Novel phagocytosis assay combining synthetic cell death switch and phagocytosis reporting system

Abstract

The present invention relates to a recombinant expression vector encoding an inducible cell death switch, a pH stable fluorophore and a pH sensitive fluorophore. Furthermore, the invention relates to a cell comprising said recombinant expression vector and to the use of said cell in an in vitro phagocytosis assay.

Description

Technical Field

[0001] The present invention relates to a recombinant expression vector encoding an inducible cell death switch, a pH-stable fluorophore and a pH-sensitive fluorophore. In addition, the present invention relates to a cell comprising the recombinant expression vector and the use of the cell in an in vitro phagocytosis assay. Background Art

[0002] Maintaining the functional state of any organ requires rapid and efficient clearance of pathogens and cellular debris. Macrophages, as part of the immune system, maintain tissue homeostasis by binding, engulfing, and digesting such particles through a process called phagocytosis. In the brain, microglia perform the primary job of apoptotic cell clearance. Dysfunction of macrophages and microglia results in inappropriate clearance and has been associated with a variety of autoimmune and neurodegenerative diseases, including systemic lupus erythematosus, Alzheimer's disease, and Parkinson's disease.

[0003] In view of this important role of macrophages and microglia, multiple phagocytosis assays are currently used. Commonly used substrates are opsonized red blood cells, yeast particles, Escherichia coli bioparticles, amyloid beta plaques, myelin or cell debris. The substrate is usually labeled with pH-sensitive dye pHrodo, and when pH decreases in the phagolysosome, the fluorescence intensity of pHrodo increases, and thus indicates phagocytosis. The substrate is usually incubated with phagocytic cells for a fixed time. Then separate phagocytic cells and measure the internalized substrate by, for example, ELISA (if the substrate has a specific epitope), in a plate reader (for example, for RBC) or by flow cytometry (for example, for fluorescently labeled fragments). Time-lapse imaging during incubation with phagocytic cells is used to observe and quantify phagocytosis.

[0004] There are two major limitations of current phagocytosis assays. The first is high experimental variability due to variable efficiencies in generating apoptotic cells and modifying them by labeling, making it difficult to quantify and compare results. The second is the lack of temporal flexibility. The assay is started when all components are mixed, which is undesirable in more complex multicellular in vitro models. Therefore, we identified the following needs for an improved phagocytosis assay.

[0005] Need for precise and specific generation of apoptotic cells

[0006] Cell debris from apoptotic cells is one of the most important substrates for phagocytosis, since cell clearance constitutes a large part of the activity of macrophages and microglia under both physiological and pathological conditions. However, protocols for obtaining apoptotic cells (e.g., by incubation with staurosporine, a bacterial product) may result in different proportions of dead cells. Another concern is that the product may contain other forms of cell debris (e.g., necrotic cells, leaked DNA, and other components).

[0007] Furthermore, when a substrate is labeled, its surface is modified (eg by the addition of a fluorescent dye), which may affect substrate recognition by phagocytes.

[0008] In order to obtain consistent and physiologically important apoptotic cell substrates, apoptosis should ideally be induced with high efficiency, specificity, reproducibility, and without the need for subsequent modification / external labeling.

[0009] Need to intrinsically mark apoptotic cells

[0010] Importantly, in order to clearly identify engulfment events, it is necessary to unambiguously label both engulfed and non-engulfed apoptotic cells. Commonly used pHrodo dyes emit weak fluorescence in non-engulfed cells and, after engulfment, emit strong signals in labeled cells that are taken up and inside lysosomes at low pH. Due to variable labeling efficiencies and occasional detachment of the dye from the surface, the pHrodo signal is variable between experiments, and the threshold between high and low signals needs to be adjusted for each experiment.

[0011] On-demand apoptosis gene switches are needed to gain assay flexibility

[0012] Finally, in current assays, apoptotic cells (or other substrates) need to be generated and labeled prior to contact with phagocytes. Thus, phagocytosis starts immediately after the two components are mixed. However, for some complex assays (spheroids, organoids, organotypic structure models), multiple cell types need to be seeded together, often within a 3D matrix, and the cells require several days to align and self-organize. Therefore, if the phagocytosis assay needs to be performed several days after seeding all components, conventional assays cannot be used. In this case, there needs to be an apoptotic switch (on the system) in which the phagocytic substrate can be generated at the desired time point.

[0013] Novelty and advantages of the present invention over established methods

[0014] The inventors have developed a novel phagocytosis assay that combines two features: 1) on-demand cell-specific apoptosis and 2) phagocytosis-specific fluorescent reporters. First, the inventors used a method in which apoptotic cells of any cell type can be generated on demand at any desired time point with high efficiency and reproducibility. The inventors utilized an inducible caspase 9 construct first described in Straathof, KC et al. An inducible Caspase 9 safety switch for T-cell therapy. Blood. 2005; 105 (11): 4247-54 and disclosed in WO2011 / 146862A1, in which the dimerization domain of caspase 9 was replaced with the FKBP12-F36V dimerization domain. This replacement resulted in dimerization of caspase 9 only after the addition of the small molecule AP20187, which subsequently triggered the apoptotic cascade.

[0015] Secondly, the inventors constitutively co-expressed a fusion construct including mCherry and a GFP-based pH-sensitive Superecliptic pHluorin (SEP; Sankaranarayanan, S et al. The Use of pHluorins for Optical Measurements of Presynaptic Activity. Biophys. J. 2000; 79(4): 2199-208) in the same cell. At pH 7.4, the reporter emits green (SEP) and red (mCherry) fluorescence when excited at 488nm and 580nm, respectively. At acidic pH, SEP fluorescence is quenched and the reporter emits only red fluorescence. With this genetically encoded reporter, we are able to distinguish engulfed cells from non-engulfed cells with high accuracy without the need for external modification of cells. This novel construct iCaspase9-SEP-mCherry ( Figure 1 ) can be transfected or transduced into mammalian cells (cell lines, primary cells, iPSCs) and expressed transiently or stably.

[0016] Phagocytosis assays can be performed in 2D or 3D several days after seeding a variety of cell types, including inducible substrate cells (e.g., H4-iCaspase9-SEP-mCherry cells or Jurkat-iCaspase9-SEP-mCherry cells) and phagocytic cells (e.g., iPSC-derived microglia or macrophages). Summary of the invention

[0017] In one aspect, the present invention provides a recombinant expression vector encoding an inducible cell death switch, a pH-stable fluorophore, and a pH-sensitive fluorophore.

[0018] In one aspect, the recombinant expression vector is a viral vector. In one aspect, the viral vector is a lentiviral vector.

