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WO2025099670A1 - Procédé de test d'activité in vitro sur des tumeurs solides de cellules et/ou d'agents actifs - Google Patents

Procédé de test d'activité in vitro sur des tumeurs solides de cellules et/ou d'agents actifs Download PDF

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Publication number
WO2025099670A1
WO2025099670A1 PCT/IB2024/061105 IB2024061105W WO2025099670A1 WO 2025099670 A1 WO2025099670 A1 WO 2025099670A1 IB 2024061105 W IB2024061105 W IB 2024061105W WO 2025099670 A1 WO2025099670 A1 WO 2025099670A1
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Prior art keywords
cells
target cells
microwells
microchannel
gel
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English (en)
Inventor
Massimo Bocchi
Andrea FAENZA
Alice BETTELLI
Alice MORELLINI
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Cellply SRL
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Cellply SRL
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system

Definitions

  • cytotoxicity and cytokine release assays that reveal interactions between immune cells and target cells. These tests can typically be performed for 4-72 hours.
  • Cytotoxicity assays used in cell therapy are based on the ability to keep the viability of the target cells in the long term. It is therefore critical to keep ideal conditions and minimize noise in the measurement due to the unwanted and unrelated death of target cells and at the same time ensure that the effector cells are kept in an environment where they can exert their cytotoxic activity against the target cells, with minimal alteration of the surrounding environment.
  • target cells are cells from solid tumours
  • the object of the present invention is a method for assays that evaluate the effect of an active, be it a molecule or a cell, on a target cell that is a cell that grows in adhesion, or that forms clusters or 3D structures.
  • Figure 1 Inverted open microwell system, perspective view (A), vertical section (B) and top view (C); detail of an embodiment of the inverted open microwell system, with two parallel and superimposed channels (D).
  • Figure 2 Embodiments of co-cultures in an inverted open microwell system, with two parallel and superimposed channels.
  • Liquid/ air A, comparative
  • liquid/ air + beads B, comparative
  • gel/ air C, comparative
  • liquid/ gel D, according to the invention
  • Figure 3 A, B, C Steps of the method for loading the inverted open microwell system, with two parallel and superimposed channels.
  • Figure 4 representative photographs of microwell solid tumor cell loading experiments. Melanoma cells in GelMA 6% (A), Glioblastoma cells in Ultimatrix 1:1 (B).
  • Figure 5 Vitality of target cells, in the presence or absence of effector cells. Representative photograph of microwells containing (solid line) or not containing (dashed line) effector cells (A); expression of PI marker over time measured in individual wells in the presence (solid line) or absence (dashed line) of effector cells (B); expression of cytoplasmic marker (CMAC) over time measured on individual target cells, cultured in the presence (solid line) or absence (dashed line) of effector cells (C).
  • A Representative photograph of microwells containing (solid line) or not containing (dashed line) effector cells
  • A expression of PI marker over time measured in individual wells in the presence (solid line) or absence (dashed line) of effector cells
  • B expression of cytoplasmic marker (CMAC) over time measured on individual target cells, cultured in the presence (solid line) or absence (dashed line) of effector cells (C).
  • CMAC cytoplasmic marker
  • Figure 6 representative photographs of microwells containing co-cultures of effector cells (T lymphocytes) and solid tumor target cells (A); expression of the percentage of dead target cells measured on two different microchannels containing the co-culture of effector and target cells (T cells) or only target cells (Control) (B); colour map representing a matrix of 720 microwells, where for each element of the matrix it is reported the percentage of dead target cells within the microwell on two different microchannels containing the co-culture of effector and target cells (T cells) or only target cells (Control) (C).
  • Figure 7 Embodiments of the lower channel 23 of the inverted open microwell system, with two parallel and superimposed channels, with liquid/ gel interface or gellike structure (detail).
  • A volume of the channel 23 entirely occupied by a sheet of already solidified elastomer integrated in the construction step in the device;
  • B volume of the channel 23 partially occupied by a plurality of sheets of already solidified elastomer inserted in the construction step, or, if shaped, in the assembly step;
  • C volume of the channel 23 partially occupied by a plurality of sheets of already solidified elastomer, and by an adhesive material.
  • Interference coupling means here a cooperation between two elements, whereby said two elements can be regarded as joined.
  • said two elements in this case a tip and a vertical channel
  • a fluid loaded into said tip and released into said vertical channel is forced to move within the channel, said interference coupling being such as to prevent the passage of fluid, i.e. said interference coupling is such as to seal the two elements together.
