WO2024100056A1 - Dispositif microfluidique - Google Patents

Dispositif microfluidique Download PDF

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Publication number
WO2024100056A1
WO2024100056A1 PCT/EP2023/081023 EP2023081023W WO2024100056A1 WO 2024100056 A1 WO2024100056 A1 WO 2024100056A1 EP 2023081023 W EP2023081023 W EP 2023081023W WO 2024100056 A1 WO2024100056 A1 WO 2024100056A1
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Prior art keywords
cells
carbohydrate
coating
interest
binding protein
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PCT/EP2023/081023
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English (en)
Inventor
Rui Pedro Rijo da Costa Carvalho
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Lumicks Ca Holding B.V.
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Publication of WO2024100056A1 publication Critical patent/WO2024100056A1/fr

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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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/415—Assays involving biological materials from specific organisms or of a specific nature from plants
    • G01N2333/42—Lectins, e.g. concanavalin, phytohaemagglutinin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4724—Lectins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates

Definitions

  • the invention relates to devices suitable for measuring biological processes, for example for measuring cellular avidity.
  • the invention also relates to processes for making and using the devices.
  • Microfluidics is an area of research that addresses and exploits the fluid dynamics at millimeter-to-submillimeter scale.
  • the emerging and rapid development of microfluidic technology has presented an ideal solution for the problem of mimicking an in vivo like microenvironment. This has led to microfluidics-based setups being developed for a large range of applications in almost all scientific disciplines including chemistry, biochemistry and biophysics.
  • Microfluidics have also been used in cell biology applications.
  • One of the benefits of microfluidic devices is the possibility of integrating analytical methods to produce information from the cell models.
  • Cell avidity evaluates the overall strength of cellular interactions by defining the total intercellular force between multiple parallel interactions, including co-receptor binding, TCR clustering, cell adhesion proteins, and even orientations and valencies. Cell avidity therefore provides a complete and physiologically relevant picture reflecting the interaction between cells, for example target cells and effector cells.
  • a microfluidic device comprising a monolayer of target cells.
  • Robust and confluent cell monolayers are crucial to be able to do reliable and accurate cell avidity experiments.
  • production of said monolayers should be quick and reproducible, preferably using devices that have a good storability.
  • the terms “a,” “an,” “the,” and “said” means one or more.
  • the words “a,” “an,” “the,” or “said” may mean one or more than one.
  • another may mean at least a second or more.
  • the present application describes a process for preparing a microfluidic device comprising a coated surface, the process comprising the steps of:
  • the present application also describes a process for preparing a microfluidic device comprising a coated surface, the process comprising the steps of:
  • the present application describes a process for preparing a microfluidic device comprising a coated surface, the process comprising the steps of:
  • the present application also describes a process for preparing a microfluidic device comprising a coated surface, the process comprising the steps of:
  • the surface is activated before a coating is applied.
  • the surface is chemically activated, for example with sodium hydroxide. Activation can be done to facilitate application of a coating.
  • the processes as described herein can be stopped after the microfluidic device comprising a carbohydrate-coated surface have been provided.
  • An advantage of the present invention is that the microfluidic device comprising a carbohydrate-coated surface can be stored for long periods of time, because the carbohydrate-coated surfaces have excellent anti-fouling properties and do not lose their activation over time.
  • the processes as described herein can be resumed after the respective storage without loss of quality of the final end product, e.g. a microfluidic device comprising a coated surface or a microfluidic device comprising a surface comprising cells.
  • a further advantage of the processes as described herein is their speed. In processes for preparing a microfluidic device comprising a coated surface as described in the prior art, the coating process takes hours, while the processes for preparing a microfluidic device comprising a coated surface as described herein can be performed in 1 hour or less.
  • a surface on which a coating has been applied is known herein as a "coated surface.”
  • the term “single-coated surface” as used herein means a surface that comprises at least a coating layer comprising at least a carbohydrate. In an embodiment the single-coated surface comprises at least a carbohydrate as described herein.
  • the term “multiple-coated surface” as used herein means a surface that comprises at least two coating layers. In an embodiment the multiple-coated surface comprises a first coating layer comprising at least a carbohydrate and at least a second coating layer comprising at least a carbohydrate-binding protein.
  • the multiple-coated surface may comprise additional coating layers. Said additional coating layers may be below the first coating layer (/.e.
  • the surface may be coated multiple times with the first coating layer and/or multiple times with the second coating layer. In other words, the coating process can be repeated numerous times until the desired order of coating layers is achieved. In a preferred embodiment the surface does not comprise any additional coating layers next to the first coating layer and the second coating layer.
  • the first coating comprises more than one type of carbohydrate, i.e. a mixture of carbohydrates.
  • the second coating comprises more than one type of carbohydrate-binding protein, i.e. a mixture of carbohydrate-binding proteins.
  • the first coating comprises more than one type of carbohydrate and the second coating also comprises more than one type of carbohydrate-binding protein.
  • the cells form a monolayer on the coated surface.
  • the surface is a glass surface, a plastic surface, a metal surface, a ceramic surface or any combination thereof.
  • the surface is a glass surface, a plastic surface, a ceramic surface or any combination thereof.
  • the microfluidic device may be prepared from any suitable material including plastic, glass, ceramics, metal or any combination thereof.
  • the plastic is non-cytotoxic.
  • the plastic may include natural polymers and/or synthetic polymers.
  • the cell monolayer shows a high quality.
  • Quality can be measured for example by looking at the confluency of the cell monolayer.
  • the confluency should be 50% or higher, preferably 60% or higher, more preferably 70% or higher.
  • Quality can also be measured for example by looking at the dumpiness of the cell monolayer.
  • the cell monolayer does not show dumpiness.
  • Quality can also be measured for example by looking at the stability and/or robustness of the cell monolayer. Stability and/or robustness can be measured for example by measuring the resistance of the cell monolayer when subjected to shear flow force and/or acoustic force and/or centrifugal force.
  • the cells are cultured. Culturing of the cells may take place before, simultaneously with and/or after contacting the cells with the coated surface and allowing the cells to attach to the coated surface to provide a microfluidic device comprising a surface comprising cells.
  • the carbohydrate is selected from the group consisting of mannose, fucose, xylose, arabinose, acetylglucosamine, fructose, sialic acid, glucuronic acid, galactose, N-acetylgalactosamine, any derivative and mixture thereof.
  • the carbohydrate comprised in the first coating is selected from the group consisting of mannose, fucose, xylose, arabinose, acetylglucosamine, fructose, sialic acid, glucuronic acid, galactose, N-acetylgalactosamine, any derivative and mixture thereof.
  • the carbohydrate is carbohydrate-R, wherein R is a functional group that is capable of reacting with an OH-group on the surface. In an embodiment the carbohydrate is carbohydrate-R, wherein R is a functional group that is capable of forming a bond with an OH-group on the surface. In an embodiment R is selected from the group consisting of phosphate, silane, quinone and catechol.
  • An example of a derivative of mannose and an example of a carbohydrate-R is mannose-6-R, wherein R is selected from the group consisting of phosphate, silane, quinone and catechol. So, in an embodiment the carbohydrate is mannose-6-R, wherein R is selected from the group consisting of phosphate, silane, quinone and catechol. In an embodiment the carbohydrate comprised in the first coating is mannose-6-R, wherein R is selected from the group consisting of phosphate, silane, quinone and catechol.
  • the carbohydrate-binding protein is a lectin.
  • the lectin is selected from the group consisting of concanavalin A (ConA), lentil lectins (LCH), snowdrop lectins (GNA), proteoglycans, type II transmembrane receptor lectins, collectins, selectins, natural killer lectins, macrophage mannose receptor (MMR) lectins, type I receptor lectins, tetranectins, polycystins, attractins, CTLD/acidic neck lectins, endosialins and any combination thereof.
  • one or more active domains of a carbohydrate-binding protein may also be used in any of the processes for preparing a microfluidic device as described herein.
  • An "active domain” refers to an amino acid sequence found within the carbohydrate-binding protein that, in itself, provides function according to one or more properties of the carbohydrate-binding protein, such as providing structural support to cells and/or for attaching cells and/or for attaching a carbohydrate.
  • the peptide that includes an active domain of a carbohydrate-binding protein can have a "core sequence" of amino acid residues, and optionally one or more additional amino acid residues that flank (/.e., on the C-terminus, N-terminus, or both) the core sequence.
  • the one or more additional amino acids that flank the core sequence can correspond to the wild-type sequence in the relevant region of the protein, or can comprise one or more amino acid(s) that diverge from the wild-type sequence (e.g., a "variant amino acid sequence").
  • the variant amino sequence can be one that enhances properties of the peptide, such as providing enhanced ligand interaction.
  • a "peptide” is a short polymer of 25 or less amino acids linked by peptide bonds.
  • a "polypeptide” is a polymer of more than 25 amino acids linked by peptide bonds and which includes full length proteins.
  • a peptide having an active portion of a carbohydrate-binding protein can be synthesized by solid phase peptide synthesis (SPPS) techniques using standard techniques, such as Fmoc synthesis.
  • SPPS solid phase peptide synthesis
  • the first coating layer comprising the carbohydrate may comprise other excipients and/or components as well.
  • the carbohydrate is present in the first coating layer in an amount of at least 10% (w/w) (weight percentage of the carbohydrate based on the total weight of the first coating layer comprising the carbohydrate), at least 20% (w/w), at least 30% (w/w), at least 40% (w/w), at least 50% (w/w), at least 60% (w/w), at least 70% (w/w), at least 80% (w/w), at least 90% (w/w) or at least 100% (w/w).
  • the second coating layer comprising the carbohydrate-binding protein may comprise other excipients and/or components as well.
  • the carbohydrate-binding protein is present in the second coating layer in an amount of at least 10% (w/w) (weight percentage of the carbohydrate-binding protein based on the total weight of the second coating layer comprising the carbohydrate-binding protein), at least 20% (w/w), at least 30% (w/w), at least 40% (w/w), at least 50% (w/w), at least 60% (w/w), at least 70% (w/w), at least 80% (w/w), at least 90% (w/w) or at least 100% (w/w).
  • the first (/.e. carbohydrate comprising) and second (/.e. carbohydrate-binding protein comprising) coating can be prepared to have a desired thickness.
  • the coated layers have a thickness in the range of about 2 nm to about 20 nm.
  • the first and second coating can be prepared to have a desired density and/or concentration.
  • the coating process involves placing the coating materials in contact with the device surface.