[0019] In one aspect, the inducible cell death switch induces apoptosis, necroptosis, pyroptosis or ferroptosis, preferably apoptosis. On the other hand, the inducible cell death switch comprises an inducer binding domain and a signaling protein of a cell death pathway. In one aspect, the signaling protein of the cell death pathway is a pro-apoptotic protein, preferably caspase 9, or a functional fragment thereof. In one aspect, the inducer binding domain includes a dimerization domain, preferably FKBP12-F36V. In one aspect, the inducer binding domain is capable of binding an inducer, preferably a chemical dimerization inducer, most preferably AP20187. In one aspect, the inducible cell death switch is an inducible caspase 9.

[0020] In one aspect, pH stability fluorophore and pH sensitivity fluorophore have different excitation spectra and emission spectra. In a further aspect, pH stability fluorophore is from RFP family, Alexa Fluor dye, protein-based fluorophore, simple organic fluorophore or organic polymer, preferably mCherry. In one aspect, the fluorescence of pH sensitivity fluorophore changes in response to the reduction of pH, preferably wherein fluorescence is quenched or excitation spectrum / emission spectrum is shifted. On the other hand, pH sensitivity fluorophore is Superecliptic pHluorin (SEP), pHLemon, pHmScarlet, pHTomato, pHuji, LysoSensor or pH nanosensor, preferably Superecliptic pHluorin (SEP).

[0021] In one aspect, the recombinant expression vector comprises a promoter, in particular a ubiquitous promoter, a cell-specific promoter, a constitutive promoter or an inducible promoter. In one aspect, the recombinant expression vector comprises a CMV promoter. In one aspect, the recombinant expression vector comprises a polynucleotide sequence according to SEQ ID NO: 1.

[0022] According to a further aspect of the present invention, a cell is provided, which comprises the recombinant expression vector of the present invention. In one aspect, the cell is a mammalian cell, particularly a human cell. In one aspect, the cell is a stem cell. In a further aspect, the cell is a neural cell, a neuronal cell, a glial cell, an interstitial cell or a hematopoietic cell, preferably a glioma cell or a T cell. In one aspect, the cell is an H4 cell or a Jurkat cell. In one aspect, the expression of the recombinant expression vector in the cell is transient or stable.

[0023] The present invention also includes an in vitro method for evaluating phagocytosis, the method comprising the steps of: a) providing an inducible substrate cell according to the present invention; b) combining and co-culturing the inducible substrate cell with a phagocytic cell; c) inducing cell death of the inducible substrate cell; and d) detecting a fluorescent signal of the inducible substrate cell; wherein a change in the fluorescent signal of the pH-sensitive fluorophore indicates phagocytosis of the inducible substrate cell.

[0024] In one aspect, the phagocytic cells of step b) are macrophages or tissue-resident macrophages. In one aspect, the tissue-resident macrophages are microglia.

[0025] In one aspect, the inducible substrate cells and phagocytes are co-cultured in an in vitro model comprising additional cell types. In one aspect, the in vitro model is a 2D or 3D culture system. In another aspect, the in vitro model is an organ on a chip, a spheroid, or an organoid. In one aspect, the in vitro model is a neurovascular unit or a blood-brain barrier spheroid.

[0026] In one aspect, after combining the inducible substrate cells with the phagocytic cells in step b), cell death of the inducible substrate cells is induced in step c). In a further aspect, cell death of the inducible substrate cells in step c) is induced by an inducer, preferably a chemical dimerization inducer, most preferably AP20187.

[0027] In another aspect, the fluorescent signal of step d) is detected by fluorescence imaging, flow cytometry or by a fluorescence plate reader. In one aspect, the fluorescent signal in step d) is detected at several time points after induction of cell death. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 : Lentivirus vector diagram, which shows iCaspase9 connected to SEP-mCherry fluorophore by cleavable P2A linker under the control of CMV promoter. The construct also contains a puromycin selection box under the control of mPGK promoter.

[0029] Figure 2: iCaspase9-transfected cells rapidly underwent apoptosis when exposed to AP20187, whereas staurosporine caused less apoptosis after only 3 h of incubation.

[0030] Figure 3 : H4-iCaspase-SEP-mCherry cells imaged in the green channel (top) and the red channel (bottom). SEP is quenched at lower pH, while mCherry is present in all environments tested.

[0031] Figure 4 : H4-iCaspase-SEP-mCherry cells co-cultured with labeled HMC3 microglia in a 2D phagocytosis assay. At t0, apoptosis of transduced H4 cells was triggered by the addition of 10 nM AP20187, and cells were imaged for 8 h.

[0032] Figure 5 :exist Phagocytosis of H4-iCaspase-SEP-mCherry cells co-cultured with iPSC-derived microglia in 2D imaged on the device.

[0033] Figure 6 : Evoked Jurkat-iCaspase-SEP-mCherry cells by THP-1 macrophages quantified by flow cytometry.

[0034] Figure 7 : Phagocytosis of H4-iCaspase-SEP-mCherry cells by HMC3 microglia in 3D blood-brain barrier (BBB) ​​spheroids at day 2 after specific induction of apoptosis in transduced H4 cells.

[0035] Figure 8 : Phagocytosis of H4-iCaspase-SEP-mCherry cells by HMC3 microglia in a 3D neurovascular unit model 7 days after vascular self-assembly. Specific induction of apoptosis in transduced H4 cells. DETAILED DESCRIPTION

[0036] Unless otherwise defined below, the terms used herein are generally as used in the art.

[0037] As used herein, the term "recombinant expression vector" refers to a polynucleotide molecule capable of directing the expression of a polypeptide encoded by a polynucleotide sequence therein. The recombinant expression vector comprises a regulatory sequence that causes effective transcription of the encoding polynucleotide sequence. In the context of the present invention, the term "recombinant expression vector encoding an inducible cell death switch, a pH stability fluorophore, and a pH-sensitive fluorophore" includes (i) a single vector encoding all of the elements (i.e., an inducible cell death switch, a pH stability fluorophore, and a pH-sensitive fluorophore), or (ii) more than one vector, each vector encoding one or more of the elements, and collectively encoding all of the elements. Therefore, in some embodiments according to the present invention, the inducible cell death switch, the pH stability fluorophore, and the pH-sensitive fluorophore are encoded by a single vector, while in other embodiments, the inducible cell death switch, the pH stability fluorophore, and the pH-sensitive fluorophore are encoded by more than one vector.

[0038] As used herein, the term "inducible cell death switch" refers to a molecule that can cause the death of cells expressing the molecule after activation. The cell death switch comprises an inducer binding domain and a signal transduction protein of a cell death pathway. The signal transduction protein is activated by an inducer, and the inducer is added to the environment of the cell and binds to the inducer binding domain of the inducible cell death switch. Thereby specifically inducing cell death of cells expressing the inducible cell death switch. In the absence of an inducer, cells expressing the inducible cell death switch show cell death at a physiological rate, i.e., comparable to cells that do not express the inducible cell death switch. Therefore, cell death can be induced at a specific time point.