  • Connector means any tubular, cylindrical, more or less tapered, convergent or divergent element adapted to put in fluidic connection two compartments.
  • Re-perfusion means here the replacement of the medium in which the culture is found with a fresh medium.
  • Said fresh medium is the same or different from the medium in which said culture is already found.
  • said medium is culture medium.
  • it is culture medium comprising one or more drugs and/ or one or more dyes, and/or one or more labelled antibodies and/or one or more cell viability markers.
  • Fluid any substance in liquid or gaseous form.
  • “Gelling fluid” a liquid substance capable of giving rise to a solid or semi-fluid but elastic structure, for example a gel or a gel-like structure.
  • Bio sample sample comprising cells obtained from a microorganism, an animal and/or a human being, preferably human being, wherein said sample is preferably selected from the group comprising biological fluids or biopsies.
  • Said sample comprises suspended cells or is a tissue. In a preferred embodiment, it is a blood sample or a bone marrow aspirate.
  • said biological sample consists of cells in culture, such as a cell line, or a composition comprising cells in culture and cells from a patient.
  • Time-lapse imaging technique that provides for a series of shots of the same field made in a time sequence.
  • Ex vivo test performed on a tissue obtained from an organism in an environment outside the organism itself, with minimal alteration of natural conditions.
  • the object of the present invention is a method for determining the efficacy of a cell therapy or immunotherapy, wherein said method allows long-term assays, i.e. a culture 12 h, or 24 h, or 48 h, or 72 h long.
  • the method is performed in the open microwell microfluidic device described in WO2017/216739.
  • the method is performed in microwells with a volume up to 35 nl, preferably up to 31.8 nl, or up to 10 nl. In one embodiment, the method is performed in microwells having a volume up to 2.6 nl, preferably up to 0.5 nl.
  • said microwells have a diameter of 300 gm, or 250 gm, or 200 pm, 150 jim, or 100 jim, 70 jim, or less.
  • said microwells are 450 im high, or 150 im, or have a height of less than 100 im.
  • the size of the microwells is such as to contain a number of target cells greater than 5 or 10 and to ensure that the cells have a behaviour and morphology similar to those observed in normal cell culture substrates, also allowing the use of polarized cells, such as epithelial cells, hepatocytes, neurons and endothelial cells that normally require adequate space to be able to expand following polarization.
  • polarized cells such as epithelial cells, hepatocytes, neurons and endothelial cells that normally require adequate space to be able to expand following polarization.
  • the method claimed herein is performed by having a kit comprising a tip and a microfluidic device (1), Figure 1, comprising at least one microchannel (3) and an inlet region (8) comprising at least one vertical channel (18), said tip and said vertical channel (18) being dimensioned so as to produce an interference coupling between them.
  • said microfluidic device comprises a first microchannel (23) which is in communication with the external environment via an inlet region (28). Said device also comprises a second microchannel (24), which is in communication with the external environment via an inlet region (29). Said first microchannel (23) and said second microchannel (24) are parallel to each other, and are arranged one above the other. Inverted open microwells (22) are obtained in the layer separating said first from said second microchannel, said inverted open microwells putting in communication said first with said second microchannel.
  • said device is partially made of glass.
  • said layer separating said first from said second microchannel, in which the open microwells are obtained is made of polyimide.
  • Said tip is chosen from one of the commercially available tips comprising at least a proximal portion intended to cooperate with a fluid delivery system and an open tapered distal portion.
  • said distal portion of said tip and said vertical channel (18) are made of plastic and make the system sufficiently elastic to ensure the seal, avoiding gaskets.
  • the geometries of the system described below ensure that the contact between said vertical channel (18) and said tip does not take place at a single point but is distributed over a surface portion, also ensuring an effective seal. This condition is advantageously verified where the half-opening angle of said terminal portion of said tip and of said vertical channel (18) are slightly different, preferably less than 10°.
  • said vertical channel (18) is a cylinder, possibly slightly tapered downwards.
  • the inverted open microwell system with two parallel and superimposed microchannels it is possible to bring through the same culture media, or media containing cells, actives, or markers in said microwells.
  • High density target cells (100) were seeded in each of the microwells comprising culture medium (102). Effector cells (103) were then added to the culture, as schematized in Figure 2A. None was loaded into the microchannel below, so that a liquid/air interface was created at the base of the open microwell. Since the target cells are cells that grow in adhesion, they have shown the tendency to adhere to the walls of the microwell, thus being no longer visible to subsequent microscopic observations.