  • the device surface has been pretreated with a base coat.
  • the coating materials are applied to a surface and dried down or partially dried down.
  • the process of applying can be performed using any one of a variety of techniques including dipping, pouring, swabbing, siphoning, brushing, rolling, padding, ragging, painting, spraying, anodizing, electroplating, and/or laminating.
  • One exemplary method for applying a coating composition is by dip coating.
  • a typical dip coating procedure involves immersing the surface to be coated in a first coating composition, dwelling the object in the composition for a period of time, and then removing the surface from the composition.
  • Another exemplary method for applying a coating composition is flowing the coating composition into a microfluidic device in order to coat the inner surface(s) of the microfluidic device.
  • the processes as described herein also comprise the step of contacting the microfluidic device comprising a surface comprising cells with a blocking agent.
  • the blocking agent may reduce background binding to the cells.
  • suitable blocking agents include, but are not limited to, N-hydroxysuccinimide (NHS) ester reagents.
  • NHS ester reagent as defined herein is a compound comprising at least one succinimidyl group, which functional group is capable of forming an ester bond with a primary amine group on polypeptides, such as, for example, the amine groups as comprised in lysine residues, such that these amine groups are no longer available to attach to, for example, cells of interest.
  • the NHS ester reagent comprises a spacer, e.g. a polymer of a relative short length, up to about 100 Angstrom.
  • a polymer preferably is inert.
  • the polymer is polyethylene glycol (PEG). More preferably, the PEG length is up to 25 ethylene glycol units in length. Preferably, the number of units is selected from the range of 2 to 25, or from 3 to 10.
  • the NHS ester is a PEG-NHS ester.
  • Blocking may take place by providing a buffer suitable for the blocking reaction and sustaining cell viability of the cells present on the surface, e.g. phosphate, carbonatebicarbonate, HEPES or borate buffers at pH 7.2 to 8.5 for 0.5 to 4 h at room temperature or 4°C.
  • a buffer suitable for the blocking reaction e.g. phosphate, carbonatebicarbonate, HEPES or borate buffers at pH 7.2 to 8.5 for 0.5 to 4 h at room temperature or 4°C.
  • the NHS ester reagent is removed and the cells may, for example, be washed with serum containing medium or with a primary amine buffer such as Tris or glycine, which are not compatible because they compete for reaction, to quench (stop) the reaction.
  • NHS esters are known in the art, and it may be contemplated to utilize other compounds equally capable of reacting with amine groups as an alternative.
  • These compounds include, but are not limited to, isothiocyanates, isocyanates, acyl azides, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, carbodiimides, anhydrides, and fluorophenyl esters. Most of these compounds conjugate to amines by either acylation or alkylation. Without being bound by theory, as long as the compound is capable of reacting with the constituents responsible for the coating on the surface, it can be contemplated.
  • the cells are target cells or effector cells.
  • the cells are target cells.
  • the target cells are cells presenting an antigen.
  • a tumor antigen a viral antigen or a bacterial antigen.
  • Suitable target cells may include eukaryotic cells or prokaryotic cells.
  • Target cells that may be selected include fungal cells, animal cells, insect cells, mammalian cells, bacterial cells, yeasts, and protozoa.
  • the target cells are human cells.
  • the target cells are tumor cells such as human tumor cells.
  • the effector cells are human effector cells such as T cells, NK cells, B cells, dendritic cells, macrophages, or monocytes.
  • the effector cells may be genetically modified. When the genetic modification is with a CAR, cells such as CAR T cells can be obtained. Effector cells as used herein may thus also be CAR T cells.
  • extraneous DNA is removed from the cells before contacting the cells with the coated surface.
  • extraneous DNA is removed from the cells by treatment of the cells with a deoxyribonuclease (DNAse).
  • DNAse deoxyribonuclease
  • the present application also describes a process for determining cellular avidity comprising:
  • a microfluidic device comprising a surface comprising cells, wherein the surface comprises a coating comprising a carbohydrate, a coating comprising a carbohydrate-binding protein and cells attached to the coating comprising the carbohydrate-binding protein,
  • cellular avidity is defined by the overall strength of interactions occurring in a cell-to-cell contact, involving a diversity of molecules at the surfaces of the cells that interact.
  • cellular avidity is a parameter that expresses the strength of binding between cells. It involves a multitude of interactions, including a diversity of receptor-ligand pairs, among which e.g. a specific receptor-ligand interaction, occurring at the membrane surface of a cell, working jointly forming a strong bond between cells. It may also involve active signalling and processes internal to the cells such as e.g. during immune synapse formation. It is understood that the cellular avidity of a cell of a certain type is defined relative to its target cell and conditions tested.
  • target cells and cells of interest relate to two different cells which are to interact specifically with each other.
  • the target cells or the cells of interest may express a ligand on their surface and the other cell may express a receptor for that ligand.
  • control cells the same cells as the cells of interest or target cells may be utilized, but these cells do not express such a ligand or receptor or express a variant thereof which is not functional.
  • the cells attached to the coating comprising the carbohydrate-binding protein (e.g. target cells) and cells of interest (e.g. effector cells) (and control cells thereof)
  • this may involve cells that are to have a specific interaction, i.e.
  • ligand and receptor in this sense and in accordance with the invention may define their inter-relationship.
  • receptor may not be construed to be limiting in any way and is understood to mean a protein presented at the cell surface which can (specifically) interact with another protein (ligand) presented at another cell.
  • ligand and receptor are used to indicate a complementarity which is important for specific recognition between cells without restrictions on the complementary molecules that can be contemplated.
  • Receptors that may be of interest include, but are not limited to, a CAR, a TCR, a stimulatory or inhibitory co-receptor, or a receptor engaged via a bispecific antibody.
  • the cellular avidity may be determined. This is for example of interest when it is desirable to provide for a candidate CAR.
  • candidates can be selected and subsequently assessed with regard to functionality, e.g. by in vivo experiments.
  • cellular avidity is an important parameter for function, this way highly efficiently, suitable candidates can be selected.
  • the cellular avidity is determined.
  • the microfluidic device comprising a surface comprising cells is prepared by performing a process for preparing a microfluidic device comprising a surface comprising cells as described herein.
  • the cells are target cells or effector cells.
  • the cells of interest are target cells or effector cells.
  • the cells attached to the second coating i.e. the coating comprising the carbohydrate-binding protein
  • the cells of interest are effector cells.
  • the target cells are cells presenting an antigen.
  • a tumor antigen a viral antigen or a bacterial antigen.
  • Suitable target cells may include eukaryotic cells or prokaryotic cells.
  • Target cells that may be selected include fungal cells, animal cells, insect cells, mammalian cells, bacterial cells, yeasts, and protozoa.
  • the target cells are human cells.
  • the target cells are tumor cells such as human tumor cells.
  • the effector cells are human effector cells such as T cells, NK cells, B cells, dendritic cells, macrophages, or monocytes.
  • the effector cells may be genetically modified. When the genetic modification is with a CAR, cells such as CAR T cells can be obtained. Effector cells as used herein may thus also be CAR T cells.
  • the cells that are attached to the second coating are preferably attached as a monolayer.
  • the subsequent cells of interest (and control cells thereof) that are to interact with the cells attached to the coating on the surface are preferably provided in a relatively low cell density as compared with those cells, such that substantially all cells of interest (and control cells thereof) can interact with those cells. In other words, there are more cells per cell of interest. Such provides for advantageous controllable conditions when applying the force on the cells of interest or the control cells thereof.
  • control cells of the target cells or the cells of interest are provided and the cellular avidity of the control cells is determined as well.
  • control cells of the cells that are attached to the second coating and/or control cells of the cells of interest are provided and the cellular avidity of the control cells is determined as well.
  • the process for determining cellular avidity as described herein may be performed separately with cells of interest and their control cells and/or with cells that are attached to the second coating and their control cells.
  • the process for determining cellular avidity as described herein may comprise the step of incubating the cells attached to the second coating with the cells of interest and/or their control cells.
  • the process for determining cellular avidity as described herein may comprise the step of contacting cells of interest and/or their control cells with target cells. Control cells and/or cells of interest that have detached and/or remained attached (after force application as described herein) are determined and cellular avidity scores are provided for the control cells and/or cells of interest with the cells that are attached to the second coating.
  • control cells of the cells of interest alternatively control cells of the cells that are attached to the second coating may be provided attached to a surface.
  • cells of interest are contacted with the cells that are attached to the second coating and before or thereafter cells of interest are contacted with the control cells of the cells that are attached to the second coating, and in each case cells of interest that have detached and/or remained attached are determined and cellular avidity scores are provided for the cells of interest with the control cells of the cells that are attached to the second coating and with the cells that are attached to the second coating as such.
  • the cellular avidity scores obtained for cells and their respective controls can be compared and are of interest. Measuring the cellular avidity with and without control cells allows for better separate specific from non-specific binding.
  • control cells when cells of interest are used that express a specific ligand or receptor, control cells are used that are the same cells as the cells of interest but do not express the specific ligand or receptor or express a variant of the specific ligand or receptor which is not functional or not specific for the counterpart present on the cells that are attached to the second coating, e.g. the target cells. Obviously, the same is true vice versa.
  • the cellular avidity is determined by exerting a force on the cells of interest.
  • a force can be applied and controlled on cells of interest that bind/interact/attach with cells attached to a surface, e.g. target cells, such a force can be contemplated in accordance with the invention.
  • the force is an acoustic force, a shear flow force or a centrifugal force.
  • cellular avidity is measured using the z-Movi® device as available from the company LUMICKS.
  • the device makes use of an acoustic force.
  • a microfluidic device as described herein can be used in the z-Movi® device. It may even be used repeatedly.
  • cellular avidity it is to be understood that this is to express the strength of binding of cells of interest (or control cells thereof) to cells attached to a surface (or control cells thereof). It is to be understood that where it is referred to specific forces applied to cells, this may refer to average forces, e.g. such forces may not be fully homogeneous, for example over the contact surface.
  • the cellular binding avidity is determined by exerting a force on the cells of interest, e.g. effector cells, away from the cells that are attached to the second coating, e.g. target cells.
  • the force applied may be perpendicular (in the direction of z-axis) to the surface (x,y) to which the cells that are attached to the second coating are attached, for example when a centrifugal force or acoustic force is applied.
  • the force may also be lateral (x-axis or y-axis), for example when a shear force is applied.
  • the force is applied and is controlled such that a defined force is exerted on the cells of interest that interact with the cells that are attached to the second coating.