[0039] As used herein, the term "pH-stable fluorophore" refers to a fluorescent protein that emits fluorescence independently of the pH of the environment, that is, at different pH values, the fluorophore emits fluorescence with the same fluorescence spectrum. For example, when the pH of the environment is reduced, the fluorescence of the fluorophore will be maintained. Therefore, a pH-stable fluorophore expressed by a cell being engulfed will maintain its fluorescence during the maturation of the phagolysosome, which is associated with a decrease in pH.

[0040] As used herein, the term "pH-sensitive fluorophore" refers to a fluorescent protein that emits a fluorescent signal depending on the pH of the environment, that is, the fluorescence emission varies with different pH values. At different pH values, the fluorophore can emit different fluorescence spectra, or the fluorophore can exhibit increased or decreased fluorescence. The pH-sensitive fluorophore can emit a strong fluorescent signal at neutral pH, and the fluorescent signal can be quenched after acidification of the environment. For example, a pH-sensitive fluorophore expressed by a cell being engulfed can exhibit a decrease in its fluorescence during the maturation of the phagolysosome, which is associated with a decrease in pH.

[0041] The term "promoter" as used herein is defined as a polynucleotide sequence of a recombinant expression vector that controls the expression of the encoded polypeptide as commonly understood by the skilled person. The promoter recruits the transcriptional machinery of the cell to the expression vector and regulates the time and / or place of expressing the encoded polypeptide. The expression vector may contain several independent promoters that regulate the expression of different polypeptides. Therefore, different polypeptides may be under the control of different promoters, i.e., the expression of the polypeptide is regulated by a separate promoter. The promoter may be a "constitutive promoter", which is considered to provide a stable expression level under different conditions; or the promoter may be an "inducible promoter", which drives expression in response to a specific stimulus. In addition, the promoter may be a "ubiquitous promoter", which is active in a range of cell types and / or developmental stages; or the promoter may be a "cell type-specific promoter", which is active only in one or more specific cell types.

[0042] As used herein, the term "transfection" or "transfect" is defined as a process by which exogenous polynucleotide sequences are introduced into cells by non-viral methods as commonly understood by the skilled artisan. Common transfection methods include calcium phosphate, cationic polymers (such as polyethyleneimine (PEI)), magnetic beads, electroporation, and commercial lipid-based reagents such as and

[0043] The term "transduction" as used herein is defined as a process by which an exogenous polynucleotide sequence is introduced into a cell via a viral vector as commonly understood by a skilled person. Transduction generally results in stable expression of the encoded polypeptide.

[0044] As used herein, the term "in vitro model" refers to a cell culture system designed to replicate certain aspects of cell behavior found in vivo, thereby promoting the study of cell processes. The in vitro model may include a single cell type, or may include two or more cell types and extracellular matrix and / or excipient elements (e.g., microfabrication devices). Therefore, the in vitro model can be a simplified representation of different tissues or organs, or can simulate the in vivo structure and organization of a tissue or organ. Therefore, the cells of the in vitro model can be cultivated in a 2D or 3D culture system." 2D culture system "refers to an in vitro model in which cells are mainly cultivated as a monolayer (i.e., in a two-dimensional structure). And "3D culture system" refers to an in vitro model in which cells are organized in a three-dimensional structure (e.g., an organ on a chip, a spheroid, or an organoid as described below).

[0045] As used herein, the term "organ-on-a-chip" refers to a culture system on a microfluidic chip that mimics the activities, mechanisms, and physiological responses of an organ or organ system.

[0046] As used herein, the term "spheroid" or "spheroid culture system" refers to a 3D in vitro model of cells grown in suspension, in which cells aggregate and form a spheroid shape. Spheroids can provide cells with a physicochemical environment similar to that in vivo by promoting cell-cell interactions and cell-matrix interactions to overcome the limitations of traditional monolayer cell culture.

[0047] The term "organoid" as used herein refers to a 3D in vitro model that mimics its corresponding in vivo tissue or organ, such that it can be used to study various aspects of the organ in a tissue culture dish.

[0048] As used herein, the term "phagocytosis" refers to the cellular process of ingesting and eliminating particles such as microorganisms, foreign materials, cells or cell fragments. It includes the term "efferocytosis", which refers to a special phagocytic process. During the phagocytic process, the particles to be eliminated are engulfed by the cell membrane, forming a special intracellular vacuole called a phagosome. The phagosome matures into a phagolysosome, in which the engulfed particles are degraded and eliminated. The maturation of the phagosome into the phagolysosome is characterized by the acidification of the vacuole, i.e., a decrease in pH.

[0049] As used herein, the term "phagocytosis assay" refers to a cellular assay that assesses phagocytic activity. A phagocytosis assay typically includes at least one cell type that acts as a phagocytic cell, and a particle or substrate to be phagocytosed.

[0050] As used herein, the term "phagocyte" or "phagocytic cell" refers to a type of cell that exhibits phagocytic activity, ie, is capable of phagocytosis.

[0051] As used herein, the term "inducible substrate cell" refers to a cell comprising an inducible cell death switch, which, after induction of cell death, can undergo phagocytosis, ie, become a substrate for a phagocytic cell.

[0052] The present invention provides a recombinant expression vector encoding an inducible cell death switch, a pH-stable fluorophore, and a pH-sensitive fluorophore. The recombinant expression vector of the present invention may be a viral vector. The recombinant expression vector may be a lentiviral vector, an adenoviral vector, or a retroviral vector. In one embodiment, the recombinant expression vector is a lentiviral vector.

[0053] The inducible cell death switch encoded by the recombinant expression vector of the present invention may include an inducer binding domain and a signal transduction protein of a cell death pathway. After an inducer, particularly a chemical inducer, binds to the inducer binding domain, the signal transduction protein is activated, which leads to the initiation of a cell death pathway. The activation of the signal transduction protein can be achieved by dimerization. Therefore, the inducer binding domain may be a dimerization domain. After the inducer binds, the dimerization domain dimerizes with another dimerization domain, leading to the activation of a cell death signal transduction protein. The inducer binding domain may be FKBP12-F36V, as described in Straathof, KC et al., An inducible caspase 9 safety switch for T-cell therapy. Blood. 2005; 105(11): 4247-54. FKBP12-F36V is a human FK506 binding protein (FKBP12; GenBank AH002 818) comprising an F36V mutation. The polynucleotide sequence of FKBP12-F36V is shown in SEQ ID NO: 2. The amino acid sequence is shown in SEQ ID NO: 11. In one embodiment, the inducer binding domain is a dimerization domain. In another embodiment, the inducer binding domain is FKBP12-F36V. In one embodiment, the inducer binding domain is encoded by the polynucleotide sequence of SEQ ID NO: 2. In one embodiment, the inducer binding domain comprises the amino acid sequence of SEQ ID NO: 11. In one embodiment, the inducer is a chemical inducer. In a specific embodiment, the inducer is AP20187. In one embodiment, the inducer binding domain is FKBP12-F36V and the inducer is AP20187.