  • Effector cells (103) and target cells (100) were then seeded, Figure 2C, in an inverted open microwell suspended in a gelling fluid (101), and not in a liquid culture medium.
  • a gelling fluid 101
  • the ability of the cells to grow correctly at the air/ gel interface was shown, but the solution excluded the possibility of proceeding with medium change, washing, reperfusions.
  • said first channel (23) is loaded with a gelling fluid (101) that fills said first microchannel and, partially, said open microwells (Figure 3A).
  • said second microchannel is filled with a culture medium in which the cells of interest are resuspended ( Figure 3C).
  • said gelling fluid (101) is a viscoelastic substance that forms a stable three-dimensional mesh of polymers capable of retaining a fluid, for example it is 6% GelMA (methacrylated gelatin).
  • Said gelling fluid has a structure consisting of a three- dimensional mesh of polymers at 4°C and becomes liquid upon heating it. It is therefore loaded in liquid form to then solidify by cross-linking.
  • nanoclays which have good colloidal stability, are biocompatible and biodegradable, are added to said GelMA.
  • said nanoclays allow the gelation of the methacrylated gelatin following exposure to UV.
  • said nanoclays are synthetic nanoclays, for example Laponite (LAP).
  • said gelling fluid is added with laminin and/or collagen IV and/ or entactin and/ or heparan sulfate protoglycan or the like, e.g. it is Ultimatrix, R&D Systems.
  • said gelling fluid is added with nanofibres, e.g. it is Puramatrix, Coming, to facilitate cell adhesion to the gel-liquid interface surface.
  • said gelling fluid (101) is an elastomer, e.g. PDMS, e.g. Silgard 184.
  • said passage from gelling fluid to gel-like solid structure takes place by curing, increasing the temperature.
  • substances are flowed for coating said PDMS, which coating is necessary to allow the cells to adhere.
  • Said substances are for example selected from fibronectin, collagen, poly dopamine, laminin, poly- D-lysine, poly-L-lysine.
  • said gelling fluid (101) is a SEBS (styrene-ethylene-butylene- styrene), e.g. Flexdym.
  • SEBS styrene-ethylene-butylene- styrene
  • Flexdym styrene-ethylene-butylene- styrene
  • said two parallel and superimposed microchannels consist of:
  • a lower channel (23) the volume of which is at least partially occupied by a gel, or by a gel-like structure (110, 111, 112), wherein said gel or gel-like structure occupies at least the volume below each inverted open microwell (22).
  • said gel-like structure is an elastomer.
  • said elastomer is positioned as sheet or plurality of sheets (110) already hardened during the construction of the device.
  • the device is ready for use, without the need for a step of loading the gelling fluid and subsequent curing or cross-linking.
  • the fluidic seal is guaranteed by the same elastomer that adheres to the upper and lower layers of the device itself.
  • said elastomer is positioned as a hardened sheet or a plurality of sheets already hardened and suitably shaped during the assembly of the device, so that it occupies the volume of the lower channel. Also in this embodiment, the device is ready for use, without the need for a step of loading the gelling fluid and subsequent curing or cross-linking. The fluidic seal is guaranteed by the same elastomer that adheres to the upper and lower layers of the device itself.
  • the fluidic seal is guaranteed by making said sheets with a height greater than the height of the channel so that, exploiting the compressibility of the elastomer itself, the seal is guaranteed.
  • This embodiment is schematically represented in Figure 7C.
  • adhesion is favoured by surface treatments, such as plasma treatments.
  • the person skilled in the art is able to select the embodiment that best suits the purpose.
  • the most suitable embodiment is one comprising a gel, or an elastomer, not treated with coating.
  • the most suitable embodiment comprises an elastomer, treated with coating.
  • the method according to the present invention thus comprises:
  • an inverted open microwell system (1) comprising a first microchannel (23) and a second microchannel (24), in communication with the external environment via an inlet region (29).
  • Said first microchannel (23) and said second microchannel (24) are parallel to each other, and are arranged one above the other.
  • Inverted open microwells (22) are obtained in the layer separating said first from said second microchannel, said inverted open microwells putting in communication said first with said second microchannel.
  • the volume of said first channel (23) is occupied, at least in the portions below each inverted open microwell (22) by a gel or a gel-like structure (110, 111, 112);
  • an automated management system of said inverted open microwell system that comprises the following features: temperature, humidity and CO2 controlled incubator, fluid delivery system, phase contrast and fluorescence image acquisition;
  • reagents comprise: at least one effector cell population, filling buffer and/or washing solution and/or one or more drugs and/ or one or more dyes, and/ or one or more labelled antibodies and/or one or more cell viability markers;
