A human personalized in vitro immune response simulation platform

Abstract

The present invention is directed to a personalized in vitro immune response simulation system, including: a plurality of containers, each container including a culture of tissue-specific cells and endothelial cells in fluid contact with the tissue-specific cells; a liquid medium comprising at least about 25% serum or serum replacement (SR); immune cells suspended in the liquid medium; and a linkage system establishing fluidic coupling between the plurality of containers, allowing the liquid medium to flow through the linkage system, wherein the system is an isogenic system. The present invention further provides methods for preparation of such a system, and for uses thereof.

Description

[0001] A HUMAN PERSONALIZED IN VITRO IMMUNE RESPONSE SIMULATION

[0002] PLATFORM

[0003] FIELD OF THE INVENTION

[0004] The present invention is generally directed to in vitro modelling of the immune system. More specifically, the invention relates to systems and methods and especially personalized systems and methods including tissues and immune cells, for in vitro testing of the immune response in various situations.

[0005] BACKGROUND OF THE INVENTION

[0006] The immune system is essential to keeping us alive and healthy, eliminating pathogens and other entities (e.g., cancer cells) that do not “belong” in the body. This function is achieved by the joint action of the innate immune system, which attacks pathogens in a non-specific manner, and of the adaptive immune system, which learns to identify and eliminate foreign entities in a specific manner. Clearly, an understanding of how the immune system works is of monumental scientific importance. It is remarkable, then, that there are no human-relevant in vitro platforms to date that mimic the adaptive immune system in a physiological environment. The lack of such models contrasts starkly with the richness and sophistication of the current in vitro modeling landscape; indeed, modeling capabilities have achieved such high levels of clinical relevancy that the FDA now allows preclinical drug testing to be conducted entirely in “advanced human (in vitro models,” without animal experimentation.

[0007] The challenge in developing in vitro models of the adaptive immune system arises from the fact that adaptive immune cells are activated by non-autologous tissues — specifically, tissues that do not share the immune cells’ human leukocyte antigens (HLA). Consequently, any attempt to incorporate immune cells into a “generic” in vitro model containing non-autologous tissue activates the cells, which destroy the model tissue. This phenomenon resembles the well-known problem of organ rejection following transplantation.

[0008] In view of the foregoing, there is a need for developing in vitro systems for modelling the adaptive immune response in a physiological environment, to allow in vitro testing of various situations and agents that can now only be tested in live organisms.

[0009] SUMMARY OF INVENTION

[0010] The following embodiments are described and illustrated in conjunction with compositions and methods which are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above-described problems have been reduced or eliminated, while other embodiments are directed to other advantages or improvements.

[0011] In some embodiments, there is provided a personalized in vitro immune response simulation system, including: a. a plurality of containers, each container including: i. a culture of tissue-specific cells; and ii. endothelial cells in fluid contact with the tissue-specific cells; b. a liquid medium including at least about 25% serum or serum replacement (SR); c. immune cells suspended in the liquid medium; and d. a linkage system establishing fluidic coupling between the plurality of containers, allowing the liquid medium to flow through the linkage system and carry the immune cells and substances secreted from the tissue specific cells, the endothelial cells, and the immune cells, wherein: the plurality of containers include tissue-specific cells of at least two different tissues; and the system is an isogenic system with the tissue-specific cells, the endothelial cells, and the immune cells being derived from source cells obtained from a single subject.

[0012] In some embodiments, the system is a human system and the tissue-specific cells, the endothelial cells, and the immune cells are human cells.

[0013] In some embodiments, the source cells obtained from the subject are blood cells and / or skin cells.

[0014] In some embodiments, the immune cells include innate immune cells and / or adaptive immune cells. In some embodiments, the immune cells are derived from peripheral blood mononuclear cells (PBMCs) or from white blood cells (WBC)s. In some embodiments, the immune cells are not activated. In some embodiments, the immune cells are capable of remaining inactivated in the system for at least about 24, 48, or 93 hours.

[0015] In some embodiments, the at least two different tissues include at least two different tissues selected from brain, blood-brain barrier (BBB), gut, liver, kidney, lung, skin, heart, microglia, adipocytes, retinal pigment epithelium. In some embodiments, the tissue-specific cells are derived from stem cells reprogrammed from the cells obtained from the subject. In some embodiments, the stem cells are induced pluripotent stem cells (iPSCs). In some embodiments, the tissue-specific cells are capable of remaining viable in the system for at least about 24 hours, 48 hours, 93 hours.

[0016] In some embodiments, the liquid medium includes about 25%-80% serum. In some embodiments, the serum is derived from the single subject. In some embodiments, at least one container of the plurality of containers further includes a Boyden chamber including an upper chamber, a lower chamber, and a porous membrane separating the upper and the lower chamber. In some embodiments, the tissue-specific cells are cultured in the lower chamber and the endothelial cells are seeded on the porous membrane. In some embodiments, the tissue-specific cells are cultured on the porous membrane and the endothelial cells are seeded in the lower chamber. In some embodiments, the container further includes a holder holding an insert including the porous membrane and the upper chamber of the Boyden chamber, wherein the holder is configured to allow a pipetting apparatus to access both the upper and the lower chambers of the Boyden chamber. In some embodiments, the holder includes a peripheral ring including at least two arms radiating inwardly from the ring, each arm including an engaging feature configured to engage the insert such that when the insert is engaged, the upper chamber of the Boyden chamber is not centered with respect to a circumference of the container, thereby allowing direct pipetting access to the bottom of the container.

[0017] In some embodiments, at least one of the plurality of containers is configured to be replaceable.

[0018] In some embodiments, there is provided a method for preparing a personalized, in vitro immune response simulation system, the method including: a. providing a sample including source cells obtained from a subject; b. reprogramming at least a portion of the source cells to produce induced pluripotent stem cells (iPSCs); c. differentiating the iPSCs into a plurality of cultures of tissue-specific cells of at least two different tissues, and placing the cultures of tissue-specific cells in a liquid medium including at least 25% human serum or serum replacement into a plurality of containers; d. adding to each container endothelial cells from the same subject, such that the endothelial cells are in fluid contact with the tissue-specific cells; e. placing the plurality of containers in a linkage system which establishes fluidic coupling between the plurality of containers, allowing the liquid medium to flow through the linkage system; and f. adding to the liquid medium immune cells from the same subject.

[0019] In some embodiments, the source cells obtained from the subject are peripheral blood mononuclear cells (PBMCs) or white blood cells (WBC)s.

[0020] In some embodiments, the endothelial cells are obtained from the subject or from iPSCs prepared from the cells of the subject.

[0021] In some embodiments, the immune cells are derived from PBMCs or WBCs obtained from the subject or from iPSCs prepared from the cells of the subject.

[0022] In some embodiments, the serum is obtained from the sample or from the same subject.

[0023] In some embodiments, at least one of the source cells, endothelial cells, and immune cells are obtained from the subject by an isolation method including the following steps: obtaining a blood sample from the subject; separating plasma from cells of the blood sample; adding albumin to the cells; and centrifuging the cells with the albumin on a density gradient to obtain PBMCs or WBCs.

[0024] In some embodiments, the immune cells are obtained from the subject by a method including the following steps: obtaining a blood sample from the subject; and lysing red blood cells (RBC)s in the blood sample by diluting the blood sample more than about 5-fold with an RBC lysis buffer.

[0025] In some embodiments, the method further includes steps of adding to the liquid medium an agent selected from a pathogen, a toxin, a disease-causing agent, an antibiotic agent, and / or a therapeutic agent, and testing the activity of the immune cells.

[0026] In some embodiments, the method further includes adding an immune-activating agent to the liquid medium.

[0027] In some embodiments, there is provided a method for assessing immune compatibility of tissues before transplantation, the method including: a. providing the system disclosed herein wherein the subject is a transplantation recipient; b. adding to the system tissue-specific cells from a potential transplantation donor; c. evaluating the morphology and functionality of the tissue-specific cells in the system; and d. measuring the immune response of the immune cells in the system following addition of the transplantation donor cells.

[0028] In some embodiments, the method further includes removing transplantation recipient tissue-specific cells.

[0029] In some embodiments, the tissue-specific cells from a potential transplantation donor are liver cells or kidney cells.

[0030] In some embodiments, there is provided a method for evaluating a subject-specific response to a pathogen, the method including: a. providing the system disclosed herein; b. adding to the liquid medium a pathogen; c. evaluating the morphology and functionality of the tissue-specific cells in the system; and d. measuring the immune response of the immune cells in the system following addition of the pathogen.

[0031] In some embodiments, the pathogen is a bacterial pathogen.

[0032] In some embodiments, there is provided a method for evaluating a subject-specific response to an antibiotic agent, the method including: a. providing the system disclosed herein; b. adding to the liquid medium a pathogen; c. further adding to the liquid medium an antibiotic agent for treating the pathogen; d. evaluating the morphology and functionality of the tissue-specific cells in the system; and e. measuring the immune response of the immune cells in the system following addition of the pathogen and the antibiotic agent.

[0033] In some embodiments, the antibiotic agent is selected from polymyxins B & E and carbapenems.

[0034] In addition to the exemplary embodiments described above, further embodiments will become apparent by reference to the figures and by study of the following detailed descriptions.

[0035] BRIEF DESCRIPTION OF DRAWINGS

[0036] The invention will now be described in relation to certain examples and embodiments with reference to the following illustrative figures.