  • the force that is exerted on the cells of interest attached to the cells that are attached to the second coating is to be substantially equal. This can be achieved when using for example a flat surface.
  • Other suitable surface shapes may be used (e.g. a tube with exerted concentrical force or laminar flow force in the direction of the length of the tube), as long as the force exerted can be substantially equal at a defined surface area, such a surface shape may be contemplated.
  • the force required to move a cell of interest away from the cells that are attached to the second coating (/.e. a cell detachment event) can be detected optically, e.g. via microscopy or other means. Cell detachment events can be monitored and counted.
  • the process for determining cellular avidity may further comprise the step of collection of cells, for example, collection of cells that experienced a defined force.
  • the cells of interest may be provided with a photoactivatable label which may subsequently be activated by illumination with light of a suitable wavelength only in a well-defined interaction region of the device (e.g. in a center region under an (acoustic) force transducer) to photoactivate and/or switch on the dye.
  • the cells can be sorted, for example, using fluorescence activated cell sorting (FACS) and only those cells which are activated are collected thereby obtaining the cells on which defined forces have been exerted.
  • FACS fluorescence activated cell sorting
  • the target cells and cells of interest bound thereto may be trypsinized thereby obtaining both the target cells and cells of interest that remained bound thereto in a suspension.
  • attached cells of interest can also be simply collected with physical means (e.g. by scraping) from the area of interest, i.e. the surface area with a well-defined nominal force.
  • the exact forces experienced by cells may also depend on cell size and or other cell properties such as density and compressibility.
  • the force may thus be a nominal force and not the true force experienced by the cells. Since it may be hard to precisely predict the average cell size, density, compressibility, etc. of the cells, the force may have been calculated based on theory alone or may have been calibrated using test particles with specific properties (see Kamsma, D. et al. (2016). Tuning the Music: Acoustic Force Spectroscopy (AFS) 2.0. Methods, 105, 26-33).
  • the force may be a calculated or calibrated force expressed with units of N (e.g.
  • Vpp input power
  • a piezo element see Sitters, G. et al. (2014). Acoustic force spectroscopy. Nature Methods, 12(1), 47-50), as angular velocity squared (&j 2 ) in the case of centrifugal forces or as flow speed v and or as shear stress (Pa) in case of shear forces.
  • &j 2 angular velocity squared
  • Pa shear stress
  • the percentage of cells of interest that remains bound at a certain applied force is indicative of cellular avidity, i.e. the larger the percentage of cells of interest that is bound, the higher the cellular avidity, it is useful to refer to the percentage of cells.
  • a different measure which relates to cellular avidity may be used, for example the percentage of detached cells, wherein conversely a low number is indicative of a higher cellular avidity.
  • percentage the ratio of cells that remain attached or are detached divided by the total number of cells that interacted may be provided. In case of a cellular avidity plot, the area under (or above) the curve may be determined.
  • a unit that is representative of the number of cells of interest that have detached or cells of interest that have remained attached, relative to the total number of cells that have interacted.
  • Such a unit allows for ranking cellular avidities, when comparing e.g. different cells of interest.
  • Providing such a unit may be referred to as providing a cellular avidity score.
  • a preferred cellular avidity score may be the percentage of cells of interest that remains attached relative to the cells of interest that have interacted, the latter being set at 100%.
  • a cellular avidity score is preferably provided.
  • any suitable force application method may be contemplated in processes for determining cellular avidity as described herein.
  • increasing the force is well controlled.
  • the applied force is a force ramp, preferably a linear force ramp. It is understood that when a force is selected to be applied it can be a constant force applied for a defined period.
  • the forces applied may be in various forms as a function of time.
  • the applied force is an increasing force. That is, after the incubation step, an increasing force is applied for a defined period until a defined end force is reached.
  • a linear force ramp may be applied for 150 seconds resulting in a defined end force of 1000 pN.
  • the ratio or difference between the cellular avidity of cells of interest and control cells is determined.
  • the ratio of the cellular avidity between cells of interest and control cells is preferably as large as possible.
  • control cell background binding is about 15% or less and cells of interest binding is about 85% or more, for example from 85-95%.
  • the percentage difference between cellular avidity of control cells and cells of interest is at least 30%, at least 40%, at least 50%, at least 60% at least 70%, at least 80% and preferably at least 90%.
  • conditions are selected with the largest difference between the percentages. In an embodiment wherein e.g.
  • the present application also describes processes for identifying a candidate agent capable of modulating the cellular avidity between a cell of interest and a cell that is attached to the second coating, wherein in the incubation step, and cellular avidity determination step of the processes as described herein agent(s) are provided and included in these steps.
  • agent(s) capable of doing so can be identified.
  • the cells that are attached to the second coating can also be used in processes for screening of different cells of interest, by performing the processes as described herein, optionally in the presence of agent(s) as defined above, and comparing determined cellular avidities for the different cells of interest.
  • a candidate agent is provided for modulating the avidity between a cell of interest and a cell that is attached to the second coating, and wherein said agent is present in the incubation step and step of determining cellular avidity.
  • the processes for determining cellular avidity as described herein may comprise the step of providing a cell engager.
  • Cell engagers include antibodies, or the like, which are capable of binding to a target cell and a cell of interest, e.g. an effector cell.
  • Such antibodies may include single chain antibodies comprising two binding domains such as scFv domains, and include BiTEs (/.e. bispecific T-cell engagers) or the like.
  • a conventional antibody design includes heavy and light chains, with one half of the antibody (one heavy chain and one light chain) engaging with a target cell, and the other half of the antibody (another heavy chain and another light chain) engaging with an effector cell, wherein preferably, the Fc domain is made inert.
  • suitable cell engagers are widely known in the art and the current invention allows to study and/or determine cellular avidity induced by a cell engager between a target cell and an effector cell.
  • a cell engager may be provided in addition and the cellular avidity score may be determined, induced by said cell engager, between a cell of interest (e.g. an effector cell) and a target cell.
  • said cell engager has a binding region capable of binding the cell of interest (e.g. an effector cell) and a binding region capable of binding the target cell. It is understood that the incubation step in the processes as described herein can thus be performed in the presence of a cell engager to allow the cells, i.e.
  • a cell engager is provided capable of binding an effector cell and a target cell, and the cell engager is included in the incubation step. Furthermore, such processes are highly useful for screening cell engagers, e.g. by identifying cell engagers that are particular capable of binding an effector cell and a target cell.
  • processes are provided for selecting and/or sorting cells of interest, comprising the steps of the processes as described herein, and optionally in the presence of agent(s) as defined above, comprising the further step of applying the selected force on the cells of interest and subsequently selecting and/or sorting cells of interest that have detached and/or that remain attached to the cells that are attached to the second coating.
  • a process is provided comprising the steps of the processes of determining cellular avidity as described herein, wherein the process is for use in sorting and/or screening of cells of interest.
  • a microfluidic device comprising a surface comprising cells, wherein the surface comprises a coating comprising a carbohydrate, a coating comprising a carbohydrate-binding protein and cells attached to the coating comprising the carbohydrate-binding protein,
  • the microfluidic device comprising a surface comprising cells is prepared by performing a process for preparing a microfluidic device comprising a surface comprising cells as described herein.
  • processes may be of use for the screening of candidate agents as well.
  • a further process is provided for screening candidate agents for modulating cellular avidity between of cells of interest and cells attached to the coating comprising a carbohydrate-binding protein comprising the steps of:
  • a microfluidic device comprising a surface comprising cells, wherein the surface comprises a coating comprising a carbohydrate, a coating comprising a carbohydrate-binding protein and cells attached to the coating comprising the carbohydrate-binding protein,
  • the cells attached to the coating comprising the carbohydrate- binding protein are incubated with the candidate agents followed by incubation with the cells of interest.
  • the candidate agents are incubated with the cells of interest followed by incubation of the cells attached to the coating comprising the carbohydrate-binding protein with the candidate agents attached to the cells of interest.
  • the cells attached to the coating comprising the carbohydrate-binding protein are incubated with the candidate agents and the cells of interest.
  • the cellular avidity score obtained in the presence of the candidate agents is compared to the cellular avidity score that is measured in the absence of the candidate agents. Based on the comparison, candidate agents that increase or decrease cellular avidity between the cells and the cells of interest can be identified and/or selected.
  • the microfluidic device comprising a surface comprising cells is prepared by performing a process for preparing a microfluidic device comprising a surface comprising cells as described herein.
  • Cells of interest which can be selected and/or sorted and thus be obtained accordingly with processes as described herein may subsequently in a further step be admixed with a pharmaceutically acceptable buffer or otherwise pharmaceutical acceptably formulated.
  • the present application also describes a microfluidic device obtainable by a process for preparing a microfluidic device comprising a surface comprising cells as described herein.
  • the present application also describes a microfluidic device obtained by a process for preparing a microfluidic device comprising a surface comprising cells as described herein.
  • the present application also describes a microfluidic device comprising a surface, wherein the surface comprises a coating comprising a carbohydrate and a coating comprising a carbohydrate-binding protein.
  • the present application also describes a microfluidic device comprising a surface, wherein the surface comprises a first coating comprising a carbohydrate and a second coating comprising a carbohydrate-binding protein.
  • the present application also describes a microfluidic device comprising a surface, wherein the surface comprises a coating comprising a carbohydrate, a coating comprising a carbohydrate-binding protein, and cells attached to the coating comprising the carbohydrate-binding protein.
  • the present application also describes a microfluidic device comprising a surface, wherein the surface comprises a first coating comprising a polypeptide, a second coating comprising a polypeptide, and cells attached to the second coating.
  • the present application also describes a microfluidic device comprising a surface, wherein the surface comprises a first coating comprising a carbohydrate or derivative thereof, and optionally, a second coating comprising a carbohydrate-binding protein.
  • the present application also describes a microfluidic device comprising a surface, wherein the surface comprises a first coating comprising a mannose or derivative thereof, and optionally, a second coating comprising a lectin.
  • the present application also describes a microfluidic device comprising a surface, wherein the surface comprises a first coating comprising a mannose or derivative thereof, and optionally, a second coating comprising a mannose-binding lectin.
  • a microfluidic device as described herein comprises one or more internal spaces including a vessel, typically in the form of a flow channel.
  • the flow channel may be configured to convey a flow of fluid.
  • the microfluidic device as described herein may also comprise one or more compartments configured to contain one or more fluids.
  • the microfluidic device as described herein may also comprise one or more connectors for connecting purposes.