  • said method further comprises:
  • said method further comprises:
  • said first microchannel is in communication with the external environment via an inlet region (28)
  • At least one active e.g., a molecule having pharmacological activity, is also loaded.
  • said effector cells are KG-1, a human macrophage cell line.
  • said effector cells are K562, human lymphoblast cell line.
  • said effector cells are NK lymphocytes.
  • said effector cells are T lymphocytes.
  • said T or NK cells are genetically modified.
  • Said effector cells are co-cultured in the presence of target cells.
  • Said target cells are primary cells obtained from a subject, or are a cell line.
  • said target cells express the antigen recognized by said effector cells.
  • said target cells are selected from the group comprising: pancreatic cancer cells, e.g. MIA PaCa-2, or BXPC-3, melanoma cells, glioblastoma cells, ovarian cancer cells, e.g. SK-OV-3.
  • test and the time-lapse are performed for a maximum of 72 hours.
  • said marker is used for cell detection and is 7-amino-4- chloromethylcoumarin (CellTrackerTM Blue CMAC).
  • said marker is used to assess cell death, and is propidium iodide (PI).
  • said marker is Calcein AM.
  • said method provides for determining the number of effector cells and target cells present within each microwell, analysing the cytotoxic effect in subgroups of microwells having a same number of effector and target cells, i.e. a same ratio between the number of effector and target cells, and then determining a doseresponse curve that reports the viability of the target cells as the ratio between the number of effector and target cells varies.
  • said method provides for first loading the cell population comprising effector cells (or target cells) into the microwells, acquiring images of the array of microwells, and then loading the cell population comprising target cells (or effector cells).
  • said method provides for analysing only the subgroup of microwells containing an effector cell and determining the average number of target cells killed in said subgroup.
  • said method provides for determining the percentage of microwells within said subgroup in which the number of target cells killed is higher than 1 or is higher than 2, allowing the percentage of so-called "serial killer” effector cells, i.e. capable of killing more than one target cell, to be determined accordingly.
  • the gels used were 6% GelMA or 1:1 Ultimatrix.
  • FIG. 4B reports exemplary photos of some of the inverted open microwells of this experiment. The photos were acquired at the indicated times after seeding with an Olympus 1X83 inverted fluorescence microscope, magnification lOx.
  • the cells are observable under the microscope and it is therefore possible to carry out biological assays on said cells, including cytotoxicity assays by co-culture with immune cells.
  • C12 GBM cells were seeded in the second microchannel in culture medium that is FBS.
  • the effector cells were pre-labelled with Calcein AM, the target cells with CMAC.
  • the fluid was replaced so as to expose the cells to a culture medium solution containing Propidium Iodide, so as to highlight dead cells.
  • Quantification of the effector-target interaction can be observed by measuring the total fluorescence in the microwell.
  • the PI intensity remains constant in the microwells without effector cells, while it increases in the microwells with an effector cell, indicating how the presence of effector cells is able to kill the target cells and how the presence of even a single immune cell produces a detectable effect, thus making the technology suitable for observing the activity of individual immune cells.
  • the intensity of the cytoplasmic marker on the target cells is equivalent in the two groups, which means that the average number of target cells is similar.
  • panel A gathers some representative images of the microwells acquired with the Olympus 1X83 fluorescence microscope
  • panel B reports in the graph the average intensity measured in each well of the PI marker over time
  • panel C the intensity of the marker for the target cells in the presence (solid line) or in the absence (dashed line) of effector cells.
  • Example 3 quantification of cytotoxic activity on a large scale
  • melanoma target cells were seeded in a first channel and in a second channel a co-culture of melanoma target cells and T lymphocytes suitably engineered to recognize antigens present on the target cells.
  • the target cells were labelled with CMAC.
  • the fluid was replaced so as to expose the cells to a culture medium solution containing Propidium Iodide, so as to highlight dead cells. Images were subsequently acquired in time-lapse for 24h. For each time instant the quantification of the effector-target interaction can be observed by measuring the fraction of cells exhibiting both a blue and red colour, i.e.
  • FIG. 6A reports a map of 720 microwells for each channel, indicating the percentage of dead target cells for each well. The map shows the potential of the proposed method to observe biological heterogeneity within different co-cultures, presenting both microwells in which all target cells are dead, microwells in which no target cells are dead, and microwells where only a fraction of target cells are dead.