[0054] Among others, the signal transduction protein of the cell death pathway can be a signal transduction protein of an apoptotic pathway, a necroptotic pathway, a pyroptotic pathway or a ferroptotic pathway. Therefore, among others, the inducible cell death switch can induce apoptosis, necroptotic apoptosis, pyroptosis or ferroptotic death. Preferably, the inducible cell death switch induces apoptosis. The signal transduction protein can be a pro-apoptotic protein, such as a protein of the caspase signal transduction cascade. The continuous activation of caspases plays a central role in the execution of apoptosis. The signal transduction protein can be caspase 9 (Casp9; UniProtKB: P55211) or a functional fragment thereof. The term "functional fragment" used herein refers to a part of a protein that retains the biological function of the full-length protein, i.e., the functional fragment of the protein of the cell death pathway will also induce cell death. The functional fragment of caspase 9 can have an amino acid sequence as shown in SEQ ID NO: 12, or can be encoded by SEQ ID NO: 3. In its monomeric form, caspase 9 or a functional fragment of caspase 9 is inactive. By dimerization, they are activated and act as initiator caspases, activating downstream execution caspases. In one embodiment, the signaling protein of the cell death pathway is caspase 9, in particular human caspase 9, or a functional fragment thereof. In one embodiment, the protein of the cell death pathway is encoded by the sequence of SEQ ID NO: 3. In one embodiment, the protein of the cell death pathway comprises the amino acid sequence of SEQ ID NO: 12.

[0055] As used herein, the term "inducible caspase 9" or "iCasp9" refers to construct F-Casp9, also known as iCasp9 M , as described by Straathof, KC et al., the construct comprises a functional fragment of FKBP12-F36V and caspase 9. The amino acid sequence of inducible caspase 9 is shown in SEQ ID NO: 13. The polynucleotide sequence of inducible caspase 9 is shown in SEQ ID NO: 4. In one embodiment, the inducible cell death switch comprises a dimerization domain and a pro-apoptotic protein. In one embodiment, the inducible cell death switch comprises FKBP12-F36V and caspase 9. In one embodiment, the inducible cell death switch is an inducible caspase 9. In one embodiment, the inducible cell death switch is encoded by the sequence of SEQ ID NO: 4. In one embodiment, the inducible cell death switch comprises the sequence of SEQ ID NO: 13.

[0056] The recombinant expression vector of the present invention encodes a pH stability fluorophore and a pH sensitivity fluorophore. The pH stability fluorophore and the pH sensitivity fluorophore have different excitation spectra and emission spectra, that is, the fluorescence signals of each other can be distinguished by a fluorescence detection system. Therefore, in the phagocytosis assay, engulfed cells and unengulfed cells can be distinguished. The pH stability fluorophore can be mCherry, other fluorophores from the RFP family, Alexa Fluor dyes, protein-based fluorophores (such as PE), simple organic fluorophores, or organic polymers. Generally, any pH stability fluorophore with an excitation spectrum and an emission spectrum different from the pH sensitivity fluorophore can be used. mCherry is a member of the mFruits family of monomeric red fluorescent protein (mRFP). It absorbs light between 540nm-590nm and emits light in the range of 550nm-650nm. The polynucleotide sequence of mCherry is shown in SEQ ID NO:5. The amino acid sequence of mCherry is shown in SEQ ID NO:14. The pH-sensitive fluorophore can be Superecliptic pHluorin (SEP) or pHLemon in the green spectrum; pHmScarlet, pHTomato or pHuji in the red spectrum; LysoSensor; pH nanosensor (e.g., pH sensor based on QD protein FRET) or (molecular) fluorescent switch. SEP is a pH-sensitive green fluorescent protein that emits a strong green fluorescence signal at neutral pH. As the environment becomes acidic, the fluorescence signal gradually weakens, i.e., it is quenched, with a pKa of 7.2 and an apparent Hill coefficient of 1.9. The polynucleotide sequence of SEP is shown in SEQ ID NO:6. The amino acid sequence of SEP is shown in SEQ ID NO:15. Alternatively, the excitation spectrum / emission spectrum of the pH-sensitive fluorophore can vary depending on the pH of the environment. Therefore, during the phagocytic process, the excitation spectrum / emission spectrum of the pH-sensitive fluorophore can shift. In one embodiment, the pH-stable fluorophore is mCherry. In one embodiment, the pH-sensitive fluorophore is SEP. In one embodiment, the pH-stable fluorophore is mCherry and the pH-sensitive fluorophore is SEP. In one embodiment, the pH-stable fluorophore and the pH-sensitive fluorophore are connected via a linker to generate a fluorophore fusion protein. The linker can be encoded by the polynucleotide sequence of SEQ ID NO: 9. In one embodiment, mCherry and SEP are connected by the amino acid sequence of SEQ ID NO: 16.

[0057] The cell death switch and the fluorophore fusion protein can be connected by a cleavable P2A linker. In one embodiment, the cleavable P2A linker is encoded by the sequence of SEQ ID NO: 10. In one embodiment, iCaspase9 and SEP-mCherry fusion protein are connected by the amino acid sequence of SEQ ID NO: 17.

[0058] The inducible cell death switch, pH stability fluorophore and pH sensitivity fluorophore encoded by the recombinant expression vector of the present invention can be under the control of a promoter. Depending on the cell type to be transfected or transduced and / or the specific assay to be performed, the promoter can be a ubiquitous promoter, a cell type specific promoter, a constitutive promoter or an inducible promoter. For example, the immediate early gene of human cytomegalovirus (CMV), also referred to as the CMV promoter, can be used. The sequence of the CMV promoter is shown in SEQ ID NO:7. The promoter is considered to produce stable, constitutive expression of the encoded protein in a variety of cell types. Cell types that are difficult to transfect or transduce may benefit from cell type specific promoters. For example, in order to express in neurons, neuron specific promoters can be used. In addition, if undifferentiated stem cells are to be transduced or transfected with a recombinant expression vector, a cell type specific promoter may be useful. Therefore, during or after differentiation, the encoded protein will only be expressed in a specific cell type. In addition, an inducible promoter can be used to start the expression of the encoded protein at a specific time point. In one embodiment, the cell death switch, pH stability fluorophore and pH sensitivity fluorophore are under the control of a promoter. In one embodiment, the expression vector comprises a CMV promoter. In one embodiment, the cell death switch, pH stability fluorophore and pH sensitivity fluorophore are under the control of a CMV promoter.