  • the microfluidic device comprises a unitary body in which the one or more internal spaces are at least partly formed.
  • the present application also describes a cell culture device comprising a surface, wherein the surface comprises a coating comprising at least a carbohydrate.
  • the present application also describes a cell culture device comprising a surface, wherein the surface comprises a first coating comprising a carbohydrate and a second coating comprising a carbohydrate-binding protein.
  • the carbohydrate is selected from the group consisting of mannose, fucose, xylose, arabinose, acetylglucosamine, fructose, sialic acid, glucuronic acid, galactose, N-acetylgalactosamine, any derivative and mixture thereof.
  • the carbohydrate is carbohydrate-R, wherein R is a functional group that is capable of reacting with an OH-group on the surface.
  • the carbohydrate is carbohydrate-R, wherein R is a functional group that is capable of forming a bond with an OH-group on the surface.
  • R is selected from the group consisting of phosphate, silane, quinone and catechol.
  • An example of a derivative of mannose and an example of a carbohydrate-R is mannose-6- R, wherein R is selected from the group consisting of phosphate, silane, quinone and catechol. So, in an embodiment the carbohydrate is mannose-6-R, wherein R is selected from the group consisting of phosphate, silane, quinone and catechol.
  • the carbohydrate comprised in the first coating is mannose-6-R, wherein R is selected from the group consisting of phosphate, silane, quinone and catechol.
  • the carbohydrate-binding protein is a lectin.
  • the lectin is selected from the group consisting of concanavalin A (ConA), lentil lectins (LCH), snowdrop lectins (GNA), proteoglycans, type II transmembrane receptor lectins, collectins, selectins, natural killer lectins, macrophage mannose receptor (MMR) lectins, type I receptor lectins, tetranectins, polycystins, attractins, CTLD/acidic neck lectins, endosialins and any combination thereof.
  • ConA concanavalin A
  • LCH lentil lectins
  • GNA snowdrop lectins
  • proteoglycans type II transmembrane receptor lectins
  • a "cell culture device” as used herein means a receptacle that can contain media for culturing a cell or tissue.
  • the cell culture device may include glass, ceramics, metal, plastic or any combination thereof.
  • the plastic is non-cytotoxic.
  • the plastic may include natural polymers and/or synthetic polymers.
  • Exemplary cell culture devices include, but are not limited to, single and multi-well plates, including 6-well and 12-well culture plates, and smaller welled culture plates such as 96-, 384-, and 1536-well plates, culture jars, culture dishes, petri dishes, culture flasks, culture plates, culture roller bottles, culture slides, including chambered and multi-chambered culture slides, culture tubes, coverslips, cups, spinner bottles, perfusion chambers, bioreactors, and fermenters.
  • single and multi-well plates including 6-well and 12-well culture plates, and smaller welled culture plates such as 96-, 384-, and 1536-well plates, culture jars, culture dishes, petri dishes, culture flasks, culture plates, culture roller bottles, culture slides, including chambered and multi-chambered culture slides, culture tubes, coverslips, cups, spinner bottles, perfusion chambers, bioreactors, and fermenters.
  • a cell culture device as described herein may also be used in a process for determining cellular avidity as described herein.
  • a cell culture device as described herein may also be used in a process for sorting or screening cells of interest as described herein.
  • a cell culture device as described herein may also be used in a process for screening candidate agents for modulating cellular avidity between cells as described herein.
  • the carbohydrate is selected from the group consisting of mannose, fucose, xylose, arabinose, acetylglucosamine, fructose, sialic acid, glucuronic acid, galactose, N- acetylgalactosamine, any derivative and mixture thereof.
  • the carbohydrate is carbohydrate-R, wherein R is a functional group that is capable of reacting with an OH-group on the surface.
  • the carbohydrate is carbohydrate-R, wherein R is a functional group that is capable of forming a bond with an OH-group on the surface.
  • R is selected from the group consisting of phosphate, silane, quinone and catechol.
  • An example of a derivative of mannose and an example of a carbohydrate-R is mannose-6-R, wherein R is selected from the group consisting of phosphate, silane, quinone and catechol. So, in an embodiment the carbohydrate is mannose-6-R, wherein R is selected from the group consisting of phosphate, silane, quinone and catechol.
  • the carbohydrate comprised in the first coating is mannose-6-R, wherein R is selected from the group consisting of phosphate, silane, quinone and catechol.
  • the carbohydrate- binding protein is a lectin.
  • the lectin is selected from the group consisting of concanavalin A (ConA), lentil lectins (LCH), snowdrop lectins (GNA), proteoglycans, type II transmembrane receptor lectins, collectins, selectins, natural killer lectins, macrophage mannose receptor (MMR) lectins, type I receptor lectins, tetranectins, polycystins, attractins, CTLD/acidic neck lectins, endosialins and any combination thereof.
  • the microfluidic devices and/or cell culture devices as described herein can be used for cell avidity measurements and for sorting or selecting of cells of interest, they however may also be used to conduct one or more of a wide range of investigations of interactions between cells and one or more substances.
  • Such interactions to be investigated may include, but are not limited to, an effect of the substance(s) on one or more of: cell attachment, cell growth, cell survival, cell differentiation, cell apoptosis/death and multicellular structure.
  • the activity or interaction of substances on cell motility, cell migration, cell to cell interactions as well as cell-protein signal interactions are also important activities that can be studied using the microfluidic devices and/or cell culture devices as described herein.
  • Substances of interest in such investigations may include, but are not limited to, pharmaceutical compounds or other therapeutics, exosomes, nanomicelles, nanoparticles, toxins, small molecules, nucleic acids (including nucleic acid vectors), oligonucleotides (including antisense oligonucleotides), oligopeptides, proteins, ribozymes, small interfering RNAs, microRNAs, short hairpin RNAs, aptamers, viruses, and antibodies or antigen binding parts thereof.
  • nucleic acids including nucleic acid vectors
  • oligonucleotides including antisense oligonucleotides
  • oligopeptides proteins
  • ribozymes small interfering RNAs
  • microRNAs microRNAs
  • short hairpin RNAs aptamers
  • viruses and antibodies or antigen binding parts thereof.
  • the present application also describes a kit of parts comprising a microfluidic device comprising a surface, a carbohydrate, and a carbohydrate-binding protein.
  • the present application also describes a kit of parts comprising a microfluidic device comprising a surface comprising a coating comprising a carbohydrate, and a carbohydrate-binding protein.
  • a microfluidic device present in the kit of parts refers to a device having the required structural configuration of a microfluidic devices as described herein, but in which a first coating comprising a carbohydrate and/or a second coating comprising a carbohydrate-binding protein is yet to be formed.
  • kits of parts as described herein may be stored in one or more containers prior to their application and/or use.
  • kits of parts as described herein may be multi-pack kits wherein different components are stored in a plurality of containers.
  • a kit of parts as described herein may further comprise instructions for coating the surface with the carbohydrate and/or the carbohydrate-binding protein and, optionally, instructions for contacting and attaching cells with a surface coated with the carbohydrate-binding protein to provide a microfluidic device comprising a surface comprising cells.
  • the present application describes a process for preparing a microfluidic device comprising a surface comprising cells, the process comprising the steps of:
  • the present application also describes a process for preparing a microfluidic device comprising a surface comprising cells, the process comprising the steps of:
  • a microfluidic device comprising a carbohydrate-binding protein coated surface, wherein the surface optionally comprises a coating comprising a carbohydrate between the surface and the coating comprising the carbohydrate- binding protein,
  • the present application also describes a process for preparing a microfluidic device comprising a surface comprising cells, the process comprising the steps of:
  • the present application also describes a process for preparing a microfluidic device comprising a surface comprising cells, the process comprising the steps of: • providing a microfluidic device comprising a carbohydrate-binding protein coated surface, wherein the surface comprises a coating comprising a carbohydrate between the surface and the coating comprising the carbohydrate-binding protein,
  • the surface is activated before a coating is applied.
  • the surface is chemically activated, for example with sodium hydroxide. Activation can be done to facilitate application of a coating.
  • a microfluidic device comprising a surface comprising cells (e.g. target cells) as described in the prior art
  • the surface is coated with a compound to which cells (e.g. target cells) can attach.
  • the remaining compound on the surface may be prone to binding to other molecules or cells (e.g. effector cells) leading to high background binding in many assay systems.
  • An advantage of the present invention is that a microfluidic device is provided having low background binding, because any carbohydrate-binding protein remaining on the surface after contacting with cells (e.g. target cells) is coated with a carbohydrate.
  • the remaining carbohydrate-binding protein is no longer capable of binding to other molecules or cells (e.g. target cells).
  • the carbohydrate may act as a fuel source for the cells (e.g. target cells) that have attached to the surface.
  • a surface on which a coating has been applied is known herein as a "coated surface.”
  • the term “single-coated surface” as used herein means a surface that comprises at least a coating layer comprising at least a carbohydrate-binding protein. In an embodiment the single-coated surface comprises at least a carbohydrate-binding protein as described herein.
  • the term “multiple-coated surface” as used herein means a surface that comprises at least two coating layers. In an embodiment the multiple-coated surface comprises a first coating layer comprising at least a carbohydrate-binding protein and at least a second coating layer comprising at least a carbohydrate.
  • the multiple- coated surface may comprise additional coating layers. Said additional coating layers may be below the first coating layer (/.e.
  • the additional coating layer is located between the surface and the first coating layer), between the first coating layer and the second coating layer and/or on top of the second coating layer.
  • the additional coating layer is located between the surface and the first coating layer and comprises a carbohydrate.
  • Said additional coating layer located between the surface and the first coating layer may be called a precoating.
  • the surface may be coated multiple times with the precoating.
  • cells are present in between the first coating layer and the second coating layer.
  • the surface may be coated multiple times with the first coating layer and/or multiple times with the second coating layer. In other words, the coating process can be repeated numerous times until the desired order of coating layers is achieved.
  • the surface does not comprise any additional coating layers next to the first coating layer and the second coating layer.
  • the first coating comprises more than one carbohydrate-binding protein.
  • the second coating comprises more than one carbohydrate.
  • this precoating layer may comprise more than one carbohydrate.
  • the first coating comprises more than one carbohydrate-binding protein and the second coating also comprises more than one carbohydrate. With “more than one” is meant “a mixture of”.
  • the cells form a monolayer on the surface. In an embodiment the cells form a monolayer on the carbohydrate-binding protein coated surface.