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Abstract

L'objet de la présente invention est un procédé in vitro permettant de déterminer l'efficacité d'une thérapie cellulaire ou d'une immunothérapie, ledit procédé consistant à : - utiliser un système microfluidique comprenant deux microcanaux parallèles, mis en communication l'un avec l'autre par une série de micropuits ouverts inversés s'ouvrant sur lesdits deux microcanaux, le volume d'un premier canal étant occupé au moins en partie par un gel ou une structure de type gel ; - introduire, dans un second microcanal, un milieu de culture et au moins une population de cellules qui est une population de cellules cibles mises en croissance en adhérence ou en grappes ; - maintenir lesdites cellules cultivées en culture pendant au moins 12 h, ou 24 h, ou 48 h, ou 72 h ; - mesurer les paramètres d'intérêt.
PCT/IB2024/061105 2023-11-08 2024-11-08 Procédé de test d'activité in vitro sur des tumeurs solides de cellules et/ou d'agents actifs Pending WO2025099670A1 (fr)

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IT102023000023574A IT202300023574A1 (it) 2023-11-08 2023-11-08 Metodo per saggiare l’attività in vitro su tumori solidi di cellule e/o attivi
IT102023000023574 2023-11-08

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017216739A1 (fr) * 2016-06-14 2017-12-21 Cellply S.R.L. Kit et procédé de criblage
US20180172666A1 (en) * 2015-03-11 2018-06-21 Sogang University Research Foundation Hydrogel-based microfluidic chip for co-culturing cells
WO2020252225A1 (fr) * 2019-06-14 2020-12-17 University Of Connecticut Système de tumeur sur puce multigel

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180172666A1 (en) * 2015-03-11 2018-06-21 Sogang University Research Foundation Hydrogel-based microfluidic chip for co-culturing cells
WO2017216739A1 (fr) * 2016-06-14 2017-12-21 Cellply S.R.L. Kit et procédé de criblage
WO2020252225A1 (fr) * 2019-06-14 2020-12-17 University Of Connecticut Système de tumeur sur puce multigel

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
NASIM ANNABI ET AL: "Hydrogel-coated microfluidic channels for cardiomyocyte culture", LAB ON A CHIP, vol. 13, 1 January 2013 (2013-01-01), UK, pages 3569 - 3577, XP055321101, ISSN: 1473-0197, DOI: 10.1039/c3lc50252j *

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