[0037] Figs. 1A-1B show a schematic representation of a linkage system, according to some embodiments of the present invention. Fig. 1A shows a side view of container including an insert, suitable for use with the linkage system, according to some embodiments of the present invention. Fig. IB shows a top view of a linkage system including a plurality of inserts, according to some embodiments of the present invention.

[0038] Fig- 2 shows an overview of the platform preparation according to some embodiments: a donor provides a blood sample, iPSCs are generated by reprogramming of PBMCs from the blood sample, and then differentiated into 7 different isogenic tissues (6 organs plus endothelial tissue) that are used to create an isogenic platform. Once the platform is established, the donor’s immune cells are integrated into the platform to create a personalized immunized human platform. Finally, the platform is applied to examine the immune system’s response in various situations such as organ transplantation, and the presence of a foreign pathogen (e.g., antibiotic-resistant E. coli).

[0039] Figs. 3A-3E show results for derivation of 6 isogenic tissue types from iPSCs corresponding to a single donor. Figs. 3A-3B present images of the derived cells: the upper row presents differential interference contrast (DIC) microscopy images of each tissue and lower rows present the immunohistochemistry (IHC) results of tissue specific markers. Fig. 3A. induced pluripotential cells (iPSC): SSEA-4, Oct 2 / 4; gut: villin, ZO1; liver: AFC, HNF4A, albumin; kidney: E-Cad, nephrinl, cubilin, CD31. Fig. 3B. Blood-brain barrier (BBB): ZO1; brain: TBR1, P-tubulin; heart: troponin-T, actin. Figs. 3C-3E. present functional assays to ensure proper functionality of the cells obtained: Fig. 3C. Transepithelial / transendothelial electrical resistance (TEER) (BBB). Blank: medium alone; BMEC: brain microvascular endothelial cells; Fig. 3D. calcium imaging (brain), three different samples; F / FO: change in fluorescent intensity over time compared to time; Fig. 3E. Spontaneous beating (heart), three different samples.

[0040] Figs. 4A-4I show results for the immunological cells: Fig. 4A. PBMCs isolated from two donors (shown by DIC). Fig. 4B-4C. FACS identification of the immune cells in 50% serum in RPMI after a 93 hrs incubation (Fig. 4B) and with addition of non-isogenic BBB cells (Fig. 4C), showing that the cells are alive and activated (as further found by gating for cells stained with Ghost Dye®, data not shown) in the non-isogenic incubation. CD45 - a marker for white blood cells. Fig. 4D: % activated T cells out of non-activated after incubation with non-isogenic BBB tissue. PBMC were cultured with non-isogenic BBB cells or with an activating cytokine (interferon (IFN), right bar), labeled with activation markers, and analyzed by a flow cytometer. Figs. 4E-4G. PBMCs were cultured with non-isogenic and isogenic BBB tissues, which show activation and non-activation, respectively. Fig. 4E shows that the PBMCs proliferate as they become activated by IFNy (CFSE - carboxyfluorescein, a proliferation marker; CD28 488_525-40-A is an anti- CD28 antibody used for the FACS analysis; IFN - with IFNy). Proliferating cells are detected by a second peak of a lower intensity as the dye intensity decreases when the cells proliferate. As shown, PBMC proliferate after 72 hours with the activation of IFNg, as evaluated by CFSE stain in FACS. Fig. 4F shows that the ratio between the cell populations changes following activation by non-isogenic BBB cells. Fig. 4G. shows markers for early (CD69, on the left) and late (CD25, on the right) activation of the T cells. Black: isogenic, gray: non-isogenic. Fig. 4H. Functional assessment (TEER) of the BBB tissues after interactions with immune cells. Fig. 41. shows ZO-1 (a tight junction protein) staining of brain microvascular endothelial cells (BMECs) cultured for 72 hours with PBMCs. The cell morphology and ZO1 stain show that cell barrier is intact in isogenic interaction, and ZO1 expression is reduced in non- isogenic interaction.

[0041] Figs. 5A-5B show a schematic representation of top view of a holder suitable for use with containers or inserts for the linkage system, according to some embodiments of the present invention. Fig. 5A shows a top view of a holder, according to some embodiments of the present invention. Fig. 5B shows a holder with an insert, according to some embodiments of the invention. DETAILED DESCRIPTION OF THE INVENTION

[0042] In the following description, various aspects of the disclosure will be described. For the purpose of explanation, specific configurations, and details are set forth in order to provide a thorough understanding of the different aspects of the disclosure. However, it will also be apparent to one skilled in the art that the disclosure may be practiced without specific details being presented herein. Furthermore, well-known features may be omitted or simplified in order not to obscure the disclosure.

[0043] The present invention is directed to a human in vitro platform mimicking the adaptive immune system in a physiological environment. This platform addresses two issues: (i) a lack of in vitro models of the immune system; and (ii) the need for patient-specific multi-organ in vitro platforms for personalized medicine.

[0044] Existing models for adaptive immune response rely on in vivo platforms (e.g., non-human primates, xenotransplantation, or transgenic mice, which suffer from inter-species differences, ethical concerns, and low throughput), or in vitro platforms that only capture isolated aspects of the adaptive immune response, such as co-culturing mature immune cells or immune cellgenerating tissues such as bone marrow with tumors for immunotherapy experiments.

[0045] The challenge in developing in vitro models of the adaptive immune system arises from the fact that adaptive immune cells are activated by non-autologous tissues — specifically, tissues that do not share the immune cells’ human leukocyte antigens (HLA). Consequently, any attempt to incorporate immune cells into a “generic” in vitro model containing non-autologous tissue activates the cells, which destroy the model tissue. This phenomenon resembles the well-known problem of organ rejection following transplantation. In general, previous models used innate, rather than adaptive, immune cells, to minimize this issue, since the adaptive immune cells (such as T and B lymphocytes) are sensitive to non-self cells. In contrast, the system of the present invention includes the use of immune cells including adaptive immune cells, which are an important element in an immune response simulation system.

[0046] The system described in the present invention includes a platform integrating a number of tissues (for example, brain, blood-brain barrier (BBB), gut, liver, kidney, lung, skin, and heart, connected via vasculature formed by adding endothelial cells), all potentially originating from iPSCs derived from cells (e.g. blood cells) from a specific individual, which have been in vitro differentiated into specific tissue types. Advantageously, trying to solve the above issues of activation of immune cells by the tissues and thereby causing their attack by the immune cells, the donor’s own immune cells are integrated with the tissues to provide an in vitro isogenic model of the adaptive immune system in combination with other organs. As shown by the inventors, using an isogenic system, including immune cells from the same source as the tissue-specific cells, resulted in no (or less) activation of the immune cells, as compared with activation of the immune cells in non-isogenic systems (see Figs. 4B-4G).

[0047] It should be pointed out that to the best of the knowledge of the inventors, using immune cells including adaptive immune cells from the same subject has not been done in previous systems, and several examples for such systems are provided below. In contrast, as described hereinbelow, the inventors have successfully shown that using of immune cells from the same subject supports the tissue specific cells and does not cause an immune response against them.

[0048] Examples for using a mixture of cells from different sources include Sasserath et al., 2020 (Advanced science 7.13 (2020): 2000323), using a combination of three Organ-On-a-Chip systems including cardiac, skeletal muscle and liver chips together with a monocyte-derived THP-1 cell line (a human monocytic cell line derived from acute monocytic leukemia); Marzagalli et al., 2022 (Frontiers in bioengineering and biotechnology 10 (2022): 945149) using natural killer cells together with tumor cells to identify prospective anti-tumour natural killer cell phenotypes; Kwak et al., 2020 (Stem cells 38.6 (2020): 727-740), using a skin model including keratinocyte (HaCaT), human dermal primary fibroblasts, human umbilical cord cells (HUVEC)s, and human promyelocytic leukaemia cells (HL-60); Riddle et al., 2022 (Scientific Reports 12.1 (2022): 6855), using a model for inflammation including neutrophils and Human umbilical vein cells; and Vatine et al., 2017, Cell Stem Cell 20, 831-843, generating a blood-brain-barrier model by using human induced pluripotent stem cell-derived brain microvascular endothelial cells (iPSC-BMECs) from one source and astrocytes from a different source.

[0049] Accordingly, the system of the invention has the advantage of providing a personalized platform including both tissue-specific and immune cells from the same subject. As a further advantage - using a high percentage of serum from the same subject in the culture medium further ensures personalization of the system.

[0050] The platform of the invention can be used to explore fundamental biological questions, such as: understanding how different isogenic and non-isogenic tissues interact with the immune system, e.g., in organ transplantation; and identifying how various agents, such as pathogens (e.g., antibiotic-resistant E. coli, viruses), as well as antibiotic and other treatments, affect human physiology and the immune response.

[0051] The platform of the invention is the first in vitro personalized platform that enables the study of interactions between adaptive immune cells and multiple human tissues (i.e., within a complex, human-relevant system), and has a potential to replace animal models in studies involving the immune system. Potential uses of the system are described below.

