumin (OVA) peptide presentation potential of MQs (CD1 lb+ / MHC-I OVA+) and DCs (CD1 lc+ / MHC-I OVA+) was analyzed using young and old mouse immune cells after 14 days. OVA peptides were presented by young and old MQs and DCs, both on-chip and off-chip. On- chip, a dose-dependent OVA peptide presentation response was presented and a plateau of presentation was observed when a 100 micrograms / mE concentration of OVA peptide was used as the immunogen (Fig. 3D). This dosage was used for experiments and analyses.

[0089] For the OVA peptide presentation by MQs, a significant difference in OVA peptide presentation was observed between on-chip and off-chip experiments using cells from young (63.81 + / - 4.91% vs. 20.71 + / - 0.18%, p<0.001) and old mice (39.26 + / - 2.61% vs. 15.23 + / - 0.2%, p<0.001). In addition, on-chip OVA peptide presentation was significantly higher with young cells than old ones (63.81 + / - 4.91% vs. 39.26 + / - 2.61%, p<0.001), whereas no significant difference in off-chip OVA peptide presentation between young cells and old ones (20.71 + / - 0.18% vs. 15.23 + / - 0.2%, p=0.31). The same observations were made for DCs when comparing on-chip and off-chip experiments using young (65.88 + / - 10.07% vs. 21.24 + / - 0.5%, p<0.001) and old cells (42.25 + / - 2.02% vs. 16.08 + / - 0.89%, p=0.002), and on-chip OVA peptide presentation with young cells (65.88 + / - 10.07% vs. 42.25 + / - 2.02%, p=0.003) and old ones (21.24 + / - 0.5% vs. 16.08 + / - 0.89%, p=0.66) (Fig. 3D).

[0090] The confocal images of the on-chip presentation of the OVA peptide confirmed that the OVA presentation by young MQs and DCs was higher than that of old MQs and DCs (Fig. 8).

[0091] 12795415.1 Lastly, the cytokine production from MQs and DCs was assessed at day 7 after on-chip OVA peptide presentation, and a significant difference in IL-6 secretion was observed between young and old MQs (p=0.002) and DC (p<0.001). A significant difference for IL-2, IL-10, and IL-12 was not observed (Fig. 3E).

[0092] EXAMPLE 3

[0093] In this example, CD8+ T cells were isolated from the spleen of young and old C57BL / 6 mice and a negative selection of CD8+ produced a cell fraction containing 78% of CD3+ / CD8+ T cells was demonstrated, of which 34.79% and 49.53% of CD8+ T cells from young and old mice were alive, respectively (Fig. 4A). Next, OVA-specific T cell activation / proliferation and OVA peptide specificity was characterized using CFSE and OVA-tetramer, respectively, in young (Fig. 4B) and old (Fig. 4C) immune cells.

[0094] Using both young and old immune cells, the proliferation of T cells was significantly higher on-chip than off-chip (p<0.001). Also, a significantly higher on-chip proliferation of young CD8+ T cells than old CD8+ T cells (p<0.001) was observed. However, this difference was not observed off-chip (p>0.05) (Fig. 4D). Similarly, a significantly higher generation of OVA-specific CD8+ T cells on-chip compared to off-chip was observed for both young (12.3 + / - 1.25% vs. 4.0 + / - 0.83%, p<0.001) and old (9.53 + / - 0.87% vs. 3.36 + / - 0.99%, p<0.001) T cells (Fig. 4E). A significantly higher percentage of OVA-specific T cells on-chip was observed using young T cells compared to old ones (12.3 + / - 1.25% vs. 9.53 + / - 0.87%, p=0.04), whereas no significant differences were observed off-chip between young and old cells (4.0 + / - 0.83% vs. 3.36 + / - 0.99%, p=0.86) (Fig. 4E). Confocal images of on-chip activation of OVA-specific CD8+ T cells confirmed higher OVA priming of young CD8+ T cells than old CD8+ T cells (Fig. 9).

[0095] EXAMPLE 4

[0096] In this example, the antitumoral efficacy of CD8+ T cells stimulated by OVA-presenting MQs and DCs was assessed by co-culturing B16-OVA cells with activated CD8+ T cells. The viability was assessed using flow cytometry analysis of the suspension of B16-OVA / CD8+ T cells (Fig. 5A) for both young (Fig. 5B) and old immune cells (Fig. 5C). Compared to a negative control without OVA-specific CD8+ T cells (Fig. 5D), the antitumoral efficacy of young and old CD8+ T cells generated on- and off-chip (Fig. 5E-5H) was assessed.