  • the surface is a glass surface, a plastic surface, a metal surface, a ceramic surface or any combination thereof. In a preferred embodiment the surface is a glass surface, a plastic surface, a ceramic surface or any combination thereof.
  • the microfluidic device may be prepared from any suitable material including plastic, glass, ceramics, metal or any combination thereof.
  • the plastic is non-cytotoxic.
  • the plastic may include natural polymers and/or synthetic polymers.
  • the cell monolayer shows a high quality.
  • Quality can be measured for example by looking at the confluency of the cell monolayer.
  • the confluency should be 50% or higher, preferably 60% or higher, more preferably 70% or higher.
  • Quality can also be measured for example by looking at the dumpiness of the cell monolayer.
  • the cell monolayer does not show dumpiness.
  • Quality can also be measured for example by looking at the stability and/or robustness of the cell monolayer. Stability and/or robustness can be measured for example by measuring the resistance of the cell monolayer when subjected to shear flow force and/or acoustic force and/or centrifugal force.
  • the cells are cultured. Culturing of the cells may take place before, simultaneously with and/or after contacting the cells with the surface, e.g. the carbohydrate-binding protein coated surface, and allowing the cells to attach to the surface to provide a microfluidic device comprising a surface comprising cells.
  • the carbohydrate is selected from the group consisting of mannose, fucose, xylose, arabinose, acetylglucosamine, fructose, sialic acid, glucuronic acid, galactose, N-acetylgalactosamine, any derivative and any mixture thereof.
  • the carbohydrate comprised in the second coating and/or precoating is selected from the group consisting of mannose, fucose, xylose, arabinose, acetylglucosamine, fructose, sialic acid, glucuronic acid, galactose, N- acetylgalactosamine, any derivative and any mixture thereof.
  • the carbohydrate is carbohydrate-R, wherein R is polyethylene glycol (PEG).
  • R polyethylene glycol
  • the carbohydrate in the precoating is carbohydrate-R, wherein R is a chemical moiety capable of reacting with surface-bound OH groups such as phosphate or silane.
  • An example of a derivative of mannose and an example of a carbohydrate-R is mannose-6-R, wherein R is polyethylene glycol (PEG). So, in an embodiment the carbohydrate is mannose-6-R, wherein R is polyethylene glycol (PEG). In an embodiment the carbohydrate comprised in the second coating is mannose-6-R, wherein R is polyethylene glycol (PEG).
  • the carbohydrate in the precoating is mannose-6-R, wherein R is a chemical moiety capable of reacting with surface-bound OH groups such as phosphate or silane.
  • the carbohydrate comprised in the second coating differs from the carbohydrate comprised in the precoating. In an embodiment the carbohydrate comprised in the second coating is the same as the carbohydrate comprised in the precoating.
  • the carbohydrate-binding protein is a lectin.
  • the lectin is selected from the group consisting of concanavalin A (ConA), lentil lectins (LCH), snowdrop lectins (GNA), proteoglycans, type II transmembrane receptor lectins, collectins, selectins, natural killer lectins, macrophage mannose receptor (MMR) lectins, type I receptor lectins, tetranectins, polycystins, attractins, CTLD/acidic neck lectins, endosialins and any combination thereof.
  • the lectin is concanavalin A (ConA).
  • one or more active domains of a carbohydrate-binding protein may also be used in any of the processes for preparing a microfluidic device as described herein.
  • An "active domain” refers to an amino acid sequence found within the carbohydrate-binding protein that, in itself, provides function according to one or more properties of the carbohydrate-binding protein, such as providing structural support to cells and/or for attaching cells and/or for attaching a carbohydrate.
  • the peptide that includes an active domain of a carbohydrate-binding protein can have a "core sequence" of amino acid residues, and optionally one or more additional amino acid residues that flank (/.e., on the C-terminus, N-terminus, or both) the core sequence.
  • the one or more additional amino acids that flank the core sequence can correspond to the wild-type sequence in the relevant region of the protein, or can comprise one or more amino acid(s) that diverge from the wild-type sequence (e.g., a "variant amino acid sequence").
  • the variant amino sequence can be one that enhances properties of the peptide, such as providing enhanced ligand interaction.
  • a "peptide” is a short polymer of 25 or less amino acids linked by peptide bonds.
  • a "polypeptide” is a polymer of more than 25 amino acids linked by peptide bonds and which includes full length proteins.
  • a peptide having an active portion of a carbohydrate-binding protein can be synthesized by solid phase peptide synthesis (SPPS) techniques using standard techniques, such as Fmoc synthesis.
  • SPPS solid phase peptide synthesis
  • the first coating layer comprising the carbohydrate-binding protein may comprise other excipients and/or components as well.
  • the carbohydrate-binding protein is present in the first coating layer in an amount of at least 10% (w/w) (weight percentage of the carbohydrate-binding protein based on the total weight of the first coating layer comprising the carbohydrate-binding protein), at least 20% (w/w), at least 30% (w/w), at least 40% (w/w), at least 50% (w/w), at least 60% (w/w), at least 70% (w/w), at least 80% (w/w), at least 90% (w/w) or at least 100% (w/w).
  • the second coating layer comprising a carbohydrate and/or the precoating layer comprising a carbohydrate may comprise other excipients and/or components as well.
  • the carbohydrate is present in the second coating layer and/or precoating layer in an amount of at least 10% (w/w) (weight percentage of the carbohydrate based on the total weight of the second coating layer and/or precoating layer comprising the carbohydrate), at least 20% (w/w), at least 30% (w/w), at least 40% (w/w), at least 50% (w/w), at least 60% (w/w), at least 70% (w/w), at least 80% (w/w), at least 90% (w/w) or at least 100% (w/w).
  • the first (/.e. carbohydrate-binding protein comprising) and second (/.e. carbohydrate comprising) coating and any additional coating layer can be prepared to have a desired thickness.
  • the coated layers have a thickness in the range of about 2 nm to about 20 nm.
  • the first and second coating and any additional coating layer can be prepared to have a desired density and/or concentration.
  • the coating process involves placing the coating materials in contact with the device surface.
  • the coating materials are applied to a surface and dried down or partially dried down.
  • the process of applying can be performed using any one of a variety of techniques including dipping, pouring, swabbing, siphoning, brushing, rolling, padding, ragging, painting, spraying, anodizing, electroplating, and/or laminating.
  • One exemplary method for applying a coating composition is by dip coating.
  • a typical dip coating procedure involves immersing the surface to be coated in a first coating composition, dwelling the object in the composition for a period of time, and then removing the surface from the composition. After the surface has been dip coated in the coating solution, it is removed and dried or partially dried. Drying can be carried out using any suitable method, including air drying the surface.
  • Another exemplary method for applying a coating composition is flowing the coating composition into a microfluidic device in order to coat the inner surface(s) of the microfluidic device.
  • the processes as described herein also comprise the step of contacting the microfluidic device comprising a surface comprising cells with a blocking agent.
  • the blocking agent may even further reduce background binding to the cells.
  • suitable blocking agents include, but are not limited to, N- hydroxysuccinimide (NHS) ester reagents.
  • An NHS ester reagent as defined herein is a compound comprising at least one succinimidyl group, which functional group is capable of forming an ester bond with a primary amine group on polypeptides, such as, for example, the amine groups as comprised in lysine residues, such that these amine groups are no longer available to attach to, for example, cells of interest.
  • the NHS ester reagent comprises a spacer, e.g. a polymer of a relative short length, up to about 100 Angstrom.
  • a polymer preferably is inert.
  • the polymer is polyethylene glycol (PEG). More preferably, the PEG length is up to 25 ethylene glycol units in length. Preferably, the number of units is selected from the range of 2 to 25, or from 3 to 10.
  • the NHS ester is a PEG-NHS ester.
  • Blocking may take place by providing a buffer suitable for the blocking reaction and sustaining cell viability of the cells present on the surface, e.g. phosphate, carbonatebicarbonate, HEPES or borate buffers at pH 7.2 to 8.5 for 0.5 to 4 h at room temperature or 4°C.
  • a buffer suitable for the blocking reaction e.g. phosphate, carbonatebicarbonate, HEPES or borate buffers at pH 7.2 to 8.5 for 0.5 to 4 h at room temperature or 4°C.
  • the NHS ester reagent is removed and the cells may, for example, be washed with serum containing medium or with a primary amine buffer such as Tris or glycine, which are not compatible because they compete for reaction, to quench (stop) the reaction.
  • NHS esters are known in the art, and it may be contemplated to utilize other compounds equally capable of reacting with amine groups as an alternative.
  • These compounds include, but are not limited to, isothiocyanates, isocyanates, acyl azides, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, carbodiimides, anhydrides, and fluorophenyl esters. Most of these compounds conjugate to amines by either acylation or alkylation. Without being bound by theory, as long as the compound is capable of reacting with the constituents responsible for the coating on the surface, it can be contemplated.
  • the cells are target cells or effector cells.
  • the cells are target cells.
  • the target cells are cells presenting an antigen.
  • a tumor antigen a viral antigen or a bacterial antigen.
  • Suitable target cells may include eukaryotic cells or prokaryotic cells.
  • Target cells that may be selected include fungal cells, animal cells, insect cells, mammalian cells, bacterial cells, yeasts, and protozoa.
  • the target cells are human cells.
  • the target cells are tumor cells such as human tumor cells.
  • the effector cells are human effector cells such as T cells, NK cells, B cells, dendritic cells, macrophages, or monocytes.
  • the effector cells may be genetically modified. When the genetic modification is with a CAR, cells such as CAR T cells can be obtained. Effector cells as used herein may thus also be CAR T cells.
  • extraneous DNA is removed from the cells before contacting the cells with the coated surface.
  • extraneous DNA is removed from the cells by treatment of the cells with a deoxyribonuclease (DNAse).
  • DNAse deoxyribonuclease
  • the present application also describes a process for determining cellular avidity comprising:
  • a microfluidic device comprising a surface comprising cells, wherein the surface comprises a first coating comprising a carbohydrate-binding protein, cells attached to the first coating, a second coating comprising a carbohydrate and optionally a precoating comprising a carbohydrate, wherein the precoating is located between the surface and the first coating,
  • the present application also describes a process for determining cellular avidity comprising:
  • a microfluidic device comprising a surface comprising cells, wherein the surface comprises a first coating comprising a carbohydrate-binding protein, cells attached to the first coating, a second coating comprising a carbohydrate and a precoating comprising a carbohydrate, wherein the precoating is located between the surface and the first coating, • providing cells of interest,
  • the present application also describes a process for determining cellular avidity comprising:
  • cellular avidity is defined by the overall strength of interactions occurring in a cell-to-cell contact, involving a diversity of molecules at the surfaces of the cells that interact.