[0052] A human in vitro immune response simulation system

[0053] In some embodiments, there is provided a personalized in vitro immune response simulation system, including: a. a plurality of containers, each container including: i. a culture of tissue-specific cells; and ii. endothelial cells in fluid contact with the tissue-specific cells; b. a liquid medium including at least about 25% serum or serum replacement (SR); c. immune cells suspended in the liquid medium; and d. a linkage system establishing fluidic coupling between the plurality of containers, allowing the liquid medium to flow through the linkage system and carry the immune cells and substances secreted from the tissue specific cells, the endothelial cells, and the immune cells, wherein: the plurality of containers include tissue-specific cells of at least two different tissues; and the system is an isogenic system with the tissue-specific cells, the endothelial cells, and the immune cells being derived from source cells obtained from a single subject.

[0054] Reference is made to Figs. 1A and IB, which present a schematic representation of a container and a linkage system including containers, respectively, according to some embodiments of the invention.

[0055] As shown in Fig. 1A, container 100 includes an insert 102, which includes a porous membrane 103, defining in the container an upper chamber 104 and a lower chamber 106. Porous membrane 103 has a defined pore size, such as about 0.4-8 microns, allowing selective diffusion of molecules, nutrients, or cells. Porous membrane 103 may be selected from a polycarbonate membrane, a polyester (PET) membrane, a polytetrafluoroethylene (PTFE) membrane, and a cellulose acetate membrane. In some embodiments, the membrane is collagen-coated. Underneath the membrane are positioned at least two legs 108, which function to distance porous membrane 103 from the bottom of container 100, thereby creating the lower chamber 106. Further shown is tubing 110, which is part of the linkage system which links the container to other containers. Also shown in Fig. 1A are tissue specific cells seeded in the lower chamber, and endothelial cells seeded on the membrane (but this may be the other way around with endothelial cells in the bottom chamber), as well as the liquid medium which flows through the linkage system and perfuses all cells. In some embodiments, container 100 is a well of a multi-well tissue culture plate. In some embodiments, container 100 is or functions as a Boyden chamber. In some embodiments, the plurality of containers corresponds to a multi -well tissue culture plate. The container may be made of any suitable material for tissue culture. Nonlimiting examples for suitable materials include a plastic material such as polystyrene, polycarbonate, polypropylene, and polyethylene terephthalate, silicone elastomers, and derivatives thereof.

[0056] As shown in Fig. IB, linkage system 150 may be based in a multi-well tissue culture plate 152 including wells (for example, wells 153), in which cartridges 154 are placed. Cartridges 154 may correspond to insert 102 of Fig. 1A, and in this case well 153 corresponds to container 100. However, it is also possible that a container including an insert is inserted into the tissue culture well, for easy replacement of the container. In some embodiments, cartridges 154 correspond to container 100 of Fig. 1A, which further includes insert 102. Further shown is tubing 156, which serves as part of the linkage system which fluidly connects the containers. Tubing 156 may include tubing 110 shown for the container 100 in Fig. 1A.

[0057] It is appreciated that the linkage system and container of Figs. 1A-1B are shown only as an example, and other examples for suitable systems are provided below.

[0058] In some embodiments, the in vitro immune response simulation system is a human in vitro immune response simulation system.

[0059] In some embodiments, the tissue-specific cells, the endothelial cells, and the immune cells are human cells.

[0060] The term “isogenic”, as used herein, means that all cells in the system, including the tissuespecific cells, the immune cells, and the endothelial cells, are derived from the same subject, i.e., have the same genotype (except for somatic changes).

[0061] In contrast, non-isogenic systems include cells from more than one individual. In some embodiments, a non-isogenic system includes tissue-specific cells from one individual and immune cells from a different individual. In some embodiments, the non-isogenic system includes endothelial cells from one individual and tissue-specific cells and / or immune cells from a different individual. In some embodiments, the non-isogenic system includes tissue-specific cells from at least two different subjects.

[0062] The term “derived from”, as used herein with respect to the tissue-specific and immune cells, means that the source cells obtained from the subject may be the direct or indirect source of the tissue-specific and immune cells. In other words, source cells are obtained from the subject and may be further processed to produce the tissue-specific or the immune cells. Such processing may include one or more steps including but not limited to isolation of the source cells from the subject, culturing the source cells for expansion purposes, inducing pluripotency for preparing induced pluripotential stem cells (iPSCs), and differentiation of the iPSCs to tissue-specific cells. For example, according to some embodiments, immune cells are derived from peripheral blood mononuclear cells (PBMCs) obtained from the subject by isolation of specific immune cell populations and optionally by expanding the populations, while tissue-specific cells are derived from PBMCs obtained from the same subject by preparing iPSCs and differentiating them into the desired tissue, as described herein below.

[0063] The term “in fluid contact”, as used herein with reference to the interaction between the endothelial cells and the tissue-specific cells, means that the tissue-specific cells and the endothelial cells are cultured in two separate compartments (or chambers) in the respective container, allowing soluble factors and metabolites to be exchanged between the compartments while maintaining physical separation of the cell layers, e.g., preventing direct cell migration between compartments. This is usually achieved by a porous membrane having a suitable pore size (such as micron-scale pores (e g., 0.4-3 pm)) placed between the compartments, as described below (for example as in a Boyden chamber). This arrangement reproduces endothelial- parenchymal cross-talk characteristic of in vivo tissue interfaces.

[0064] The term “plurality”, as used herein, means at least two. In some embodiments, plurality means at least 3, 4, 5, 6, or 7. In some embodiments, plurality means at least 3. In some embodiments, plurality means at least 4. In some embodiments, plurality means at least 5. In some embodiments, plurality means at least 6. In some embodiments, plurality means at least 5. In some embodiments, plurality means at least 7.

[0065] In some embodiments, each one of the plurality of containers includes tissue-specific cells from a single tissue. In some embodiments, each of the plurality of containers includes tissuespecific cells from more than one tissue.

[0066] The source cells of the single subject may be any type of cells suitable for deriving tissue specific cells.

[0067] In some embodiments, the tissue-specific cells in the plurality of containers are all derived from the same source cells. In some embodiments, the tissue-specific cells and the immune cells are derived from the same source cells. In some embodiments, the tissue-specific cells are derived from different source cells than source cells from which the immune cells are derived.

[0068] In some embodiments, the source cells obtained from the subject are blood cells. In some embodiments, the source cells obtained from the subject are peripheral blood mononuclear cells (PBMC)s or white blood cells (WBC)s. In some embodiments, the source cells obtained from the subject are skin cells. In some embodiments, the source cells obtained from the subject are bone marrow cells. In some embodiments, the source cells obtained from the subject are embryonic cells. In some embodiments, the source cells obtained from the subject are stem cells.

[0069] Endothelial cells are needed in order to form vascularization of the tissue-specific (parenchymal) cell cultures in order to mimic human physiology, where blood flows through the endothelium, and tissue-specific liquid runs for each specific tissue. In some embodiments, the tissue-specific cell cultures are connected by vascularization.

[0070] Any of the cells, including the tissue-specific cells, the immune cells, and the endothelial cells, may be directly obtained from tissues of the subject, or may be derived by de-differentiating (reprogramming) of source cells obtained from the subject into induced stem cells (e.g., induced pluripotent stem cells, iPSCs), and then differentiating the iPSCs into the desired tissue type. Protocols for reprogramming and differentiation are well known in the art, and some examples are provided in the experimental section.

[0071] Accordingly, in some embodiments, the immune cells are derived from whole blood or from PBMCs or WBCs of the subject. In some embodiments, the immune cells are derived by differentiating iPSCs.

[0072] Similarly, in some embodiments, the endothelial cells are derived directly from the subject. In some embodiments, the endothelial cells are derived by differentiating iPSCs.

[0073] In some embodiments, the tissue-specific cells are derived directly from the subject. In some embodiments, the tissue-specific cells are derived by differentiating iPSCs.

[0074] In some embodiments, the tissue-specific cells and / or the endothelial cells are derived from iPSCs obtained by reprogramming of PBMCs or WBCs.

[0075] In some embodiments, the immune cells include innate immune cells and adaptive immune cells. In some embodiments, the immune cells include adaptive immune cells. In some embodiments, the immune cells are not activated. In some embodiments, the immune cells include T, B and / or NK cells. In some embodiments, the adaptive immune cells include T cells and / or B cells. In some embodiments, the immune cells include T cells. In some embodiments, the immune cells include B cells. In some embodiments, the immune cells include NK cells.

[0076] In some embodiments, the immune cells are not activated by the tissue-specific cells and / or by the endothelial cells. Activation of the immune cells may be measured by any commonly known method or assay for measuring activation of immune cells such as expression and / or secretion of activation markers such as CD69 and / or CD25, cytokines, or chemokines, level of proliferation of immune cells, etc.

[0077] In some embodiments, the immune cells activation is reduced by at least about 10%, 20%, 25%, 30%, 50%, or 70% compared to immune cells activation in a non-isogenic system. In some embodiments, the immune cells activation is reduced by at least about 20% compared to a non- isogenic system. In some embodiments, the immune cells activation is reduced by at least about 50% compared to a non-isogenic system. In some embodiments, the immune cells activation is reduced by at least about 70% compared to a non-isogenic system. The non-isogenic system for comparison is defined by the immune cells not being derived from the same subject as the tissuespecific cells.

[0078] In some embodiments, the immune cells are capable of remaining inactivated in the system for at least about 24 hours, 48 hours, 93 hours, one day, two days, three days, one week, two weeks, or one month.

[0079] In some embodiments, activation of the immune cells remains reduced for at least about 24 hours, 48 hours, 93 hours, one day, two days, three days, one week, two weeks, or one month.