[0097] A significantly higher percentage of dead B16-OVA cells was observed when OVA- specific CD8+ T cells were generated on-chip compared to off-chip for young (49.88 + / - 1.5% vs. 15.05 + / - 0.68%, p<0.001) and old (32.67 + / - 4.04% vs. 7.04 + / - 0.88%, p<0.001) immune cells (Fig. 51). Similarly, a significantly higher percentage of dead B16-OVA cells was observed with on-chip generated young CD8+ T cells compared to old ones (49.88 + / - 1.5% vs. 32.67 + / -

[0098] 12795415.1 4.04%, p<0.001), and the same trend was observed for OVA-specific CD8+ T cells generated off-chip (15.05 + / - 0.68% vs. 7.04 + / - 0.88%, p=0.01). Confocal images also confirmed more dead B16-OVA cells with young OVA-specific CD8+ T cells than old ones (Fig. 10).

[0099] EXAMPLE 5

[0100] This example illustrates an embodiment of an in vitro platform in which the immunogenicity and antitumoral efficacy of OVA peptides in a subcutaneous B16-OVA murine melanoma model was demonstrated. B16-OVA cells were inoculated and blood on day 0 was collected. The mice with OVA peptides were immunized and blood one week after inoculation was collected. Blood was also collected after two weeks. The mice were sacrificed after three weeks and blood, lymph nodes, and B16-OVA cells (Fig. 6A) were collected.

[0101] Although not significant, the tumor growth was lower in young mice compared to old ones (Fig. 6B). The percentage of circulating OVA-specific CD8+T cells is significantly higher in PBMC of young mice compared to old mice on day 5 (11.4 + / - 1.4% vs. 7.14 + / - 0.97%, p<0.001), 14 (12.8 + / - 1.31% vs. 8.16 + / - 0.78%, p<0.001), and 21 (12.44 + / - 0.75% vs. 8.75 + / - 0.89%, p<0.001) (Fig. 6C). Similarly, a significantly higher percentage of lymph node-isolated (13.35 + / - 0.87% vs. 7.66 + / - 1.99%, p<0.001) and tumor-infiltrating (27.34 + / - 2.54% vs. 13.56 + / - 1.17%, p<0.001).

[0102] OVA-specific CD8+T cells is in young mice compared to old mice was observed on day 21. Notably, it was observed that the percentages of OVA-specific CD8+T cells in young mice present in the circulation and isolated from lymph nodes at day 21 were similar to the on-chip young OVA-specific CD8+T cells (Table 1).

[0103] Table 1. Comparison for OVA-specific CD8+ T cells

[0104] On-chip young Young CD8+T Young CD8+T

[0105] CD8+T cells cells in the cells in the LN blood at day 21 circulation at day 21

[0106] Percentage of OVA-specific 12.3 12.44 13.35

[0107] CD8+T cells

[0108] EXAMPLE 6

[0109] In this example, the cell culture chamber included a 12 mm long and 5 mm wide rounded square connected to two 3 mm diameter holes providing a high surface area for APC attachment, as shown in Fig 7A. A simulation of fluidic flows in the cell culture chamber is shown in Fig. 7B.

[0110] EXAMPLE 7

[0111] 12795415.1 A design and fabrication of a microfluidic device is demonstrated in this example. The 2D computer-aided designs (CADs) of the different layers of the example device were made in CorelDraw 2021 (Corel, Canada). Three different thicknesses of poly (methylmethacrylate) (PMMA) (Sigma) sheets (3.0 mm (top layer), 0.2 mm (middle layer), and 0.2 mm (bottom layer)) were cut according to the CAD design using a laser cutter (PLS6.150D laser cutter, Universal Laser Systems). Then, the PMMA layers were treated with ozone plasma treatment for 10 minutes. Following this, the layers were rinsed with 70% isopropyl alcohol (IPA) (Sigma- Aldrich) to clean up the PMMA surfaces. Next, the PMMA layers were stacked on top of each other and held together using paper clips for 2 minutes. Afterward, excess IPA was removed from the microchannel before placing the assembled devices inside a hot air oven for 15 mins at 60 °C to finish the sealing of the device by attaching stacked PMMA layers together. After bonding, the channels of the devices were washed with 70% ethanol, followed by phosphate- buffered saline (PBS). Then, the devices were dried by passing air through the channels of the devices.