[0059] The recombinant expression vector of the present invention may further comprise a selection marker gene. The selection marker gene can be used to select cells that are successfully transfected or transduced. The selection marker gene can be an antibiotic resistance gene, such as a puromycin resistance gene (Puro). In this case, the selection marker gene can be under the control of a promoter that is active in the cell type that is transfected or transduced. The promoter can be different from the promoter that drives the expression of an inducible cell death switch, a pH stability fluorophore, and a pH sensitivity fluorophore. The promoter can be mPGK (mouse phosphoglycerate kinase), which effectively drives high expression in a variety of cell types. The sequence of the mPGK promoter is shown in SEQ ID NO:8. Alternatively, cells that are successfully transfected or transduced can be selected by fluorescence. For example, cells expressing a pH stability fluorophore can be sorted by fluorescence activated cell sorting (FACS). In one embodiment, the recombinant expression vector of the present invention comprises a selection marker gene. In one embodiment, the recombinant expression vector of the present invention comprises a puromycin resistance gene. In one embodiment, the selection marker gene is under the control of an mPGK promoter.

[0060] The recombinant expression vector of the present invention may contain an antibiotic resistance gene to select bacterial clones that produce viral vectors during vector production. In this case, the antibiotic resistance gene is under the control of a bacterial promoter. In one embodiment, the recombinant expression vector contains an antibiotic resistance gene. In one embodiment, the recombinant expression vector contains an ampicillin resistance gene.

[0061] In one embodiment, the recombinant expression vector of the present invention comprises a polynucleotide sequence according to SEQ ID NO:1.

[0062] The present invention also relates to cells comprising the recombinant expression vector of the present invention. Since cell death of such cells can be initiated at a specific time point and their phagocytic uptake is visualized by the expressed fluorophore, the cells are particularly suitable as inducible substrate cells in phagocytosis assays.

[0063] In principle, the recombinant expression vector of the present invention can be expressed in any cell type. Therefore, the cell comprising the recombinant expression vector of the present invention can be any cell type. The cell can be a cell line derived from induced pluripotent stem cells (iPSC source), derived from embryonic stem cells (ESC source) or patient-derived cells. The cell can be, for example, a nerve cell, a neuronal cell, a glial cell, an interstitial cell or a hematopoietic cell. The cell can be a healthy cell or a diseased cell. The diseased cell can be, for example, a cancer cell, such as a glioma cell. Therefore, it can be assessed that, for example, apoptotic glioma cells are engulfed by, for example, microglia.

[0064] The cell type can be selected according to the phagocytosis assay to be performed. For example, if the phagocytosis of leukemic T cells is to be studied, the cell containing the recombinant expression vector of the present invention can be a T cell.

[0065] Furthermore, the cell comprising the recombinant expression vector of the present invention may be a stem cell.By selecting a cell type specific promoter controlling the expression of the encoded polypeptide, only specific cell types will express the encoded polypeptide after differentiation.

[0066] In one embodiment, the cell comprising the recombinant expression vector of the present invention is a mammalian cell, particularly a human cell. In one embodiment, the cell comprising the recombinant expression vector of the present invention is a stem cell. In one embodiment, the cell comprising the recombinant expression vector of the present invention is a glioma cell or a T cell. In one embodiment, the cell comprising the recombinant expression vector of the present invention is an H4 cell or a Jurkat cell.

[0067] Cells can be transfected or transduced with the recombinant expression vector of the present invention. Thus, the expression of the encoded polypeptide can be transient or stable. In one embodiment, the expression of the recombinant expression vector in the cell is stable. In a further embodiment, the expression of the recombinant expression vector of the present invention in the cell is transient.

[0068] The present invention also relates to an in vitro method for evaluating phagocytosis. The in vitro method described herein provides an improved method for generating substrate cells and assessing the phagocytosis of the substrate cells. Therefore, the substrate cells according to the present invention are co-cultured with phagocytic cells, cell death of the substrate cells is induced and phagocytosis of the substrate cells by the phagocytic cells is assessed. The substrate cells express both pH-stable fluorophores and pH-sensitive fluorophores. Since phagocytosis is associated with a decrease in pH in the phagolysosome, changes in the fluorescence signal of the pH-sensitive fluorophore indicate phagocytosis of the substrate cells. The pH-stable fluorophore enables precise localization of phagocytic events.

[0069] In one embodiment, the in vitro method for evaluating phagocytosis comprises the following steps: a) providing an inducible substrate cell according to the present invention as disclosed herein (i.e., a cell comprising a recombinant expression vector of the present invention as described above); b) combining and co-culturing the inducible substrate cell with a phagocytic cell; c) inducing cell death of the inducible substrate cell; and d) detecting a fluorescent signal of the inducible substrate cell; wherein a change in the fluorescent signal of the pH-sensitive fluorophore indicates phagocytosis of the inducible substrate cell.

[0070] It should be understood that any of steps b) to d) may overlap in time, or may be performed sequentially in any order. For example, cell death of substrate cells may be induced while substrate cells and phagocytic cells are combined, i.e. steps b) and c) may occur simultaneously. Alternatively, cell death of substrate cells may be induced before or after the cells are combined. Likewise, the fluorescent signal may be detected at any time point.

[0071] Phagocytic cells can be macrophages or tissue-resident macrophages. Tissue-resident macrophages can be microglia. Since macrophages and microglia are derived from the same progenitor cells, their transcriptomes and proteomes overlap. In vitro, macrophages can be derived by differentiating macrophage / microglia precursor cells using M-CSF or GM-CSF for 7 days. Differentiation of microglia from macrophage / microglia precursor cells can be induced by M-CSF, TGF-β1 and IL-34. Microglial markers CX3CR1, P2RY12 or TMEM119 can be used to distinguish them from each other.

[0072] Inducible substrate cells and phagocytes can be co-cultured in an in vitro model comprising additional cell types. Thus, in vitro methods allow for the assessment of phagocytosis in different types of in vitro models that simulate different tissues or organs. Thus, depending on the in vitro model to be studied, various additional cell types can be employed. For example, to study the phagocytosis of neuroblastoma cells by microglia, the in vitro model can include brain endothelial cells, pericytes, and astrocytes as additional cell types. In one embodiment, inducible substrate cells and phagocytes are co-cultured in an in vitro model comprising additional cell types.

[0073] The advantage of the in vitro methods described herein is that phagocytosis of substrate cells does not necessarily start at a specific time point when substrate cells and phagocytes are combined in an in vitro model. This allows phagocytosis to be evaluated in in vitro models, which first require different cell type organizations (e.g., differentiation, migration, adhesion, connection), i.e., to establish an in vitro model, and then start phagocytosis assays. Therefore, after a period of in vitro model establishment, cell death and phagocytosis of inducible substrate cells can be induced. This is especially true for 3D in vitro models, such as on-chip organs, spheroids, or organoids. These culture systems may require several cell inoculation steps and / or specific time periods for cells to be organized.