  • cellular avidity is a parameter that expresses the strength of binding between cells. It involves a multitude of interactions, including a diversity of receptor-ligand pairs, among which e.g. a specific receptor-ligand interaction, occurring at the membrane surface of a cell, working jointly forming a strong bond between cells. It may also involve active signalling and processes internal to the cells such as e.g. during immune synapse formation. It is understood that the cellular avidity of a cell of a certain type is defined relative to its target cell and conditions tested.
  • target cells and cells of interest relate to two different cells which are to interact specifically with each other.
  • the target cells or the cells of interest may express a ligand on their surface and the other cell may express a receptor for that ligand.
  • control cells the same cells as the cells of interest or target cells may be utilized, but these cells do not express such a ligand or receptor or express a variant thereof which is not functional.
  • the cells attached to the coating comprising the carbohydrate-binding protein (e.g. target cells) and cells of interest (e.g. effector cells) (and control cells thereof)
  • this may involve cells that are to have a specific interaction, i.e.
  • ligand and receptor in this sense and in accordance with the invention may define their inter-relationship.
  • receptor may not be construed to be limiting in any way and is understood to mean a protein presented at the cell surface which can (specifically) interact with another protein (ligand) presented at another cell.
  • ligand and receptor are used to indicate a complementarity which is important for specific recognition between cells without restrictions on the complementary molecules that can be contemplated.
  • Receptors that may be of interest include, but are not limited to, a CAR, a TCR, a stimulatory or inhibitory co-receptor, or a receptor engaged via a bispecific antibody.
  • the cellular avidity may be determined. This is for example of interest when it is desirable to provide for a candidate CAR.
  • candidates can be selected and subsequently assessed with regard to functionality, e.g. by in vivo experiments.
  • cellular avidity is an important parameter for function, this way highly efficiently, suitable candidates can be selected.
  • the cellular avidity is determined.
  • the microfluidic device comprising a surface comprising cells is prepared by performing a process for preparing a microfluidic device comprising a surface comprising cells as described herein.
  • the cells are target cells or effector cells.
  • the cells of interest are target cells or effector cells.
  • the cells attached to the first coating are target cells and the cells of interest are effector cells.
  • the target cells are cells presenting an antigen.
  • a tumor antigen a viral antigen or a bacterial antigen.
  • Suitable target cells may include eukaryotic cells or prokaryotic cells.
  • Target cells that may be selected include fungal cells, animal cells, insect cells, mammalian cells, bacterial cells, yeasts, and protozoa.
  • the target cells are human cells.
  • the target cells are tumor cells such as human tumor cells.
  • the effector cells are human effector cells such as T cells, NK cells, B cells, dendritic cells, macrophages, or monocytes.
  • the effector cells may be genetically modified. When the genetic modification is with a CAR, cells such as CAR T cells can be obtained. Effector cells as used herein may thus also be CAR T cells.
  • the cells that are attached to the first coating are preferably attached as a monolayer.
  • the subsequent cells of interest (and control cells thereof) that are to interact with the cells attached to the coating on the surface are preferably provided in a relatively low cell density as compared with those cells, such that substantially all cells of interest (and control cells thereof) can interact with those cells. In other words, there are more cells per cell of interest. Such provides for advantageous controllable conditions when applying the force on the cells of interest or the control cells thereof.
  • control cells of the target cells or the cells of interest are provided and the cellular avidity of the control cells is determined as well.
  • control cells of the cells that are attached to the first coating and/or control cells of the cells of interest are provided and the cellular avidity of the control cells is determined as well.
  • the process for determining cellular avidity as described herein may be performed separately with cells of interest and their control cells and/or with cells that are attached to the first coating and their control cells.
  • the process for determining cellular avidity as described herein may comprise the step of incubating the cells attached to the first coating with the cells of interest and/or their control cells.
  • the process for determining cellular avidity as described herein may comprise the step of contacting cells of interest and/or their control cells with target cells. Control cells and/or cells of interest that have detached and/or remained attached (after force application as described herein) are determined and cellular avidity scores are provided for the control cells and/or cells of interest with the cells that are attached to the first coating.
  • control cells of the cells of interest alternatively control cells of the cells that are attached to the first coating may be provided attached to a surface.
  • cells of interest are contacted with the cells that are attached to the first coating and before or thereafter cells of interest are contacted with the control cells of the cells that are attached to the first coating, and in each case cells of interest that have detached and/or remained attached are determined and cellular avidity scores are provided for the cells of interest with the control cells of the cells that are attached to the first coating and with the cells that are attached to the first coating as such.
  • the cellular avidity scores obtained for cells and their respective controls can be compared and are of interest. Measuring the cellular avidity with and without control cells allows to better separate specific from non-specific binding.
  • control cells when cells of interest are used that express a specific ligand or receptor, control cells are used that are the same cells as the cells of interest but do not express the specific ligand or receptor or express a variant of the specific ligand or receptor which is not functional or not specific for the counterpart present on the cells that are attached to the first coating, e.g. the target cells. Obviously, the same is true vice versa.
  • the cellular avidity is determined by exerting a force on the cells of interest.
  • a force can be applied and controlled on cells of interest that bind/interact/attach with cells attached to a surface, e.g. target cells, such a force can be contemplated in accordance with the invention.
  • the force is an acoustic force, a shear flow force or a centrifugal force.
  • cellular avidity is measured using the z-Movi® device as available from the company LUMICKS.
  • the device makes use of an acoustic force.
  • a microfluidic device as described herein can be used in the z-Movi® device. It may even be used repeatedly.
  • cellular avidity it is to be understood that this is to express the strength of binding of cells of interest (or control cells thereof) to cells attached to a surface (or control cells thereof). It is to be understood that where it is referred to specific forces applied to cells, this may refer to average forces, e.g. such forces may not be fully homogeneous, for example over the contact surface.
  • the cellular binding avidity is determined by exerting a force on the cells of interest, e.g. effector cells, away from the cells that are attached to the first coating, e.g. target cells.
  • the force applied may be perpendicular (in the direction of z-axis) to the surface (x,y) to which the cells that are attached to the first coating are attached, for example when a centrifugal force or acoustic force is applied.
  • the force may also be lateral (x-axis or y-axis), for example when a shear force is applied.
  • the force is applied and is controlled such that a defined force is exerted on the cells of interest that interact with the cells that are attached to the first coating.
  • the force that is exerted on the cells of interest attached to the cells that are attached to the first coating is to be substantially equal. This can be achieved when using for example a flat surface.
  • Other suitable surface shapes may be used (e.g. a tube with exerted concentrical force or laminar flow force in the direction of the length of the tube), as long as the force exerted can be substantially equal at a defined surface area, such a surface shape may be contemplated.
  • the force required to move a cell of interest away from the cells that are attached to the first coating (/.e. a cell detachment event) can be detected optically, e.g. via microscopy or other means. Cell detachment events can be monitored and counted.
  • the process for determining cellular avidity may further comprise the step of collection of cells, for example, collection of cells that experienced a defined force.
  • the cells of interest may be provided with a photoactivatable label which may subsequently be activated by illumination with light of a suitable wavelength only in a well-defined interaction region of the device (e.g. in a center region under an (acoustic) force transducer) to photoactivate and/or switch on the dye.
  • the cells can be sorted, for example, using fluorescence activated cell sorting (FACS) and only those cells which are activated are collected thereby obtaining the cells on which defined forces have been exerted.
  • FACS fluorescence activated cell sorting
  • the target cells and cells of interest bound thereto may be trypsinized thereby obtaining both the target cells and cells of interest that remained bound thereto in a suspension.
  • attached cells of interest can also be simply collected with physical means (e.g. by scraping) from the area of interest, i.e. the surface area with a well-defined nominal force.
  • the exact forces experienced by cells may also depend on cell size and or other cell properties such as density and compressibility.
  • the force may thus be a nominal force and not the true force experienced by the cells. Since it may be hard to precisely predict the average cell size, density, compressibility, etc. of the cells, the force may have been calculated based on theory alone or may have been calibrated using test particles with specific properties (see Kamsma, D. et al. (2016). Tuning the Music: Acoustic Force Spectroscopy (AFS) 2.0. Methods, 105, 26-33).
  • the force may be a calculated or calibrated force expressed with units of N (e.g.
  • Vpp input power
  • a piezo element see Sitters, G. et al. (2014). Acoustic force spectroscopy. Nature Methods, 12(1), 47-50), as angular velocity squared (&j 2 ) in the case of centrifugal forces or as flow speed v and or as shear stress (Pa) in case of shear forces.
  • &j 2 angular velocity squared
  • Pa shear stress
  • the percentage of cells of interest that remains bound at a certain applied force is indicative of cellular avidity, i.e. the larger the percentage of cells of interest that is bound, the higher the cellular avidity, it is useful to refer to the percentage of cells.
  • a different measure which relates to cellular avidity may be used, for example the percentage of detached cells, wherein conversely a low number is indicative of a higher cellular avidity.
  • percentage the ratio of cells that remain attached or are detached divided by the total number of cells that interacted may be provided. In case of a cellular avidity plot, the area under (or above) the curve may be determined.
  • a unit that is representative of the number of cells of interest that have detached or cells of interest that have remained attached, relative to the total number of cells that have interacted.
  • Such a unit allows for ranking cellular avidities, when comparing e.g. different cells of interest.
  • Providing such a unit may be referred to as providing a cellular avidity score.
  • a preferred cellular avidity score may be the percentage of cells of interest that remains attached relative to the cells of interest that have interacted, the latter being set at 100%.
  • a cellular avidity score is preferably provided.
  • any suitable force application method may be contemplated in processes for determining cellular avidity as described herein.
  • increasing the force is well controlled.
  • the applied force is a force ramp, preferably a linear force ramp. It is understood that when a force is selected to be applied it can be a constant force applied for a defined period.
  • the forces applied may be in various forms as a function of time.
  • the applied force is an increasing force. That is, after the incubation step, an increasing force is applied for a defined period until a defined end force is reached.
  • a linear force ramp may be applied for 150 seconds resulting in a defined end force of 1000 pN.
  • the ratio or difference between the cellular avidity of cells of interest and control cells is determined.
  • the ratio of the cellular avidity between cells of interest and control cells is preferably as large as possible.