[0112] Monocytes from mice bone marrow were then isolated as follows. Young and old C57BL / 6 female mice (The Jackson Laboratory) were kept in a pathogen-free environment for at least 3 days. C57BL / 6 mice were euthanized to obtain the femur and tibia. All animal procedures were conducted under the supervision of the Institute's Committee on Use and Care of Animals (ICUCA) committee of the Lundquist Institute (#32706-02). The femurs and the tibia were harvested, followed by flushing the bone marrow with sterile PBS using a 30-gauge needle and a 5 mL syringe on a 40 micromolar cell strainer (Coming). The bone marrow was disintegrated using the plunger of the syringe and then passed through the cell strainer to collect the solution of PBS and bone marrow inside a sterile 50 mL Falcone tube. Then, the cell suspension was centrifuged at 400g for 10 mins at 4 °C before discarding the supernatant to separate the monocyte pellet from the populations of the cells. After that, the cell pellets were ready for differentiation into MQs and DCs.

[0113] Macrophages and dendritic cells were differentiated as follows. The freshly prepared monocytes (~ 2xl06) were plated in a petri dish and incubated for 7 days at 37 °C and 5% CO2. To generate MQs, the bone marrow cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) + GlutaMax (Fisher Scientific), IX nonessential amino acid (Thermo Fisher Scientific), 1 mM sodium pyruvate (Cytiva), 100 micromolar beta- mercaptoethanol (Sigma- Aldrich), and mouse macrophage colony-stimulating factor (M-CSF) (5 ng / mL) (Miltenyi Biotec). To generate DCs, the bone marrow cells cultured in Roswell Park Memorial Institute (RPMI) (Cytiva) media, IX non-essential amino acid, 1 mM sodium pyruvate, 100 micromolar beta-mercaptoethanol, and mouse granulocyte-macrophage colony- stimulating factor (GM-CSF)

[0114] 12795415.1 (5 ng / mL) (GenScript). The differentiation of monocytes to MQs (CD 11b) and DCs (CD l ie) were confirmed with flow cytometry by using Anti-CDl lb Mouse Monoclonal Antibody (Brilliant Violet 711-A) (BioLegend, 101241) and Anti-CDl 1c mouse monoclonal antibody (FITC) (BioLegend, 117305), respectively.

[0115] Macrophages and dendritic cells using OVA peptide were stimulated on-chip as follows. The microfluidic devices were coated with 50 micrograms / mL of fibronectin and incubated for 2 hours at 37 °C and 5% CO2 before washing with sterile PBS twice. MQs and DCs were trypsinized from the petri dish, followed by centrifugation. Then, the cells were resuspended into associated media to obtain the cell suspension of around IxlO6cells / mL. To prepare the cells for the experiment, we mixed MQs and DCs with their respective media in a 1:1 ratio. Then, 100 ng / mL of OVA (257-264), Peptide Fragment (OVA-I) peptide (Anaspec), and 250 ng / mL of lipopolysaccharide (LPS) were added to the cell-containing solution. Finally, seeded the cells were seeded inside the microchannels of the device. A 200-microliter pipette was used to transfer a volume of 20 microliter cell suspension to seed the cells. The devices were incubated for 7 days at 37 °C and 5% CO2 before naive CD8+ T cell inoculation. The presentation of the OVA-I by MHC-I was characterized by flow cytometry using iTAg Tetramer / PE - H-2 Kb OVA (NIH core facility).

[0116] Naive CD8+ T cells from mice spleen were isolated as follows. Old and young C57BL / 6 mice were euthanized under the ICUCA committee of the Lundquist Institute (#32706-02) to obtain spleens. The process involved taking four spleens and cutting them into small pieces using scissors and a scalpel on a petri dish under sterile conditions. CD8+ T cell media was prepared by adding RPMI, IX nonessential amino acid, 1 mM sodium pyruvate, and 100 micromolar beta-mercaptoethanol. After adding CD8+ T cell media into a petri dish containing minced spleens, the cells were transferred into a centrifuge tube with a pipette. The supernatant was discarded after centrifugation, followed by resuspending the cell pellet in the CD 8+ T cell media. The cell suspension was passed through the 70 micromolar cell strainer to remove clumps or debris. The naive CD8+ T cells were isolated by negative selection from the total cell populations using an EasySep™ Mouse CD8+ T Cell Isolation Kit (STEMCELL Technologies). The isolated naive CD8+ T cells were washed twice before counting. The freshly isolated naive CD8+ T cells were stained anti-mouse CD8a (APC) (BioLegend) to assess purification efficiency.