[0074] It is further envisioned that the in vitro method for evaluating phagocytosis can be carried out in an in vitro model established by stem cell differentiation. The recombinant expression vector of the present invention under the control of a cell type-specific promoter can be transduced into undifferentiated stem cells. After differentiation and establishment of an in vitro model derived from stem cells, cell death switches, pH stability fluorophores, and pH sensitivity fluorophores will only be expressed in cell types in which the cell type-specific promoter is active. This enables phagocytosis to be studied in a culture system derived from stem cells.

[0075] Therefore, in one embodiment, in the case of an in vitro model of cells, cell death of the inducible substrate cells can be induced after a period of time. In one embodiment, after the inducible substrate cells are combined with phagocytes, cell death of the inducible substrate cells is induced (i.e., steps b and c of the in vitro method of evaluating phagocytosis). In a further embodiment, cell death of the inducible substrate cells is induced after about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, or about 6 hours of combining and co-culturing the inducible substrate cells and phagocytes. In a further embodiment, cell death of the inducible substrate cells is induced after about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days of combining and co-culturing the inducible substrate cells and phagocytes.

[0076] As described above, cell death of inducible substrate cells can be induced at any specific time point selected according to the in vitro model used or the preference of the person performing the in vitro method. Cell death is induced by the presence of an inducer, such as a chemical inducer, in the environment of the inducible substrate cell. Therefore, the inducer can be added to the cell culture medium at any selected time point. The inducer binds to the inducer binding domain of the cell death switch and activates the signal transduction protein of the cell death pathway. The activated signal transduction protein initiates the corresponding cell death pathway in the inducible substrate cell, causing its cell death. Different inducers can be used according to the cell death switch used in the inducible substrate cell and its inducer binding domain. The inducer binding domain can be a dimerization domain. Therefore, the inducer can be a dimerization inducer. The inducible cell death switch can include FKBP12-F36V as a dimerization domain. Therefore, the chemical dimerization inducer can be AP20187 (Formula I).

[0077]

[0078] AP20187 (CAS No. 195514-80-8) is a non-toxic synthetic FK506 analog that is modified to reduce interaction with endogenous FKBP while enhancing binding to FKBP12-F36V. Binding of AP20187 to the inducer binding domain of inducible caspase 9 leads to dimerization and activation of caspase 9, thereby initiating apoptosis. In one embodiment, in step c), cell death of inducible substrate cells is induced by a chemical dimerization inducer. In one embodiment, in step c), cell death of inducible substrate cells is induced by AP20187. In one embodiment, in step c), cell death of inducible substrate cells is induced by 10 nM AP20187.

[0079] As described herein, changes in the fluorescence signal of the pH-sensitive fluorophore indicate phagocytosis of the inducible substrate cell, while the pH-stable fluorophore enables localization of the cell. The fluorescence signal of the inducible substrate cell can be detected by fluorescence imaging, flow cytometry, or a fluorescence plate reader.

[0080] The fluorescence signal of the pH stability fluorophore and / or pH sensitivity fluorophore of substrate cells can be detected at any time point of the in vitro method described herein. Fluorescence signal can be detected before and after the cell death of the induced substrate cells. In addition, fluorescence signal can be detected at several time points during the phagocytosis assay. Thus the time course study of phagocytosis can be carried out. In one embodiment, the fluorescence signal of the pH stability fluorophore and / or pH sensitivity fluorophore is detected before the cell death of the induced substrate cells. In a further embodiment, the fluorescence signal of the pH stability fluorophore and / or pH sensitivity fluorophore is detected at several time points after the cell death of the induced substrate cells. In one embodiment, the fluorescence signal of the pH stability fluorophore and / or pH sensitivity fluorophore is detected every 5 minutes, every 10 minutes, every 15 minutes, every 20 minutes or every 30 minutes after the induction of cell death. In a further embodiment, the fluorescence signal of the pH stability fluorophore and / or pH sensitivity fluorophore is detected after about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours and / or about 8 hours after the induction of cell death.

[0081] Examples

[0082] The iCaspase9-SEP-mCherry construct (SEQ ID NO: 1; Figure 1) were transduced into the cell lines H4 (neuroblastoma) and Jurkat (T lymphocytes), and HEK293 were transfected with iCaspase9 constructs. H4 and Jurkat clones with stable expression of the constructs were selected. Cell lines HMC3 (microglia) or iPSC-derived macrophages or microglia (produced in-house as described in WO2020 / 239714A1) were used as phagocytic cells.

[0083] Example 1: Efficient on-demand generation of apoptotic cells.

[0084] HEK293 cells were transfected with 2 μg of Caspase9 construct by nuclear transfection (Amaxa 4D nuclear transfection instrument from Lonza, program CM-130) and seeded in 24-well plates. The next day, 100 nM AP20187 was added to the cells and analyzed after 1 h, 2 h, or 3 h. As a control, the cells were incubated with 2.5 μM staurosporine for 3 h. For analysis, cells were harvested and labeled with annexin V-BV421 and the viability dye APCeF780 and analyzed by flow cytometry. Apoptotic cells were judged as annexin V positive and viability dye negative.

[0085] HEK293 cells transfected with iCaspase9 undergo rapid and sustained apoptosis after treatment with 100 nM AP20187. After 1 h, 88% of iCaspase9-transduced cells were apoptotic, compared to 36% apoptotic cells after 3 h of staurosporine treatment ( Figure 2 ). Untransfected cells showed basal apoptosis levels between 10%-35%.

[0086] In conclusion, induction of apoptosis with AP20187 in cells expressing iCaspase9 is faster and more efficient than the standard method of inducing apoptosis with staurosporine.

[0087] Example 2: Accurate quantification of phagocytosis with increased dynamic range.

[0088] 2.1. pH-sensitive dual fluorescence indicators

[0089] H4 cells were transduced with a lentiviral vector containing iCaspase-SEP-mCherry and expanded in medium containing 1 μg / ml puromycin to select transduced cells. Cells were placed in different pH solutions (Intracellular pH Calibration Buffer Kit, Invitrogen) and imaged on an inverted fluorescence microscope (Leica).

[0090] At a physiological pH of 7.5, mCherry and SEP were co-expressed and co-localized in transduced cells ( Figure 3The presence of mCherry fluorescence is independent of pH ( Figure 3 , lower figure), while the SEP fluorescence signal is strong at physiological pH 7.4, but is quenched under acidic conditions ( Figure 3 , above).