[0080] In some embodiments, the system further includes activated immune cells and / or agents which activate immune cells.

[0081] The system of the invention is capable of including a large number of different tissuespecific cell cultures. In some embodiments, the plurality of containers include tissue-specific cells from at least about 3, 4, 5, 6, 7, 10, 20, 100, 500, or 1000 different tissues. In some embodiments, the at least two different tissues are at least about 3 different tissues. In some embodiments, the at least two different tissues include at least about 5 different tissues. In some embodiments, the at least two different tissues include at least about 6 different tissues. In some embodiments, the at least two different tissues include about 3-10 different tissues. In some embodiments, the at least two different tissues include about 3-7 different tissues. In some embodiments, the at least two different tissues include 6 different tissues.

[0082] The tissue-specific cells may be of any cell type desired to be studied in the system. Such tissue-specific cell types may be produced from iPSCs, as explained above, or obtained directly from the subject. Non-limiting examples for tissue-specific cell types include brain, blood-brain barrier (BBB), gut, liver, kidney, lung, skin, heart, microglia, adipocytes, retinal pigment epithelium (RPE).

[0083] In some embodiments, the at least two different tissues are selected from kidney, liver, gut, brain, BBB, heart, and endothelium. In some embodiments, the at least two different tissues include kidney tissue. In some embodiments, the at least two different tissues include liver tissue. In some embodiments, the at least two different tissues include brain tissue. In some embodiments, the at least two different tissues include cardiac (heart) tissue. In some embodiments, the at least two different tissues include BBB tissue. In some embodiments, the at least two different tissues include gut tissue. In some embodiments, the at least two different tissues include endothelium tissue. In some embodiments, the at least two different tissues include kidney, liver, gut, brain, BBB, heart, and endothelium.

[0084] In some embodiments, immune-related organs are not included in the tissue-specific cell types. Nonlimiting examples for immune-related organs include thymus, bone marrow, and spleen. In some embodiments, immune-related organs are included in the tissue-specific cell types.

[0085] In some embodiments, the tissue-specific cells are capable of remaining viable in the system for at least about 24 hours, 48 hours, 93 hours, one day, two days, three days, one week, two weeks, or one month.

[0086] The liquid medium may be any cell culture suitable medium, such as DMEM or RPMI. The serum may be a human serum or a solution imitating a human serum (such as a serum replacement, SR).

[0087] The term “serum replacement” or SR, as used herein means an artificial composition that is used instead of a human serum in cell cultures. Some nonlimiting examples include Physiologix™ XF Serum Replacement (Nucleus Biologies), Sigma’s Serum Replacement 3, and CTS™ Immune Cell Serum Replacement (SR).

[0088] In some embodiments, the liquid medium includes at least about 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 95% serum. In some embodiments, the liquid medium includes at least about 30% serum. In some embodiments, the liquid medium includes at least about 50% serum. In some embodiments, the liquid medium includes at least about 70% serum. In some embodiments, the liquid medium is 100% serum. In some embodiments, the liquid medium includes about 25%-80% serum. In some embodiments, the liquid medium includes about 30%- 60% serum.

[0089] It is appreciated that finding a suitable medium for the cells may also be challenging, since it should support the various cell types of the system and not cause activation of the immune cells. Using serum obtained from the same subject involves some challenges including high cost and limited amounts. However, as shown in Fig. 4, 50% serum from the same subject was able to support iPSC-derived BBB tissue cultures for at least 93 hours.

[0090] In some embodiments, the serum is a human serum.

[0091] In some embodiments, the serum is obtained from the single subject (i.e., autologous, from the same subject from which the cells are obtained).

[0092] In some embodiments, the serum is autologous and is present in an effective amount to suppress activation of the immune cells.

[0093] The personalized in vitro immune response simulation system may be shaped in any form or shape which includes separate containers for the separate tissue-specific cell cultures, which are interconnected, i.e., having connections between the separate containers which allow medium carrying immune cells and substances to flow between the containers, and vasculature connecting the tissues to form.

[0094] In some embodiments, the system is a microfluidic device. Some nonlimiting examples for a microfluidic device include a multi-channel microfluidic device, organ-on-chip device, hydrogel-based microfluidic device, electro-microfluidic system, and microvascular network.

[0095] In some embodiments, the system is a linkage system.

[0096] The fluidic coupling may be carried out by any number of ways, such as, but not limited to, microfluidic channels, pumps and valves, tubing and connectors, porous membranes, gravity- driven flow, or electroosmotic flow.

[0097] The term “fluidic coupling”, as used herein, relates to the integration of fluid flow systems that connect different components or chambers within a microfluidic device, for mimicking physiological conditions in tissue culture and organ-on-chip systems. Fluidic coupling ensures the controlled delivery of nutrients, oxygen, and signaling molecules while also enabling waste removal, thereby maintaining cell viability and functionality in microenvironments.

[0098] The term “linkage system”, as used herein, means a structure that facilitates fluidic coupling by providing a controlled network that interconnects two or more containers including cells via microfluidic channels, pumps, and recirculating media, allowing communication through biochemical signals, metabolites, or drugs, just as the bloodstream links organs in the human body. The linkage system enables inter-organ crosstalk in vitro — a key step toward replicating wholebody physiology and pharmacokinetics on a micro-scale platform.

[0099] To maximize the linkage process, an automated pipetting apparatus can be used. The pipetting apparatus is adding medium to the vasculature and parenchymal compartments to ensure that there is no medium evaporation. Moreover, it is set to maximize the linkages depending on the amount of samples and organs that are linked.

[0100] Accordingly, in some embodiments, the system further includes an automated pipetting apparatus configured for adding the liquid medium to the plurality of containers to thereby prevent evaporation.

[0101] Some specific, non-limiting, examples for relevant systems can be found in earlier work by the inventors, briefly described below.

[0102] One example for implementing the platform described herein is by using a previously developed platform (an “organ chip”), which includes human tissues grown in a microfluidic chip, with the capacity to closely mimic organ functionality by recapitulating multicellular architectures, vascular-parenchymal tissue interfaces, chemical gradients, mechanical cues, and vascular perfusion of the body (Leung et al., Nature Reviews Methods Primers 2.1 (2022): 33; Low, et al., Nature Reviews Drug Discovery 20.5 (2021): 345-361; Ingber et al., Nature Reviews Genetics 23.8 (2022): 467-491; Zhang, Nature Reviews Materials 3.8 (2018): 257-278).

[0103] The feasibility of producing an organ chip including tissue from a specific donor has been demonstrated by reprogramming somatic cells obtained from the specific donor to a pluripotent stage (induced pluripotent stem cells; iPSCs) and then differentiating them into a desired tissue type (Sela, Advanced Materials (2023): 2304654; Vatine, Cell stem cell 24.6 (2019): 995-1005; Palasantzas, Trends in Genetics (2023); Fanizza, Journal of Tissue Engineering 13 (2022): 20417314221095339).

[0104] An additional improvement which could be further used is a multi -organ system previously developed by the inventors, which includes multiple linked vascularized organ chips, producing functional outputs that correlated to clinical data (Herland et al. Nature biomedical engineering 4.4 (2020): 421-436; Novak et al. Nature biomedical engineering 4.4 (2020): 407-420; Maoz et al. Nature biotechnology 36.9 (2018): 865-874; WO2023 / 275871; and W02022 / 097150).

[0105] In some embodiments, at least one of the plurality of containers further includes (or functions as) a Boyden chamber including an upper chamber (e.g. chamber 104 shown in Fig. 1), a lower chamber (e.g. chamber 106 shown in Fig. 1), and a porous membrane (e.g. membrane 103 shown in Fig. 1) separating the upper and the lower chamber. A well-known example for a Boyden chamber is a Transwell™ system. A Boyden chamber is a laboratory apparatus used mainly in cell migration assays, consisting of two compartments separated by a porous membrane, which allows cells to migrate from an upper compartment into a lower compartment containing chemoattractants. The Boyden chamber may be created in the container (such as tissue culture well) by adding into the container an insert (such as insert 102 depicted in Fig. 1), which includes the upper chamber and the membrane, and the lower chamber is created in the container by the membrane which separates it from the upper chamber.

[0106] In some embodiments, each of the plurality of containers includes (or functions as) a Boyden chamber, with the Boyden chamber including: (i) a porous membrane having a defined pore sizes allowing selective diffusion of molecules, nutrients, or cells; (ii) an upper chamber above the membrane for culturing the endothelial cells; and (iii) a lower chamber below the membrane for culturing the tissue specific (parenchymal) cells.

[0107] The insert may be of any shape suitable for holding the membrane in place between the upper chamber and the lower chamber. Further, the insert may be prepared from any suitable material that does not interact with the liquid medium. In some embodiments, the insert is a made of a plastic material such as polystyrene, polycarbonate, polypropylene, and polyethylene terephthalate, silicone elastomers, and derivatives thereof.

[0108] The upper chamber and the lower chamber are of a suitable size for growing the respective cells.

[0109] The membrane may be any suitable membrane. In some embodiments, the membrane is selected from a polycarbonate membrane, a polyester (PET) membrane, a polytetrafluoroethylene (PTFE) membrane, and a cellulose acetate membrane. In some embodiments, the membrane is collagen-coated.