[0117] Naive CD8+ T cells were activated on-chip as follows. The concentration of the naive CD8+ was IxlO6cells / mL before seeding into the device. To seed the naive CD8+ T cells, 20 microliters of the cell suspension volume was added to the microchannel of the device that already contained pre-seeded MQs and DCs. This was accomplished using a 200-microliter

[0118] 12795415.1 pipette. Then, the naive CD8+ T cells were placed over a population of MQs and DCs and incubated at 37 °C and 5% CCL for 2 days. This process allowed the CD8+ T cells to become activated.

[0119] Activated CD8+ T-cells were prepared as follows. After co-culturing with matured MQs and DCs, activated CD8+ T cells were isolated from the device. To determine the presence of ova-specific T cells, the activated CD8+ T cells were stained with an OVA-tetramer (R-PE) (NIH core facility). The stained cells were then analyzed using flow cytometry. An antitumoral assay was accomplished. The B16-OVA cells (Sigma- Aldrich) were grown in DMEM (Fisher Scientific), supplemented with 1% penicillin-streptomycin (Caisson Labs) and 10% fetal bovine serum (FBS) (Life Technologies). The cells were then trypsinized with TripleE (Fisher Scientific) and seeded in 6-well plates at a concentration of 0.3xl06cells / mL. The cells were allowed to grow until they reached the confluency. The isolated and activated CD8+ T cells, with a cell concentration of IxlO6cells / mL, were added to each well of the 6-well plates and co-cultured for two days. After two days, the flow cytometry and the confocal imaging were used to quantify cancer cell viability.

[0120] In vivo animal studies were performed as follows. The B16-OVA cells were trypsinized with TripleE before reaching 80% confluence. After counting the cells, IxlO5cells were injected subcutaneously into the right flank of both old (aged 6-8 weeks) and young C57BL / 6 mice. The injection was carried out using a 30-gauge needle and 100 microliters of PBS. After cell injection, the growth of the tumors was monitored every three days, and tumor size was measured using a caliper. Once the tumors reached their optimal size (20 mm3), the mice were immunized with 100 microliters of free OVA peptide or PBS (negative control).

[0121] Enzyme-linked immunosorbent assays (ELISA) were performed as follows. IL-6, IL-10, IL- 12, TNF-alpha, and IFN-gamma ELISA kits (BioLegend) were used to quantify the levels of the different cytokines in the media collected after 7 days from the device according to the manufacturer’s protocol. In the 96- well plate of the ELISA kit, the diluted standards and the samples were added into their respective wells, with a blank well as the negative control. The plates were incubated at room temperature to allow the binding of IL-2, IL-6, IL- 10, and IL- 12 with the captured antibody on the precoated kit. After incubation, plates were washed several times to remove unbound proteins and debris. The substrate solutions were added to each well, followed by incubation as recommended by the instructions from the kit. This process allowed for colorimetric reactions, which were proportional to the IL-2, IL-6, IL- 10, and IL- 12 amounts. After the development of colors, the stop solution was added to terminate the colorimetric reaction. Finally, the absorption from each well was measured using the plate reader to quantify the concentration of the IL-2, IL-6, IL- 10, and IL- 12 samples.

[0122] 12795415.1 Flow cytometry experiments were performed as follows. The differentiated MQs and DCs, matured MQs and DCs, naive CD8+ T cells, activated CD8+ T cells, in vitro B16-OVA cells after the killing assay, and CD8+ T cells from in vivo models from old and young mice have been analyzed through flow cytometry (FACSCanto II flow cytometer, BD Biosciences). Cells were collected and prepared for cell suspension with corresponding media. The cells were labeled with antibodies specific to the receptors of the cells (e.g., Anti-CDl lb Mouse Monoclonal Antibody, Anti-CDl 1c mouse monoclonal antibody, iTAg Tetramer / PE - H-2 Kb OVA, PE-tetramer), followed by incubation for 60 mins at 37 °C and 5% CO2. After the incubation, the cells were washed twice with the flow cytometry buffer to remove any unbound antibodies. Then, cells were fixed at 4% PFA solution (Santa Cruz Biotechnology). After preparing the cells, they were placed onto flow cytometry plates along with various controls, including unstained cells, stained beads with individual fluorochromes, and then the flow cytometry analysis was performed. After acquiring the data, the analysis was performed using FlowJo software with proper gating strategies to check the expression levels of the receptors.