[0091] H4-iCaspase-SEP-mCherry expresses mCherry constitutively fused to SEP and exhibits quenching of the SEP fluorophore in acidic environments. Therefore, this construct is a suitable fluorescent pH sensor.

[0092] 2.2. Evaluation of microglial phagocytosis of H4 cells in a 2D model

[0093] H4 cells were transduced with a lentiviral vector containing iCaspase-SEP-mCherry, single cell sorting was performed for SEP and mCherry double positive cells, and amplified in a medium containing 1 μg / ml puromycin to select stably transduced cells. HMC3 or iPSC-derived microglia (Reich, M et al. Alzheimer's Risk GeneTREM2Determines Functional Properties of New Type of Human iPSC-DerivedMicroglia.2021; 11:617860) were seeded in a 96-well imaging plate, and. In order to visualize the morphology of microglia during the phagocytosis assay, cells were stained with far-red viability dye (eBioscience). H4-iCaspase-SEP-mCherry cells were added the next day and left to attach for 3 hours. For the control wells, 1 μM cytochalasin D (a fungal toxin that binds to actin filaments and inhibits actin polymerization / phagocytosis) was added 30 minutes before the start of time-lapse imaging. At t0, apoptosis was induced by adding 10 nM AP20187 to the culture medium and time-lapse imaging was immediately started on an inverted fluorescence microscope (Leica) or incucyte (Sartorius) with a 10x objective and 1 image every 10 min for 8 h or longer.

[0094] Apoptotic H4-iCaspase-SEP-mCherry was actively engulfed by co-cultured HMC3 microglia ( Figure 4 ). The mCherry signal indicates transduced H4 cells, and the SEP signal allows determination of the precise time point of engulfment by microglia (loss of the SEP signal indicates phagocytosis). Figure 4The highlighted examples show co-localization of microglia (arrows) with apoptotic H4 cells at 2h40 after the onset of apoptosis (upper row), and phagocytosis of H4 cells as evidenced by loss of SEP signal at 3h10 after the onset of apoptosis (lower row).

[0095] Figure 5 Phagocytosis of apoptotic H4-iCaspase-SEP-mCherry by iPSC-derived microglia is shown. The number of engulfed cells per region was determined by automated image quantification using incucyte software by the following steps: 1) segmenting individual cells, 2) identifying all H4-iCaspase-SEP-mCherry cells that were positive for mCherry (red fluorescence), and 3) identifying cells that were negative for SEP (green fluorescence) from these cells as engulfed cells. Apoptotic cells were engulfed by iPSC-derived microglia over a 12 hour period until a plateau was reached. Addition of cytochalasin D reduced the onset and total amount of engulfed apoptotic cells, and no phagocytosis was observed without inducing apoptosis ( Figure 5 ).

[0096] The rapid and efficient induction of apoptosis results in rapid phagocytosis that can be precisely and reproducibly quantified.

[0097] 2.3. Evaluation of macrophage efferocytosis of Jurkat cells in a 2D model

[0098] Jurkat cells were transduced with a lentiviral vector containing iCaspase-SEP-mCherry, sorted by FACS for SEP and mCherry expression, and expanded in medium containing 1 μg / ml puromycin to select stably transduced cells. For pretreatment, Jurkat cells were incubated with GAS6 (an efferocytosis enhancer) or cytochalasin D (an efferocytosis inhibitor), then exposed to 10 nM AP20187 and incubated with THP-1 macrophages. As a control, Jurkat cells were treated with staurosporine and labeled with pHrodo (a red pH-sensitive fluorophore). Using flow cytometry, efferocytosis was quantified by gating on mCherry-positive and SEP-negative cells (apoptotic cells taken up by THP1) and pHrodo-high cells in control samples.

[0099] When using the iCaspase9-SEP-mCherry construct, the range between the highest uptake (GAS6 pretreatment) and lowest uptake (cytochalasin D pretreatment) of apoptotic cells by macrophages was 2-3 fold higher than that of staurosporine-treated and pHrodo-labeled Jurkat cells ( Figure 6 ). This increased range may be caused by a decrease in fluorescence background and / or an increase in efferocytosis.

[0100] Compared to traditional substrates, iCaspase-SEP-mCherry transduced apoptotic cells induce stronger phagocytosis / efferocytosis, providing a higher dynamic range. This allows for better sorting of different regulators of phagocytosis compared to traditional methods, increasing the value of this assay.

[0101] Example 3: Application of a novel phagocytosis assay in a complex 3D co-culture model

[0102] 3.1. Use of novel phagocytosis assay in 3D BBB spheroids

[0103] H4 cells were transduced with a lentiviral vector containing iCaspase-SEP-mCherry, single cells were sorted for SEP and mCherry double positive cells, and expanded in medium containing 1 μg / ml puromycin to select stably transduced cells. H4-iCaspase-SEP-mCherry and HMC3 microglia were co-inoculated with primary human brain endothelial cells, pericytes and astrocytes (Sciencell) to form blood-brain barrier (BBB) ​​spheroids, as described by Simonneau, C et al. Investigating receptor-mediated antibody transcytosis using blood-brainbarrier organoid arrays. Fluids Barriers CNS. 2021; 18(1): 43. In brief, cells were combined and seeded into glass-bottom GRI3D microplates (SUN Biosciences) at a ratio of 1:1:1:1:1. After culturing for 2 days in EGM2 medium (Lonza) with astrocyte growth supplement (Sciencell), time-lapse experiments were performed as described above (for Figure 4 ): At t0, apoptosis was induced by adding 100 nM AP20187 to the culture medium, and time-lapse imaging was immediately started on an inverted fluorescence microscope (Leica) using autofocus, 10x objective, and 1 image every 10 min for 8 h or longer.

[0104] Spheroids containing H4-iCaspase-SEP-mCherry gradually lost SEP signals after AP20187 treatment, indicating active phagocytosis within the spheroid core ( Figure 7 ). In untreated spheroids, SEP and mCherry fluorescence remained stable over 8 h.

[0105] iCaspase9-SEP-mCherry transduced H4 cells were successfully integrated into the high-throughput 3D in vitro model. After 2 days, apoptosis and subsequent phagocytosis were triggered after the 3D structure of the complex cell model was established. This assay cannot be compared with other phagocytosis assays because in other known assays, substrate generation (apoptotic cells) cannot be triggered in a cell-specific manner after cell seeding.

[0106] 3.2. Use of the novel phagocytosis assay in the 3D neurovascular unit model.