[0110] The membrane may have a pore size relevant to the use. In some embodiments, the membrane has a pore size of about 0.4-8 microns. In some embodiments, the membrane has a pore size of about 0.4-3 microns.

[0111] Specific examples for suitable parameters for inserts, membranes, and chambers are provided in W02022 / 097150.

[0112] In some embodiments, the tissue-specific cells are cultured in the lower chamber of the Boyden chamber and the endothelial cells are seeded on the membrane, in the upper chamber. In some embodiments, the endothelial cells are cultured in the lower chamber of the Boyden chamber and the tissue-specific cells are seeded on the membrane, in the upper chamber.

[0113] Reference is made to Figs. 5A-5B depicting an optional feature of the system, which is a unique holder for placing an insert (such as insert 102 shown in Fig. 1A) in a container (e.g., container 100 shown in Fig. 1A) that may be a tissue culture well (e.g., well 153 shown in Fig. IB). The purpose of the holder is to position the insert at one side of the container (i.e., not centered with respect to the container circumference) so that a pipetting system may comfortably access both the upper chamber and the lower chamber (which is the bottom of the container or of the tissue culture well), so as to easily add ingredients (e.g. medium, supplements, cells) to both the upper chamber and the lower chamber.

[0114] As shown in Fig. 5A, the holder 200 includes a peripheral ring 210 having an inner circumference, with two opposing support arms 220a and 220b extending inwardly from the inner circumference of the ring 210. The support arms are spaced apart circumferentially by an angular offset such that the can support an insert placed on top of the support arms. Each support arm terminates at its free end in an engagement feature 230a and 230b, respectively, which projects upward from the plane of the ring. These engagement features define retaining shoulders or hooklike formations configured to engage, support, or interlock with an insert as defined above, such as insert 102 of Fig. 1A, thereby securing the insert in a fixed or semi-fixed position within the peripheral ring. Fig. 5B shows a holder 250 (which may be the same as holder 200) holding an insert 260, such as insert 102 of Fig. 1A, so that insert 260 is not centered with respect to the peripheral ring 210. Holder 250 holding an insert may be placed in a container such as a well of a multi -well tissue culture plate (e.g., well 153 of Fig. IB), resulting in the insert not being centered with respect to the wells of the plate, thereby allowing direct access to the bottom of the well by a pipetting system, without the need to remove the insert, e.g., for adding or replacing media. The circumference of holder 200 or holder 250 is similar in size to the circumference of the container, so that the holder securely fits within the container, thereby defining the position of the insert within the container.

[0115] The holder may be made from any material suitable for tissue culture work, including plastic materials such as polystyrene, polycarbonate, polypropylene, and polyethylene terephthalate, silicone elastomers, and derivatives thereof.

[0116] In some embodiments, the upper chamber of the Boyden chamber is not centered with respect to a circumference of the container, leaving space on at least one side of the membrane, to allow pipetting apparatus used with the system to access to both upper chamber and the lower chamber.

[0117] In some embodiments, the container further includes a holder (such as holder 200 of Fig. 5A) configured to allow a pipetting apparatus to access both the upper and the lower chambers of the Boyden chamber. In some embodiments, the holder holds an insert including the porous membrane (such as membrane 103 of Fig. 1A) and the upper chamber of the Boyden chamber (such as upper chamber 104 of Fig. 1A).

[0118] In some embodiments, the holder includes a peripheral ring including at least two arms radiating inwardly from the ring, each arm including an engaging feature.

[0119] In some embodiments, the engaging features of the arms are configured to engage the insert such that when the insert is engaged, the upper chamber of the Boyden chamber is not centered with respect to a circumference of the container, thereby allowing direct pipetting access to the bottom of the container.

[0120] Additionally, in some embodiments, the system is modular, meaning that at least one of the containers is configured to be replaceable with a different container. In some embodiments, more than one, or all containers are configured to be replaceable with different containers. For example, a container may be detachable, and replaceable by a different container.

[0121] In some embodiments, at least one of the containers is replaceable with a different container including a different tissue. The different tissue may be, e.g., the same tissue from a different subject, a different tissue of the same subject, or the same tissue but in a different situation of the subject, e.g. taken at a different age, or at a different physiological situation.

[0122] A method for preparing a personalized in vitro immune response simulation system

[0123] In some embodiments, there is provided a method for preparing a personalized in vitro immune response simulation system, the method including: a. providing a sample including source cells obtained from a subject; b. reprogramming at least a portion of the source cells to produce induced pluripotent stem cells (iPSCs); c. differentiating the iPSCs into a plurality of cultures of tissue-specific cells of at least two different tissues, and placing the cultures of tissue-specific cells in a liquid medium including at least 25% human serum or serum replacement (SR) into a plurality of containers; d. adding to each container endothelial cells from the same subject, such that the endothelial cells are in fluid contact with the tissue-specific cells; e. placing the plurality of containers in a linkage system which establishes fluidic coupling between the plurality of containers, allowing the liquid medium to flow through the linkage system; and f. adding to the liquid medium immune cells from the same subject.

[0124] Definitions and embodiments mentioned above and which may be relevant to the present embodiments also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated. For terms used herein, unless stated otherwise, their definition and embodiments are intended to be the same as above (mutatis mutandis).

[0125] An example for a scheme for preparing the system as described herein is provided in Fig. 2.

[0126] It is appreciated that the order of steps in the method is not necessarily fixed, for examples, the different cell types are not necessarily added in the order above, with possibly immune cells added before adding the tissue-specific cells or the endothelial cells.

[0127] It is appreciated that, as above, the source cells obtained from the subject may be any type of cells, as explained in more detail above.

[0128] In some embodiments, the cells obtained from the subject are PBMCs or WBCs.

[0129] In some embodiments, at least one of the source cells, endothelial cells, and immune cells are obtained from the subject by an isolation method including the following steps: obtaining a blood sample from the subject; separating plasma from cells of the blood sample; adding albumin to the cells; and centrifuging the cells with the albumin on a density gradient to obtain PBMCs or WBCs.

[0130] In some embodiments, the albumin is a bovine serum albumin (BSA). In some embodiments, the albumin is a human serum albumin. In some embodiments, the albumin is human albumin.

[0131] The amount of albumin added is based on the amount of plasma removed from the sample. More specifically, the amount of albumin added is calculated based on the volume of plasma to be restored, the target plasma density (df= 1.025 g / mL), the density of PBS (dj = 1.005 g / mL), and the partial specific volume of BSA (i.e., the volume BSA occupies per gram of protein). A calculation is provided in the examples.

[0132] In some embodiments, serum is added to the isolated cells to a concentration of about 2%. In some embodiments, the serum is a fetal calf serum or a fetal bovine serum human.

[0133] In some embodiments, the cells obtained are viable for at least 72 hours in culture.

[0134] In some embodiments, at least one of the plurality of containers includes a Boyden chamber including an upper chamber, a lower chamber, and a porous membrane separating the upper and the lower chamber, and the method further includes a further step of adding the tissue-specific cultures to the lower chamber and seeding the endothelial cells on the porous membrane.

[0135] In some embodiments, at least one of the plurality of containers includes a Boyden chamber including an upper chamber, a lower chamber, and a porous membrane separating the upper and the lower chamber, and the method further includes a further step of adding the endothelial cells to the lower chamber and seeding the tissue-specific cells on the porous membrane.

[0136] In some embodiments, the endothelial cells are directly obtained from the subject. In some embodiments, the endothelial cells are derived from iPSCs prepared from the source cells of the subject.

[0137] In some embodiments, the immune cells are derived from PBMCs or WBCs obtained from the subject. In some embodiments, the immune cells are derived from iPSCs prepared from the source cells of the subject.

[0138] In some embodiments, the immune cells are obtained from the subject by a method including the following steps: obtaining a blood sample from the subject; and lysing red blood cells (RBC)s in the blood sample to enrich for white blood cells (WBC). In some embodiments, the lysis step is repeated twice.

[0139] In some embodiments, the step of lysing the RBCs includes diluting blood in an RBC lysis buffer more than about 5 fold. In some embodiments, the step of lysing the red blood cells includes diluting blood in an RBC lysis buffer about 10 fold. RBC lysis buffer typically includes ammonium chloride that causes osmotic shock to the anucleated red blood cells, making it easier to isolate white blood cells

[0140] In some embodiments, the obtained immune cells are viable for at least 72 hours in culture.

[0141] In some embodiments, the human serum is obtained from the same sample or from the same subject as the cells.

[0142] In some embodiments, the method further includes a step of adding activated immune cells and / or agents which activate immune cells to the liquid medium.

[0143] In some embodiments, the method further includes a step, prior to step (a), or preparing a set of interconnected containers configured to allow a medium to flow through all containers, such as a microfluidic device (e.g., an organ-on-chip device). In some embodiments, at least one of the containers is configured to be replaceable.

[0144] Uses of the system

[0145] After the system is set, it may be used for a variety of purposes. This is generally done by providing a system as disclosed herein, or preparing a system by the methods disclosed herein, and then adding different agents and testing the resulting immune response and response of the tissues of the system. Such testing may also include replacing tissues by tissues from a different subject, as explained above.

[0146] Some example for uses are provided below.

[0147] Assessment of immune response prior to transplantation

[0148] In order to study the effect of transplantation, the system may include a tissue (e.g., kidney or liver) from a different subject. The immune response of the system to the different tissue is evaluated and simulates a situation of transplantation, e.g., graft rejection.