  • control cell background binding is about 15% or less and cells of interest binding is about 85% or more, for example from 85-95%.
  • the percentage difference between cellular avidity of control cells and cells of interest is at least 30%, at least 40%, at least 50%, at least 60% at least 70%, at least 80% and preferably at least 90%.
  • conditions are selected with the largest difference between the percentages. In an embodiment wherein e.g.
  • the present application also describes processes for identifying a candidate agent capable of modulating the cellular avidity between a cell of interest and a cell that is attached to the first coating, wherein in the incubation step, and cellular avidity determination step of the processes as described herein agent(s) are provided and included in these steps.
  • agent(s) capable of doing so can be identified.
  • the cells that are attached to the first coating can also be used in processes for screening of different cells of interest, by performing the processes as described herein, optionally in the presence of agent(s) as defined above, and comparing determined cellular avidities for the different cells of interest.
  • a candidate agent is provided for modulating the avidity between a cell of interest and a cell that is attached to the second coating, and wherein said agent is present in the incubation step and step of determining cellular avidity.
  • the processes for determining cellular avidity as described herein may comprise the step of providing a cell engager.
  • Cell engagers include antibodies, or the like, which are capable of binding to a target cell and a cell of interest, e.g. an effector cell.
  • Such antibodies may include single chain antibodies comprising two binding domains such as scFv domains, and include BiTEs (/.e. bispecific T-cell engagers) or the like.
  • a conventional antibody design includes heavy and light chains, with one half of the antibody (one heavy chain and one light chain) engaging with a target cell, and the other half of the antibody (another heavy chain and another light chain) engaging with an effector cell, wherein preferably, the Fc domain is made inert.
  • suitable cell engagers are widely known in the art and the current invention allows to study and/or determine cellular avidity induced by a cell engager between a target cell and an effector cell.
  • a cell engager may be provided in addition and the cellular avidity score may be determined, induced by said cell engager, between a cell of interest (e.g. an effector cell) and a target cell.
  • said cell engager has a binding region capable of binding the cell of interest (e.g. an effector cell) and a binding region capable of binding the target cell. It is understood that the incubation step in the processes as described herein can thus be performed in the presence of a cell engager to allow the cells, i.e.
  • a cell engager is provided capable of binding an effector cell and a target cell, and the cell engager is included in the incubation step. Furthermore, such processes are highly useful for screening cell engagers, e.g. by identifying cell engagers that are particular capable of binding an effector cell and a target cell.
  • processes are provided for selecting and/or sorting cells of interest, comprising the steps of the processes as described herein, and optionally in the presence of agent(s) as defined above, comprising the further step of applying the selected force on the cells of interest and subsequently selecting and/or sorting cells of interest that have detached and/or that remain attached to the cells that are attached to the first coating.
  • a process is provided comprising the steps of the processes of determining cellular avidity as described herein, wherein the process is for use in sorting and/or screening of cells of interest.
  • a microfluidic device comprising a surface comprising cells, wherein the surface comprises a first coating comprising a carbohydrate-binding protein, cells attached to the first coating, a second coating comprising a carbohydrate and optionally a precoating comprising a carbohydrate, wherein the precoating is located between the surface and the first coating,
  • the microfluidic device comprising a surface comprising cells is prepared by performing a process for preparing a microfluidic device comprising a surface comprising cells as described herein.
  • processes may be of use for the screening of candidate agents as well.
  • a further process is provided for screening candidate agents for modulating cellular avidity between of cells of interest and cells attached to a coating comprising a carbohydrate-binding protein comprising the steps of:
  • a microfluidic device comprising a surface comprising cells, wherein the surface comprises a first coating comprising a carbohydrate-binding protein, cells attached to the first coating, a second coating comprising a carbohydrate and optionally a precoating comprising a carbohydrate, wherein the precoating is located between the surface and the first coating,
  • the cells attached to the first coating are incubated with the candidate agents followed by incubation with the cells of interest.
  • the candidate agents are incubated with the cells of interest followed by incubation of the cells attached to the first coating with the candidate agents attached to the cells of interest.
  • the cells attached to the first coating are incubated with the candidate agents and the cells of interest.
  • the cellular avidity score obtained in the presence of the candidate agents is compared to the cellular avidity score that is measured in the absence of the candidate agents. Based on the comparison, candidate agents that increase or decrease cellular avidity between the cells and the cells of interest can be identified and/or selected.
  • the microfluidic device comprising a surface comprising cells is prepared by performing a process for preparing a microfluidic device comprising a surface comprising cells as described herein.
  • Cells of interest which can be selected and/or sorted and thus be obtained accordingly with processes as described herein may subsequently in a further step be admixed with a pharmaceutically acceptable buffer or otherwise pharmaceutical acceptably formulated.
  • the present application also describes a microfluidic device obtainable by a process for preparing a microfluidic device comprising a surface comprising cells as described herein.
  • the present application also describes a microfluidic device obtained by a process for preparing a microfluidic device comprising a surface comprising cells as described herein.
  • the present application also describes a microfluidic device comprising a surface, wherein the surface comprises a coating comprising a carbohydrate-binding protein.
  • the present application also describes a microfluidic device comprising a surface, wherein the surface comprises a coating comprising a lectin.
  • the present application also describes a microfluidic device comprising a surface, wherein the surface comprises a coating comprising concanavalin A.
  • the present application also describes a microfluidic device comprising a surface, wherein the surface comprises a coating comprising a carbohydrate-binding protein, cells and a coating comprising a carbohydrate.
  • the present application also describes a microfluidic device comprising a surface, wherein the surface comprises a first coating comprising a carbohydrate-binding protein, cells and a second coating comprising a carbohydrate.
  • the present application also describes a microfluidic device comprising a surface, wherein the surface comprises a precoating comprising a carbohydrate, a coating comprising a carbohydrate-binding protein, and cells attached to the coating comprising the carbohydrate-binding protein.
  • the present application also describes a microfluidic device comprising a surface, wherein the surface comprises a first coating comprising a carbohydrate-binding protein, cells attached to the first coating, a second coating comprising a carbohydrate, and optionally, a precoating comprising a carbohydrate, wherein the precoating is located between the surface and the first coating.
  • the present application also describes a microfluidic device comprising a surface, wherein the surface comprises a first coating comprising a carbohydrate-binding protein, cells attached to the first coating, a second coating comprising a carbohydrate, and a precoating comprising a carbohydrate, wherein the precoating is located between the surface and the first coating.
  • the present application also describes a microfluidic device comprising a surface, wherein the surface comprises a first coating comprising a lectin, cells attached to the first coating, and a second coating comprising a mannose or derivative thereof.
  • the present application also describes a microfluidic device comprising a surface, wherein the surface comprises a first coating comprising a lectin, cells attached to the first coating, a second coating comprising a mannose or derivative thereof, and a precoating comprising a mannose or derivative thereof, wherein the precoating is located between the surface and the first coating.
  • a microfluidic device as described herein comprises one or more internal spaces including a vessel, typically in the form of a flow channel.
  • the flow channel may be configured to convey a flow of fluid.
  • the microfluidic device as described herein may also comprise one or more compartments configured to contain one or more fluids.
  • the microfluidic device as described herein may also comprise one or more connectors for connecting purposes.
  • the microfluidic device comprises a unitary body in which the one or more internal spaces are at least partly formed.
  • the present application also describes a cell culture device comprising a surface, wherein the surface comprises a coating comprising at least a carbohydrate-binding protein.
  • the present application also describes a cell culture device comprising a surface, wherein the surface comprises a first coating comprising a carbohydrate-binding protein and a second coating comprising a carbohydrate.
  • the present application also describes a cell culture device comprising a surface, wherein the surface comprises a first coating comprising a carbohydrate-binding protein, cells attached to the first coating, and a second coating comprising a carbohydrate.
  • the carbohydrate is selected from the group consisting of mannose, fucose, xylose, arabinose, acetylglucosamine, fructose, sialic acid, glucuronic acid, galactose, N- acetylgalactosamine, any derivative and mixture thereof.
  • the carbohydrate is carbohydrate-R, wherein R is polyethylene glycol (PEG).
  • R polyethylene glycol
  • An example of a derivative of mannose and an example of a carbohydrate-R is mannose-6-R, wherein R is polyethylene glycol (PEG). So, in an embodiment the carbohydrate is mannose-6- R, wherein R is polyethylene glycol (PEG).
  • the carbohydrate comprised in the second coating is mannose-6-R, wherein R is polyethylene glycol (PEG).
  • the carbohydrate-binding protein is a lectin.
  • the lectin is selected from the group consisting of concanavalin A (ConA), lentil lectins (LCH), snowdrop lectins (GNA), proteoglycans, type II transmembrane receptor lectins, collectins, selectins, natural killer lectins, macrophage mannose receptor (MMR) lectins, type I receptor lectins, tetranectins, polycystins, attractins, CTLD/acidic neck lectins, endosialins and any combination thereof.
  • the cell culture device also comprises a precoating comprising a carbohydrate.
  • a "cell culture device” as used herein means a receptacle that can contain media for culturing a cell or tissue.
  • the cell culture device may include glass, ceramics, metal, plastic or any combination thereof.
  • the plastic is non-cytotoxic.
  • the plastic may include natural polymers and/or synthetic polymers.
  • Exemplary cell culture devices include, but are not limited to, single and multi-well plates, including 6-well and 12-well culture plates, and smaller welled culture plates such as 96-, 384-, and 1536-well plates, culture jars, culture dishes, petri dishes, culture flasks, culture plates, culture roller bottles, culture slides, including chambered and multi-chambered culture slides, culture tubes, coverslips, cups, spinner bottles, perfusion chambers, bioreactors, and fermenters.
  • single and multi-well plates including 6-well and 12-well culture plates, and smaller welled culture plates such as 96-, 384-, and 1536-well plates, culture jars, culture dishes, petri dishes, culture flasks, culture plates, culture roller bottles, culture slides, including chambered and multi-chambered culture slides, culture tubes, coverslips, cups, spinner bottles, perfusion chambers, bioreactors, and fermenters.
  • a cell culture device as described herein may also be used in a process for determining cellular avidity as described herein.
  • a cell culture device as described herein may also be used in a process for sorting or screening cells of interest as described herein.
  • a cell culture device as described herein may also be used in a process for screening candidate agents for modulating cellular avidity between cells as described herein.