[0123] Confocal analysis experiments were performed as follows. All of the confocal imaging (LSM 800 confocal microscope, Zeiss) was performed on the microfluidic device and 6-well plates. The cells were initially stained with fluorescent markers corresponding to cell type, livecell stains, iTAg Tetramer / PE - H-2 Kb OVA, PE-tetramer, Calcein acetoxymethyl (Calcein- AM) (Thermo Fisher Scientific), and Ethidium Homodimer- 1 (EthH) (Thermo Fisher Scientific) with proper incubation as per the staining protocol. After mounting the samples on the microscope stage, imaging was performed, and data was acquired. All the images were analyzed using ImageJ open-access software.

[0124] While several embodiments of the present disclosure have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the present disclosure. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the teachings of the present disclosure is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the

[0125] 12795415.1 appended claims and equivalents thereto, the disclosure may be practiced otherwise than as specifically described and claimed. The present disclosure is directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the scope of the present disclosure.

[0126] In cases where the present specification and a document incorporated by reference include conflicting and / or inconsistent disclosure, the present specification shall control. If two or more documents incorporated by reference include conflicting and / or inconsistent disclosure with respect to each other, then the document having the later effective date shall control.

[0127] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0128] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0129] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0130] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.”

[0131] 12795415.1 As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0132] When the word “about” is used herein in reference to a number, it should be understood that still another embodiment of the disclosure includes that number not modified by the presence of the word “about.”

[0133] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0134] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0135] 12795415.1

Claims

CLAIMSWhat is claimed is:

1. A method, comprising: exposing target cells attached to a surface of a chamber to a plurality of immune cells, wherein at least some of the immune cells attach to the target cells; removing at least some of the non-attached immune cells from the chamber; culturing the target cells and the immune cells together in the chamber to activate at least some of the immune cells; and removing at least some of the non-activated immune cells from the chamber.

2. The method of claim 1, wherein the target cells comprise antigen presenting cells.

3. The method of any one of claims 1 or 2, wherein the target cells comprise monocytes.

4. The method of any one of claims 1-3, wherein the target cells comprise one or more types of target cells.

5. The method of any one of claims 1-4, wherein the target cells cover at least 30% of the surface of the chamber.

6. The method of any one of claims 1-5, wherein the target cells are pathogens.

7. The method of any one of claims 1-6, wherein the target cells are pathogens contained in other cells.

8. The method of any one of claim 1-7, wherein the target cells are target cells that originate another type of cell.

9. The method of any one of claims 1-8, wherein the target cells are target cells that originate one or more types of cells.

10. The method of any one of claims 1-9, wherein the immune cells comprise T cells.12795415.

111. The method of any one of claims 1-10, wherein the immune cells comprise immune cells able to target cancer.

12. The method of any one of claims 1-11, wherein the immune cells comprise immune cells able to target pathogens.

13. The method of any one of claims 1-12, wherein the immune cells comprise immune cells able to target other immune cells.

14. The method of any one of claims 1-13, further comprising flushing fluid through the chamber to remove at least some of the non-attached immune cells from the chamber.

15. The method of any one of claims 1-14, the chamber defining a volume, the method further comprising flushing a volume of at least 20 microliters of fluid through the chamber that is the same or greater than the volume of the chamber.

16. The method of any one of claims 1-15, the chamber defining a volume, the method further comprising flushing a volume of fluid through the chamber at a fluid flow speed between 100 nL / min and 1,000 microliter s / min.

17. The method of any one of claims 1-16, further comprising flushing fluid through the chamber a plurality of times to remove at least some of the non-attached immune cells from the chamber.

18. The method of any one of claims 1-17, further comprising flushing fluid through the chamber at least three times to remove at least some of the non-attached immune cells from the chamber.

19. The method of any one of claims 1-18, further comprising culturing the immune cells to at least double the number of immune cells.

20. The method of any one of claims 1-19, further comprising obtaining a secreted product from the immune cells.12795415.