[0149] In some embodiments, there is provided method for assessing immune compatibility of tissues before transplantation, the method including:

[0150] - providing the system of the invention wherein the subject is a transplantation recipient;

[0151] - adding to the system tissue-specific cells from a potential transplantation donor;

[0152] - evaluating the morphology and functionality of the tissue-specific cells in the system; and

[0153] - measuring the immune response of the immune cells in the system following addition of the transplantation donor cells.

[0154] The results of the evaluation are used to assess the chances of successful transplantation, e.g., when the morphology and functionality of the tissue-specific cells is within normal range, and when the immune response following addition of the donor cells is low, then transplantation is likely to succeed. And when the morphology and functionality of the tissue-specific cells is not within normal range, and the immune response following addition of the donor cells is high, then transplantation is not likely to succeed.

[0155] In some embodiments, adding the transplantation donor cells further includes removing tissue specific cells of the transplantation recipient, such that the donor cells replace the recipient cells. In some embodiments, the removed transplantation recipient cells are from the same tissue as the added transplantation donor cells.

[0156] In some embodiments, adding the transplantation donor cells and removing transplantation recipient tissue specific cells includes replacing the container including the recipient cells with a container including the donor cells. As stated above, the containers may be detachable and replaceable.

[0157] In some embodiments the transplantation donor tissue-specific cells are from liver. In some embodiments the transplantation donor tissue-specific cells are from kidney.

[0158] Evaluating the morphology of the tissue-specific cells may be conducted by may be conducted by any suitable method known in the art. Some nonlimiting examples include brightfield / phase-contrast microscopy, differential interference contrast (DIC) microscopy, fluorescence microscopy with specific stainings for cellular structures or proteins, confocal microscopy, and live-cell imaging. Examples for testing morphological features of tissue-specific cells in the system are provided in Figs. 3A-3B.

[0159] Evaluating the functionality of the tissue specific cells may be conducted by any suitable method known in the art for evaluation of the functionality of specific cell types. Some nonlimiting examples include Transepithelial / transendothelial electrical resistance (TEER) for BBB cells; calcium imaging for neurons; spontaneous beating for cardiomyocytes; metabolic (e.g. CYP activity), synthetic (e.g., urea production) and secretory (e.g., albumin secretion) activities for liver cells; and ion transport, drug secretion, reabsorption of glucose and amino acids and TEER for gap junction function for kidney cells. Examples for evaluating the functionality of tissue-specific cells in the system are provided in Figs. 3C-3E.

[0160] Measuring the immune response of the immune cells may be conducted by any suitable method known in the art. Some nonlimiting examples include immune cell proliferation, expression of activation markers (e.g. CD69, CD25), cytokine and chemokine secretion (e.g., IL- 2, IFN-y, TNF-a, IL-6, IL- 10), e.g. by ELISA, bead assays, and qPCR.

[0161] Evaluating response to pathogens

[0162] In some embodiments, there is provided a method for evaluating a subject-specific response to a pathogen, the method including:

[0163] - providing the system of the invention;

[0164] - adding to the liquid medium a pathogen;

[0165] - evaluating the morphology and functionality of the tissue-specific cells in the system; and

[0166] - measuring the immune response of the immune cells in the system following addition of the pathogen.

[0167] In some embodiments, the pathogen is a bacterial pathogen. In some embodiments, the pathogen is an antibiotic-resistant E coli.

[0168] Evaluating response to antibiotic agents

[0169] In some embodiments, there is provided a method for evaluating a subject-specific response to an antibiotic agent, the method including:

[0170] - providing the system of the invention;

[0171] - adding to the liquid medium a pathogen;

[0172] - further adding to the liquid medium an antibiotic agent for treating the pathogen;

[0173] - evaluating the morphology and functionality of the tissue-specific cells in the system; and

[0174] - measuring the immune response of the immune cells in the system following addition of the pathogen and the antibiotic agent.

[0175] In some embodiments, the antibiotic agent is selected from polymyxins B & E and carbapenems.

[0176] In some embodiments, EC50 of the antibiotic agent is measured.

[0177] The system may be used in many more applications, which generally involve providing the system of the invention, which is a personalized simulation of a subject’s body and adding to the system various agents for testing, while measuring parameters related to the tissue-specific cells and immune cells activation, as described above. Some additional examples include: a) Personalized drug screening and therapeutic assays: examining how a specific patient’s own immune system and tissues respond to specific threats, diseases, and treatments. b) Gender-based medicine: The female immune system is known to be about 3 times more potent than the male one, but the reason remains unknown. The platform provides a tool to investigate the source of these differences (e.g., by creating a “male model” vs. a “female model”). c) Immunotherapy: The success of an immunotherapy regimen depends on many variables that are challenging to predict and mimic. The platform enables various regimens to be tested and optimized on a patient-specific model before being administered to the patient, d) Autoimmune disease: The fact that the platform is created from the patient's own cells (iPSCs) allows studying how the immune system reacts to tissues that “suffer” from autoimmune disease (e.g., diabetes, multiple sclerosis, lupus), and then use the system to optimize treatment.

[0178] The agents may be any agent desired to be tested by the system. Non-limiting examples include bacteria, viruses, other microorganisms, toxins, drugs, antibiotics, various therapies such as immunotherapy or chemotherapy, small molecules (e.g. drug candidates), etc.

[0179] Accordingly, in some embodiments, the method disclosed above further includes the steps of adding to the liquid medium an agent selected from a pathogen, a toxin, a disease-causing agent, an antibiotic agent, and / or a therapeutic agent, and testing the activity of the immune cells.

[0180] In some embodiments, the methods further include adding an immune-activating agent to the liquid medium.

[0181] Immune responses and responses of other tissues in the system may be assessed by any known method, including methods disclosed herein. Some non-limiting examples include enzyme- linked immunosorbent assay (ELISA), and flow cytometry.

[0182] In some embodiments, the subject is afflicted with a disease. In some embodiments, the disease is an autoimmune disease. In some embodiments, the disease is cancer.

[0183] Definitions and embodiments mentioned above and which may be relevant to the present pointed out or explicitly repeated. For terms used herein, unless stated otherwise, their definition and embodiments are intended to be the same as above (mutatis mutandis).

[0184] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.

[0185] The term "a" and "an" refers to one or to more than one (i.e., to at least one, or to one or more) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0186] The term "about", when referring to a measurable value such as an amount, a ratio, and the like, is meant to encompass variations of ±10% of the indicated value, as such variations are also suitable to perform the disclosed invention. Any numerical values appearing in the application are intended to be construed as if preceded by “about”, unless indicated otherwise.

[0187] While certain embodiments of the invention have been illustrated and described, it will be clear that the invention is not limited to the embodiments described herein. Numerous modifications, changes, variations, substitutions, and equivalents will be apparent to those skilled in the art without departing from the spirit and scope of the present invention as described by the claims, which follow.

[0188] The following examples are presented in order to more fully illustrate some embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

[0189] EXAMPLES

[0190] Materials and Methods

[0191] Establishing the platform

[0192] Establishing multiple iPSC-derived, isogenic tissues.

[0193] Peripheral blood mononuclear cells (PBMCs) from 4 specific donors (about 50ml samples, a total of about 50X106PBMCs) are isolated and reprogrammed into induced pluripotent stem cells (iPSC)s as described in Rabinski et al, Stem cells Res. 2021 Mar:51 : 102178.

[0194] The PBMCs isolation from whole blood was according to standard protocols. In this case, plasma was separated from cells, the cells were supplemented with BSA and diluted 1 :2-1 :4 with PBS, and fetal bovine serum (FBS) was added to a final concentration of 2% prior to loading on a density gradient (e.g., Ficoll-Paque™). The addition of BSA and FBS was used to adjust for removal of the plasma and significantly improved yield, purity, and viability of the PBMCs (data not shown).

[0195] The amount of BSA required was calculated based on the volume of plasma removed using Equation 1. This equation accounts for the volume of plasma to be restored (k , the target plasma density 1.025 g / mL), the density of the PBS (dj = 1.005 g / mL), and the partial specific volume of BSA (V = 0.733 mL / g), which reflects the volume BSA occupies per gram of protein. Equation 1. Calculation of solvent based on target density

[0196] _ Vfdf- dt) > Vj(1.025 - 1.005)

[0197] X~ l - 7(dr) “ 1 - 0.733(1.025)

[0198] The iPSCs are then differentiated into the target functional tissues: first passage (gut, liver, kidney), central nervous system (brain, including BBB vasculature), and cardiovascular system (heart and endothelium), and their functionality is confirmed by relevant tests including Transepithelial / transendothelial electrical resistance (TEER) for BBB, calcium imaging for neurons, and spontaneous beating for cardiomyocytes. It is noted that in some cases (based on need) immune-related organs such as thymus, bone marrow, and spleen may not be integrated into the platform, e.g., when the focus is on developing an in vitro system that can accommodate exogenous immune cells, as opposed to generating immune cells endogenously. However, the organs included are highly significant for drug development. The differentiation protocols for each tissue are briefly summarized in Table 1, based on the following publications: Workman et al., Cellular and Molecular Gastroenterology and Hepatology, vol. 5, no. 4, pp. 669-677. e2, 2018 (gut), Siller et al., Stem Cell Reports, vol. 4, no. 5, pp. 939- 952, 2015 (liver), Takasato et al., Nat Protoc, vol. 11, no. 9, pp. 1681-1692, 2016 (kidney), Qi et al., Nat Biotechnol, vol. 35, no. 2, pp. 154-163, 2017 (brain), Neal et al., Stem Cell Reports, vol. 12, no. 6, pp. 1380-1388, Jun. 2019 (BBB), Lian et al., Nat Protoc, vol. 8, no. 1, pp. 162-175,

[0199] 2013 (heart), and Edri et al., Advanced Materials, vol. 31, no. 1, Art. no. 1, 2019 (endothelium).