[0107] H4 cells were transduced with a lentiviral vector containing iCaspase-SEP-mCherry, single cell sorting was performed for SEP and mCherry double positive cells, and amplified in a medium containing 1 μg / ml puromycin to select stably transduced cells. H4-iCaspase-SEP-mCherry and HMC3 microglia were co-inoculated with primary human brain endothelial cells, pericytes and astrocytes (Sciencell) at a ratio of 1:1:4:0.5:1, and self-assembled into neurovascular units in the fibrin gel matrix of a microfluidic chip (AIM Biotech). Cells were seeded into microfluidic chips as described in Campisi, M et al. 3D Self-Organized Human Blood-Brain Barrier in a Microfluidic Chip. Methods Mol Biol. 2021; 2258: 205-219, and vascular networks were grown in EGM2 medium (Lonza) with astrocyte growth supplement (Sciencell) for more than 7 days. At t0, apoptosis was induced by adding 10 nM AP20187 to the culture medium, and then after 30 min or 2 h the chips were fixed in 4% PFA, counterstained with DAPI and imaged on an inverted fluorescence microscope.

[0108] After 7 days in culture, H4-iCaspase-SEP-mCherry cells surrounded vascular structures. Within the 3D matrix of the microfluidic chip, apoptosis of transduced cells was efficiently induced within 30 minutes. Single-cell resolution in this setting allowed identification of single instances of SEP signal loss in apoptotic cells after 2 hours ( Figure 8 ), indicating local acidification due to phagocytosis.

[0109] iCaspase9-SEP-mCherry transduced H4 cells were successfully integrated into the complex 3D in vitro model. Apoptosis and subsequent phagocytosis were triggered after 7 days of culture. Single phagocytosis events were observed. The system will further allow the colocalization of phagocytosis events to single microglia. Other phagocytosis assays do not have the capability to trigger substrate generation (apoptotic cells) on demand and in a cell-specific manner, so we cannot compare our results with other phagocytosis assays.

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Claims

1. A recombinant expression vector encoding an inducible cell death switch, a pH-stable fluorophore and a pH-sensitive fluorophore.

2. The recombinant expression vector according to claim 1, wherein the recombinant expression vector is a viral vector. The recombinant expression vector according to claim 2 , wherein the viral vector is a lentiviral vector.

4. The recombinant expression vector according to any one of the preceding claims, wherein the inducible cell death switch induces apoptosis, necroptosis, pyroptosis or ferroptosis, preferably apoptosis.

5. The recombinant expression vector according to any one of the preceding claims, wherein the inducible cell death switch comprises an inducer binding domain and a signaling protein of a cell death pathway. 6 . The recombinant expression vector according to claim 5 , wherein the signal transduction protein of the cell death pathway is a pro-apoptotic protein, preferably caspase 9, or a functional fragment thereof.

7. The recombinant expression vector according to any one of claims 5 or 6, wherein the inducer binding domain comprises a dimerization domain, preferably FKBP12-F36V.

8. The recombinant expression vector according to any one of claims 5 to 7, wherein the inducer binding domain is capable of binding an inducer, preferably a chemical dimerization inducer, most preferably AP20187.

9. The recombinant expression vector according to any one of the preceding claims, wherein the inducible cell death switch is inducible caspase 9.

10. The recombinant expression vector of any one of the preceding claims, wherein the pH-stable fluorophore and the pH-sensitive fluorophore have different excitation and emission spectra.

11. The recombinant expression vector according to any one of the preceding claims, wherein the pH-stable fluorophore is from the RFP family, Alexa Fluor dyes, protein-based fluorophores, simple organic fluorophores or organic polymers, preferably mCherry.

12. The recombinant expression vector according to any one of the preceding claims, wherein the fluorescence signal of the pH-sensitive fluorophore changes in response to a decrease in pH, preferably wherein the fluorescence signal is quenched or the excitation / emission spectrum is shifted.

13. The recombinant expression vector according to any one of the preceding claims, wherein the pH-sensitive fluorophore is Superecliptic pHluorin (SEP), pHLemon, pHmScarlet, pHTomato, pHuji, LysoSensor or a pH nanosensor, preferably Superecliptic pHluorin (SEP).

14. The recombinant expression vector according to any one of the preceding claims, wherein the recombinant expression vector comprises a promoter, in particular a ubiquitous promoter, a cell-specific promoter, a constitutive promoter or an inducible promoter.

15. The recombinant expression vector according to any one of the preceding claims, wherein the recombinant expression vector comprises a CMV promoter.

16. The recombinant expression vector according to any one of the preceding claims, wherein the recombinant expression vector comprises a polynucleotide sequence according to SEQ ID NO:

1.

17. A cell comprising the recombinant expression vector according to any one of the preceding claims.

18. The cell according to claim 17, wherein the cell is a mammalian cell, in particular a human cell.

19. The cell according to claim 17 or 18, wherein the cell is a stem cell.

20. The cell according to claim 17 or 18, wherein the cell is a neural cell, a neuronal cell, a glial cell, a mesenchymal cell or a hematopoietic cell, preferably a glioma cell or a T cell.

21. The cell according to any one of claims 17, 18 or 20, wherein the cell is an H4 cell or a Jurkat cell.

22. The cell according to any one of claims 17 to 21, wherein expression of the recombinant expression vector in the cell is transient or stable.

23. An in vitro method for assessing phagocytosis, the in vitro method comprising the steps of: a) providing an inducible substrate cell according to any one of claims 17 to 22; b) combining and co-culturing the inducible substrate cells with phagocytes; c) inducing cell death of said inducible substrate cells; and d) detecting the fluorescence signal of the inducible substrate cells; A change in the fluorescence signal of the pH-sensitive fluorophore indicates cellular phagocytosis of the inducible substrate.

24. The method of claim 23, wherein the phagocytic cells of step b) are macrophages or tissue-resident macrophages.

25. The method of claim 24, wherein the tissue-resident macrophages are microglia.

26. The method according to any one of claims 23 to 25, wherein the inducible substrate cells and the phagocytic cells are co-cultured in an in vitro model comprising an additional cell type.

27. The method of claim 26, wherein the in vitro model is a 2D or 3D culture system.

28. The method of claim 26 or 27, wherein the in vitro model is an organ chip, a spheroid or an organoid.

29. The method according to any one of claims 26 to 28, wherein the in vitro model is a neurovascular unit or a blood-brain barrier spheroid.

30. The method of any one of claims 23 to 29, wherein after combining the inducible substrate cells with the phagocytic cells in step b), cell death of the inducible substrate cells is induced in step c).

31. The method according to any one of claims 23 to 30, wherein in step c) cell death of the inducible substrate cells is induced by an inducer, preferably by a chemical dimerization inducer, most preferably by AP20187.

32. The method according to any one of claims 23 to 31, wherein the fluorescent signal of step d) is detected by fluorescence imaging, flow cytometry or by a fluorescence plate reader.

33. The method according to any one of claims 23 to 32, wherein the fluorescent signal in step d) is detected at several time points after induction of cell death.

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