121. The method of claim 20, wherein the secreted product comprises antibodies.

22. The method of any one of claims 1-20, wherein the target cells comprise cells obtained from a subject.

23. The method of any one of claims 1-21, wherein the immune cells comprise cells obtained from a subject.

24. The method of any one of claims 21 or 22, wherein the subject is human.

25. The method of any one of claims 21-23, wherein the subject is at least 40 years old.

26. The method of any one of claims 19-25 further comprising administering the secreted product to the subject.

27. The method of any one of claims 1-26, further comprising exposing the activated immune cells to dissociated cells.

28. The method of any one of claims 1-27, wherein the target cells comprise target cells attached to more than one surface of the chamber.

29. The method of any one of claims 1-28, further comprising exposing the activated immune cells to cancer cells.

30. The method of any one of claims 1-29, further comprising removing at least some of the activated immune cells from the chamber.

31. The method of any one of claims 1-30, further comprising sorting and / or counting at least some of the activated immune cells using flow cytometry.

32. The method of any one of claims 1-31, further comprising sequencing at least some of the activated immune cells.12795415.

133. The method of any one of claims 1-32, further comprising exposing the target cells and / or the immune cells to an agent suspected of altering an immune response of the immune cells to the target cells.

34. The method of any one of claims 1-33, further comprising keeping at least some of the activated immune cells and target cells inside the chamber.

35. The method of any one of claims 1-34, wherein culturing the target cells and the immune cells comprises recirculating the immune cells within the chamber.

36. The method of any one of claims 1-35, further comprising determining immune cell characteristics using optical imaging.

37. The method of claim 36, wherein immune cell characteristics comprise immune cell size, shape, and / or function.

38. The method of any one of claims 36 or 37, wherein optical imaging of immune cells comprises imaging the cells inside and / or outside the chamber.

39. The method of any one of claims 1-38, wherein culturing the target cells and the immune cells comprises incubating the cells inside the chamber.

40. The method of any one of claims 1-39, wherein the chamber is a polymer.

41. The method of any one of claims 1-40, wherein the chamber is coated with a glycoprotein.

42. The method of any one of claims 1-41, wherein the chamber has at least one dimension smaller than 1 mm.

43. The method of any one of claims 1-42, wherein the plurality of immune cells is a suspension of cells.

44. The method of claim 43, wherein the suspension is cells is at least IxlO6cells / mL.12795415.1- l-45. The method of any one of claims 1-44, further comprising culturing the target cells and the immune cells together in the chamber for at least 4 days.

46. A method, comprising: exposing target cells attached to a surface of a chamber to a plurality of immune cells, wherein at least some of the immune cells attach to the target cells; flushing at least some of the non-attached immune cells from the chamber at least two times; and activating the immune cells within the chamber via exposure to the target cells.

47. A method, comprising: exposing antigen-presenting cells attached to a surface of a chamber to a plurality of T cells, wherein at least some of the T cells attach to the antigen-presenting cells, and wherein the T cells are from a human subject at least 40 years old; flushing at least some of the non-attached T cells from the chamber; activating the T cells within the chamber via exposure to the antigen-presenting cells; and culturing the activated T cells.

48. The method of claim 47, further comprising administering the cultured activated T cells to the subject.

49. The method of any one of claims 47 or 48, further comprising determining cancer.

50. The method of any one of claims 47-49, further comprising determining infectious diseases.

51. The method of any one of claims 47-50, further comprising determining inflammatory diseases.

52. The method of any one of claims 47-51, further comprising determining neurodegenerative disorders.

53. The method of any one of claims 47-52, further comprising determining organ transplantation rejection in the subject.12795415.

154. The method of any one of claims 47-53, further comprising determining aging-related changes to the subject.

55. The method of any one of claims 47-54, wherein the chamber has at least one dimension smaller than 1 mm.

56. A method, comprising: exposing antigen-presenting cells attached to a surface of a chamber to a plurality of immune cells, wherein at least some of the immune cells attach to the antigen- presenting cells; removing at least some of the non-attached immune cells from the chamber; and activating at least some of the immune cells within the chamber via exposure to the antigen-presenting cells.

57. The method of claim 56, further comprising recovering at least some of the activated T cells from the chamber.

58. The method of any one of claims 56 or 57, wherein at least some of the immune cells are T cells.

59. The method of any one of claims 56-58, wherein at least some of the immune cells are human cells.

60. The method of any one of claims 56-59, further comprising determining a response of the immune cells within the chamber.

61. The method of claim 60, wherein the response comprises an age-related immune response.

62. The method of any one of claims 60 or 61, wherein the response comprises an antitumoral response.12795415.1

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