[0200] Briefly, in Table 1 each phase corresponds to a certain period of time, indicated by days in culture (denoted by “D”) in which certain agents are provided (indicated in the boxes) and the indicated structures develop. The Markers column indicates the markers used for determining successful differentiation, and the Assays column describes the functional assays used to assess functionality of the tissue.

[0201] Table 1: Summary of differentiation protocols

[0202] Developing and establishing a fully isogenic platform

[0203] This is done as described in previous work by the inventors (Herland et al. Nature biomedical engineering 4.4 (2020): 421-436; Novak et al. Nature biomedical engineering 4.4 (2020): 407-420), in which organ chips were linked into a complex system mimicking organ-organ interactions, in combination with a modular “Insert-Chip” developed by the inventors (Rauti et al. APL bioengineering 5.2 (2021)). An automated linkage system is also added, allowing “plug and play” integration of different organ chips. The automated linkage system and the insert-chip have been developed to avoid the challenges associated with standard use of microfluidic chips — including risks of bubble generation, the need for a complex pump and tubing system, and difficulty accessing the tissue. The Insert-Chip is modified to fit the automated linkage system, and the platform is characterized to ensure stability, robustness, and modularity.

[0204] The tissues developed are integrated into the platform, creating vascularized organ chips (each including one tissue), in which parenchymal (functional) tissues are seeded on the bottom side of a membrane (e.g. a polycarbonate, polyester (PET), polytetrafluoroethylene (PTFE), cellulose acetate, or collagen coated membrane having a pore size of about 3-8 microns) or on the bottom side of the tissue culture well, and the vasculature (iPSC-derived endothelial cells) is seeded on top.

[0205] The need for an ideal medium that supports multiple different tissues, and that does not activate the immune cells, is answered in the present application by using the subject’s own serum (“isogenic medium”).

[0206] In terms of medium, the Insert-Chip has two compartments (endothelial and parenchymal compartments) to mimic the in vivo physiology, where blood flows through the endothelium, and tissue-specific liquid runs for each specific tissue (e.g., cerebrospinal fluid (CSF) and CNS interstitial fluid (ISF) is perfused in the brain, intracellular fluid (ICF) in other tissues). This feature allows perfusing serum on top of the endothelium, while perfusing tissue-specific medium on the specific tissue.

[0207] Incorporating the immune system into the platform

[0208] Since preparation of iPSCs from PBMCs and development of tissue-specific cells from the iPSCs requires several months, to prepare an isogenic system an additional sample must be obtained from the same subject for preparing isogenic immune cells.

[0209] The donor’s PBMCs or WBCs are integrated into the platform and the cells’ viability is assessed. Isolated PBMCs have been cultured in the donor’s serum, confirming that they remain viable and are not activated by the medium. This is done by the following steps:

[0210] 1. Isolating and characterizing the immune cells: a ficoll gradient (e.g. Ficoll-Paque™) is used to isolate PBMCs from a whole blood sample (usually up to 50ml per donation), or RBC lysis buffer (e.g., Cytek Biosciences RBC lysis buffer) is used to isolate WBC-depleted RBC by diluting the blood 10 fold in the RBC lysis buffer. Next, flow cytometry is used to analyze and quantify the different cell types.

[0211] 2. Optimizing the medium: PBMCs are cultured in 50% serum of the same blood sample. Flow cytometry is used for analyzing surviving populations and activation state overtime, by using biomarkers from the list presented in Table 2.

[0212] Table 2: biomarkers for following PBMC populations

[0213] The immune system is integrated into the platform by adding the isolated cells into the medium, and the immune cells’ viability in the system is examined over 96 hours.

[0214] The assessment of the morphology and functionality of the tissues integrated into the platform are also evaluated over time after immune system incorporation. The response of each of the organs in the platform is assessed by using platforms from different donors and comparing the results.

[0215] Implementations of the platform

[0216] Characterization of immune cells interaction with organ systems The major immunological interactions between the adaptive immune system and the different types of human vascularized (i.e., including endothelial cells) tissues is characterized, as well as the difference in responses to isogenic and to non-isogenic tissues.

[0217] Immune response to isogenic tissues: The immune response to isogenic tissues (having an identical genotype, including identical HLA antigens) is characterized (e.g., by a cytometric analysis, such as by flow cytometry), verifying that the immune cells are indeed not activated in the presence of the isogenic tissues. Controls that activate immune cells are used (e.g., interferon- y or Pan-malarial Antigen (PMA) / lipopolysaccharide (LPS)), as well as incorporating the cells into a non-isogenic system.

[0218] To this end, HLA antigens of all tissues in the platform are characterized by high-resolution HLA typing (such as by sequencing (e.g., next generation sequencing (NGS)), and the immune response is monitored at different time points after adding the PBMCs (after 4, 8, 24, 48, 72, and 96 hours) by testing cytokine and chemokine secretion by ELISA, and identifying activation state of the immune cells by the flow cytometry of markers from Table 2. The response of the vascularized tissues is also monitored for morphological and functional changes and for cytokine and chemokine release.

[0219] Immune response to non-isogenic tissues: The immunological response to non-isogenic tissues is assessed to show that the platform does activate a non-isogenic immune system. This is done as above, except instead of integrating an immune system that comes from the same donor who provided the iPSCs, an immune system from a different donor is used.

[0220] Immune response in organ transplantation: the immunological response following transplantation of a (non-isogenic) organ is characterized by the platform. To this end, a single non-isogenic tissue is introduced into the isogenic platform, and the immune response as well as the effects on each individual tissue may be characterized. This is done by replacing one of the isogenic tissues with a non-isogenic tissue. The characterization includes assessing the immune cells response to a non-isogenic tissue, the response of each of the tissues in this situation, and the effect of the cytokine and chemokine release associated with immune-system activation on the immunological response as an integrated platform.

[0221] Specifically, kidney are liver are separately transplanted into the platform. First, for a control, an isogenic platform not including immune cells is generated, then one of the tissues is replaced with a non-isogenic tissue. “Immunological” and physiological response (e.g., cytokine and chemokine release, morpho-logical and functional changes) are then monitored. The process is then repeated but the immune system is added before the replacement of the tissue and the response is measured and compared to the control. This experiment may be done with different levels of HLA similarity, to test the effect.

[0222] Studying patient-specific responses to pathogens and treatments

[0223] The platform is applied for assessing patient-specific immunological response to bacterial pathogens (antibiotic-resistant E. coli), with and without antibiotic treatment. It should be noted that since PBMCs are used, additional components of the immune system, such as the innate immune system (e.g., monocytes and neutrophils) are also assessed by the platform.

[0224] Assessing activation of the innate and adaptive immune systems as a result of bacterial infection: Innate immune cells (monocytes and neutrophils) are isolated and characterized from whole blood. The innate immune cells are then activated with lipopolysaccharide (LPS, a major component of the outer membrane of Gram-negative bacteria). LPS will also be added to the platform as a control. Bacteria, such as the pathogenic E. coli strain ST131 (e.g., O25b:H4 or O16:H5) are introduced into the platform via the medium, and dynamics of bacterial progression, innate and adaptive immune responses, and physiological response are monitored. To identify these interactions, samples of medium and immune cells are obtained from the platform at different time points (1, 4, 8, 24, 48, 72, 96 hours) and the activation state of the different immune cells over time in response to the presence of the bacteria is assessed by using methods mentioned above such as flow cytometry, cytokine and chemokine identification, mass-spectrometry.

[0225] Evaluating systemic response to bacterial infection: As opposed to assessing the immune response to infection, here the systemic response is assessed as well, including assessment of immune response (flow cytometry, cytokines), and assessment of the morphology and functionality of the tissues integrated into the platform. The response of each of the organs in the platform is assessed by using platforms from different donors and comparing the results.

[0226] Evaluating systemic response to antibiotic treatment: the design of the experiments is similar to the response to bacterial infection design, with the addition of antibiotics. Antibiotics (such as polymyxins B & E and carbapenems) are introduced to a platform “infected” with E. coli, and responses to treatment, including, e.g., side effects, allergic reactions, nephrotoxicity, are assessed. Since liver and kidney tissues are included in the platform and will metabolize and filter the drug, the EC50 is identified over 72 hours. Pharmacokinetics / pharmacodynamics (PK / PD) is monitored to identify whether and how the antibiotic affects the bacteria, and what physiological effects it induces on each one of the tissues.

[0227] Example 1: Establishing multiple iPSC-derived, isogenic tissues

[0228] Two iPSC lines were established from two PBMC donors as described above. Isogenic tissues: neurons, brain microvasculature (BBB), cardiomyocytes, hepatocytes, podocytes, kidney epithelial cells, and gut epithelial cells were successfully prepared from one of the donors, as described above and in Table 1. Figs. 3A-3B present validation of the tissues by morphology and cellular markers, and Figs. 3C-3E present functional characterization of the tissues prepared by commonly used methods: showing BBB function by a Transepithelial / transendothelial electrical resistance (TEER) test (Fig. 3C), e.g., as done by Renous, Noa, et al. "Spatial trans-epithelial electrical resistance (S-TEER) integrated in organs-on-chips." Lab on a Chip 22.1 (2022): 71-79); functional characterization of the neuronal cells by calcium imaging (Fig. 3D); and functional characterization of the cardiac cells by measuring spontaneous beating (Fig. 3E).

[0229] Example 2: Developing and establishing a fully isogenic platform

[0230] An iPSC-derived BBB-on-a-Chip (Sela, Advanced Materials (2023): 2304654) was previously integrated into a preliminary version of the system, showing that the donor serum supports the viability and functionality of the tissue. Serum from the donor who donated the PBMCs was used to culture the BBB for 5 days, and BBB viability and functionality were assessed and confirmed, as shown in Figs. 4A-4H. This shows that serum from the same donor is capable of supporting functional BBB cells (e.g., Fig. 4B). Further, while the use of non-isogenic systems (including PBMCs from a different donor) leads to increased levels of activated T cells (Fig. 4D), when testing isogenic response (immune (PBMC) cells from the same donor as the BBB tissue) the results clearly show that an isogenic system does not activate the immune system compared to two controls (Fig. 4G, non-isogenic cells, and interferon-y stimulation shown in Fig. 4D). In summary, it is found that when co-culturing the isogenic PBMCs or WBCs with isogenic tissues, there is no immune response, whereas, when co-culturing the PBMCs or WBCs with non-isogenic tissues, there is an immune response.

[0231] Finally, Fig. 4H shows a decreased BBB functionality in an non-isogenic system compared to an isogenic system by a TEER analysis. As shown, in the isogenic model (left), the TEER gradually increases over time while in the non- isogenic model (right), there is an increase of TEER after 24h, but then the TEER drops, as the co-culture progresses. Additionally, as shown in Fig. 41, staining of brain microvascular endothelial cells (BMECs) cultured for 72 hours with PBMCs for the tight junction protein ZO-1 showed the cell barrier is intact in an isogenic interaction, while ZO1 expression is reduced in the non- isogenic interaction.

Claims

CLAIMSWhat is claimed is:

1. A personalized in vitro immune response simulation system, comprising: a. a plurality of containers, each container comprising: i. a culture of tissue-specific cells; and ii. endothelial cells in fluid contact with the tissue-specific cells; b. a liquid medium comprising at least about 25% serum or serum replacement (SR); c. immune cells suspended in the liquid medium; and d. a linkage system establishing fluidic coupling between the plurality of containers, allowing the liquid medium to flow through the linkage system and carry the immune cells and substances secreted from the tissue specific cells, the endothelial cells, and the immune cells, wherein: the plurality of containers comprise tissue-specific cells of at least two different tissues; and the system is an isogenic system with the tissue-specific cells, the endothelial cells, and the immune cells being derived from source cells obtained from a single subject.

2. The system of claim 1, wherein the system is a human system and the tissue-specific cells, the endothelial cells, and the immune cells are human cells.

3. The system of any one of claim 1 or 2, wherein the source cells obtained from the subject are blood cells and / or skin cells.

4. The system of any one of claims 1-3, wherein the immune cells comprise innate immune cells and / or adaptive immune cells.

5. The system of any one of claims 1-4, wherein the immune cells are derived from peripheral blood mononuclear cells (PBMCs) or from white blood cells (WBC)s.

6. The system of any one of claims 1-5, wherein the immune cells are not activated.

7. The system of any one of claims 1-5, wherein the immune cells are capable of remaining inactivated in the system for at least about 24, 48, or 93 hours.

8. The system of any one of claims 1-7, wherein the at least two different tissues are at least 3 different tissues.

9. The system of any one of claims 1-8, wherein the at least two different tissues comprise at least two different tissues selected from brain, blood-brain barrier (BBB), gut, liver, kidney, lung, skin, heart, microglia, adipocytes, retinal pigment epithelium.

10. The system of any one of claims 1-9, wherein the tissue-specific cells are derived from stem cells reprogrammed from the cells obtained from the subject.

11. The system of claim 10, wherein the stem cells are induced pluripotent stem cells (iPSCs).

12. The system of any one of claims 1-11, wherein the tissue-specific cells are capable of remaining viable in the system for at least about 24 hours, 48 hours, 93 hours.

13. The system of any one of claims 1-12, wherein the liquid medium comprises about 25%-80% serum.

14. The system of one of claims 1-13, wherein the serum is derived from the single subject.

15. The system of any one of claims 1-14, wherein at least one container of the plurality of containers further comprises a Boyden chamber comprising an upper chamber, a lower chamber, and a porous membrane separating the upper and the lower chamber.

16. The system of claim 15, wherein the tissue-specific cells are cultured in the lower chamber; and the endothelial cells are seeded on the porous membrane.

17. The system of claim 15 or 16, wherein the container further comprises a holder holding an insert comprising the porous membrane and the upper chamber of the Boyden chamber, wherein the holder is configured to allow a pipetting apparatus to access both the upper and the lower chambers of the Boyden chamber.

18. The system of claim 17, wherein the holder comprises a peripheral ring comprising at least two arms radiating inwardly from the ring, each arm comprising an engaging feature configured to engage the insert such that when the insert is engaged, the upper chamber of the Boyden chamber is not centered with respect to a circumference of the container, thereby allowing direct pipetting access to the bottom of the container.

19. The system of any one of claims 1-18, wherein at least one of the plurality of containers is configured to be replaceable.

20. A method for preparing a personalized, in vitro immune response simulation system, the method comprising: a. providing a sample comprising source cells obtained from a subject; b. reprogramming at least a portion of the source cells to produce induced pluripotent stem cells (iPSCs); c. differentiating the iPSCs into a plurality of cultures of tissue-specific cells of at least two different tissues, and placing the cultures of tissue-specific cells in a liquid medium comprising at least 25% human serum or serum replacement into a plurality of containers; d. adding to each container endothelial cells from the same subject, such that the endothelial cells are in fluid contact with the tissue-specific cells; e. placing the plurality of containers in a linkage system which establishes fluidic coupling between the plurality of containers, allowing the liquid medium to flow through the linkage system; and f. adding to the liquid medium immune cells from the same subject.

21. The method of claim 20, wherein the source cells obtained from the subject are peripheral blood mononuclear cells (PBMCs) or white blood cells (WBC)s.

22. The method of claim 20 or 21, wherein the endothelial cells are obtained from the subject or from iPSCs prepared from the cells of the subject.

23. The method of any one of claims 20-22, wherein the immune cells are derived from PBMCs or WBCs obtained from the subject or from iPSCs prepared from the cells of the subject.

24. The method of any one of claims 20-23, wherein the serum is obtained from the sample or from the same subject.

25. The method of any one of claims 20-24, wherein at least one of the source cells, endothelial cells, and immune cells are obtained from the subject by an isolation method comprising the following steps: obtaining a blood sample from the subject; separating plasma from cells of the blood sample; adding albumin to the cells; and centrifuging the cells with the albumin on a density gradient to obtain PBMCs or WBCs.

26. The method of any one of claims 20-25, wherein the immune cells are obtained from the subject by a method comprising the following steps: obtaining a blood sample from the subject; and lysing red blood cells (RBC)s in the blood sample by diluting the blood sample more than about 5-fold with an RBC lysis buffer.

27. The method of any one of claims 20-26, further comprising steps of adding to the liquid medium an agent selected from a pathogen, a toxin, a disease-causing agent, an antibiotic agent, and / or a therapeutic agent, and testing the activity of the immune cells.

28. The method of claim 27, further comprising adding an immune-activating agent to the liquid medium.

29. A method for assessing immune compatibility of tissues before transplantation, the method comprising: a. providing the system of any one of claims 1-19 wherein the subject is a transplantation recipient; a. adding to the system tissue-specific cells from a potential transplantation donor; b. evaluating the morphology and functionality of the tissue-specific cells in the system; and c. measuring the immune response of the immune cells in the system following addition of the transplantation donor cells.

30. The method of claim 29, wherein the method further comprises removing transplantation recipient tissue-specific cells.

31. The method of claim 29 or 30, wherein the tissue-specific cells from a potential transplantation donor are liver cells or kidney cells.

32. A method for evaluating a subject-specific response to a pathogen, the method comprising: a. providing the system of any one of claims 1-19; b. adding to the liquid medium a pathogen; c. evaluating the morphology and functionality of the tissue-specific cells in the system; and d. measuring the immune response of the immune cells in the system following addition of the pathogen.

33. The method of claim 32, wherein the pathogen is a bacterial pathogen.

34. A method for evaluating a subject-specific response to an antibiotic agent, the method comprising: a. providing the system of any one of claims 1-19; b. adding to the liquid medium a pathogen; c. further adding to the liquid medium an antibiotic agent for treating the pathogen; d. evaluating the morphology and functionality of the tissue-specific cells in the system; ande. measuring the immune response of the immune cells in the system following addition of the pathogen and the antibiotic agent.

35. The method of claim 34, wherein the antibiotic agent is selected from polymyxins B & E and carbapenems.

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