  • the carbohydrate is selected from the group consisting of mannose, fucose, xylose, arabinose, acetylglucosamine, fructose, sialic acid, glucuronic acid, galactose, N- acetylgalactosamine, any derivative and mixtures thereof.
  • the carbohydrate is carbohydrate-R, wherein R is polyethylene glycol (PEG).
  • R polyethylene glycol
  • An example of a derivative of mannose and an example of a carbohydrate-R is mannose-6-R, wherein R is polyethylene glycol (PEG). So, in an embodiment the carbohydrate is mannose-6- R, wherein R is polyethylene glycol (PEG).
  • the carbohydrate comprised in the second coating is mannose-6-R, wherein R is polyethylene glycol (PEG).
  • R polyethylene glycol
  • the carbohydrate in the precoating is carbohydrate-R, wherein R is a chemical moiety capable of reacting with surface-bound OH groups such as phosphate or silane.
  • the carbohydrate comprised in the second coating is mannose-6-R, wherein R is polyethylene glycol (PEG).
  • the carbohydrate- binding protein is a lectin.
  • the lectin is selected from the group consisting of concanavalin A (ConA), lentil lectins (LCH), snowdrop lectins (GNA), proteoglycans, type II transmembrane receptor lectins, collectins, selectins, natural killer lectins, macrophage mannose receptor (MMR) lectins, type I receptor lectins, tetranectins, polycystins, attractins, CTLD/acidic neck lectins, endosialins and any combination thereof.
  • the microfluidic devices and/or cell culture devices as described herein can be used for cell avidity measurements and for sorting or selecting of cells of interest, they however may also be used to conduct one or more of a wide range of investigations of interactions between cells and one or more substances.
  • Such interactions to be investigated may include, but are not limited to, an effect of the substance(s) on one or more of: cell attachment, cell growth, cell survival, cell differentiation, cell apoptosis/death and multicellular structure.
  • the activity or interaction of substances on cell motility, cell migration, cell to cell interactions as well as cell-protein signal interactions are also important activities that can be studied using the microfluidic devices and/or cell culture devices as described herein.
  • Substances of interest in such investigations may include, but are not limited to, pharmaceutical compounds or other therapeutics, exosomes, nanomicelles, nanoparticles, toxins, small molecules, nucleic acids (including nucleic acid vectors), oligonucleotides (including antisense oligonucleotides), oligopeptides, proteins, ribozymes, small interfering RNAs, microRNAs, short hairpin RNAs, aptamers, viruses, and antibodies or antigen binding parts thereof.
  • nucleic acids including nucleic acid vectors
  • oligonucleotides including antisense oligonucleotides
  • oligopeptides proteins
  • ribozymes small interfering RNAs
  • microRNAs microRNAs
  • short hairpin RNAs aptamers
  • viruses and antibodies or antigen binding parts thereof.
  • the present application also describes a kit of parts comprising a microfluidic device comprising a surface, a carbohydrate, and a carbohydrate-binding protein.
  • the present application also describes a kit of parts comprising a microfluidic device comprising a surface comprising a coating comprising a carbohydrate-binding protein, and a carbohydrate.
  • a microfluidic device present in the kit of parts refers to a device having the required structural configuration of a microfluidic devices as described herein, but in which a first coating comprising a carbohydrate-binding protein and/or a second coating comprising a carbohydrate is yet to be formed.
  • the kit may additionally comprise the carbohydrate present in the precoating.
  • kits of parts as described herein may be stored in one or more containers prior to their application and/or use.
  • kits of parts as described herein may be multi-pack kits wherein different components are stored in a plurality of containers.
  • a kit of parts as described herein may further comprise instructions for coating the surface with the carbohydrate and/or the carbohydrate-binding protein and, optionally, instructions for contacting and attaching cells with a surface coated with the carbohydrate-binding protein to provide a microfluidic device comprising a surface comprising cells.
  • the present application describes a process for preparing a microfluidic device comprising a surface comprising cells, the process comprising the steps of:
  • the present application also describes a process for preparing a microfluidic device comprising a surface comprising cells, the process comprising the steps of:
  • a microfluidic device comprising a carbohydrate-binding protein coated surface, wherein the surface optionally comprises a coating comprising a carbohydrate between the surface and the coating comprising the carbohydrate- binding protein,
  • the present application also describes a process in accordance with any one of the two previously described processes, wherein the carbohydrate is selected from the group consisting of mannose, fucose, xylose, arabinose, acetylglucosamine, fructose, sialic acid, glucuronic acid, galactose, N-acetylgalactosamine, any derivative and mixture thereof.
  • the present application also describes a process in accordance with any one of the previously described processes, wherein the carbohydrate-binding protein is a lectin.
  • the present application also describes a process in accordance with the previously described process, wherein the lectin is selected from the group consisting of concanavalin A (ConA), lentil lectins (LCH), snowdrop lectins (GNA), proteoglycans, type II transmembrane receptor lectins, collectins, selectins, natural killer lectins, macrophage mannose receptor (MMR) lectins, type I receptor lectins, tetranectins, polycystins, attractins, CTLD/acidic neck lectins, endosialins and any combination thereof.
  • ConA concanavalin A
  • LCH lentil lectins
  • GDA snowdrop lectins
  • proteoglycans type II transmembrane receptor lectins
  • collectins collectins, selectins, natural killer lectins, macrophage mannose receptor (MMR) lectins, type I receptor lectins,
  • the present application also describes a process in accordance with any one of the previously described processes, wherein the cells are target cells or effector cells.
  • the present application also describes a process for determining cellular avidity comprising:
  • a microfluidic device comprising a surface comprising cells, wherein the surface comprises a first coating comprising a carbohydrate-binding protein, cells attached to the first coating, a second coating comprising a carbohydrate and optionally a precoating comprising a carbohydrate, wherein the precoating is located between the surface and the first coating,
  • the present application also describes a process in accordance with the previously described process, wherein the cells attached to the first coating are target cells and the cells of interest are effector cells.
  • the present application also describes a process in accordance with any one of the two previously described processes, wherein control cells of the target cells or the cells of interest are provided and the cellular avidity of the control cells is determined as well.
  • the present application also describes a process in accordance with any one of the three previously described processes, wherein the cellular avidity is determined by exerting a force on the cells of interest.
  • the present application also describes a process in accordance with the previously described process, wherein the force is an acoustic force, a shear flow force or a centrifugal force.
  • the present application also describes a process in accordance with any one of the five previously described processes, wherein the microfluidic device comprising a surface comprising cells is prepared by performing a process according to any of the first six described processes.
  • the present application also describes a microfluidic device comprising a surface, wherein the surface comprises:
  • a precoating comprising a carbohydrate, wherein the precoating is located between the surface and the first coating.
  • the present application also describes a kit of parts comprising:
  • a microfluidic device comprising a surface
  • kits of parts comprising: • a microfluidic device comprising a surface comprising a coating comprising a carbohydrate-binding protein, and
  • the present application describes a process for preparing a microfluidic device comprising a coated surface, the process comprising the steps of:
  • the present application describes a process for preparing a microfluidic device comprising a coated surface, the process comprising the steps of:
  • the present application also describes a process for preparing a microfluidic device comprising a coated surface, the process comprising the steps of:
  • a microfluidic device comprising a carbohydrate-binding protein coated surface, wherein the surface optionally comprises a coating comprising a carbohydrate or derivative thereof between the surface and the coating comprising the carbohydrate-binding protein,
  • the present application also describes a process for preparing a microfluidic device comprising a coated surface, the process comprising the steps of:
  • a microfluidic device comprising a carbohydrate-binding protein coated surface, wherein the surface comprises a coating comprising a carbohydrate or derivative thereof between the surface and the coating comprising the carbohydrate-binding protein,
  • the present application also describes a process for preparing a microfluidic device comprising a surface comprising cells, the process comprising the steps of:
  • the surface is activated before a coating is applied.
  • the surface is chemically activated, for example with sodium hydroxide. Activation can be done to facilitate application of a coating.
  • a microfluidic device comprising a surface comprising cells (e.g. target cells) as described in the prior art
  • the surface is coated with a compound to which cells (e.g. target cells) can attach.
  • Subsequent contacting of the cells with the coated surface generally leads to cell monolayers with a high confluency. High confluency leads to increased nutrient consumption and decreased ability of keeping the cells of the monolayer alive for extended periods of time, especially without introduction of fresh nutrient which is considered to be impractical.
  • a microfluidic device comprising a surface coated with a carbohydrate-binding protein, wherein the coated surface before the addition of cells is coated with a carbohydrate or derivative thereof, for example a mix of carbohydrates or derivatives thereof as described herein. Coating with the carbohydrate or derivative thereof enables control of how many carbohydrate-binding proteins are available for binding to the cells. Thus, confluency of the cell monolayer can be manipulated to a desirable level.
  • a mix of carbohydrates or derivatives thereof such as for example a mix of mannose or mannose-6-polyethylene glycol (mannose-6-PEG) with mannose-6-R, wherein R is selected from the group consisting of a polyethylene glycol- NHS ester, a polyethylene glycol-antibody, a polyethylene-maleimide, a polyethylene- SPAC and a polyethylene-SPOCQ gives maximum flexibility with regard to modulation of the attachment rate of cells onto the coated surface, because mannose or mannose- 6-PEG have a blocking effect on the attachment of cells to the surface coated with the carbohydrate-binding protein, while mannose-6-R, wherein R is selected from the group consisting of a polyethylene glycol-NHS ester, a polyethylene glycol-antibody, a polyethylene-maleimide, a polyethylene-SPAC and a polyethylene-SPOCQ, has the opposite effect on the attachment of cells to the surface.
  • a surface on which a coating has been applied is known herein as a "coated surface.”
  • the term “single-coated surface” as used herein means a surface that comprises at least a coating layer comprising at least a carbohydrate-binding protein. In an embodiment the single-coated surface comprises at least a carbohydrate-binding protein as described herein.
  • the term “multiple-coated surface” as used herein means a surface that comprises at least two coating layers. In an embodiment the multiple-coated surface comprises a first coating layer comprising at least a carbohydrate-binding protein and at least a second coating layer comprising at least a carbohydrate or derivative thereof, for example a carbohydrate or derivative thereof as described herein.
  • the multiple-coated surface may comprise additional coating layers.
  • An additional coating layer located between the surface and the first coating layer may be called a precoating.
  • the multiple-coated surface comprises a precoating.
  • the surface may be coated multiple times with the precoating.
  • the precoating comprises a carbohydrate or derivative thereof.
  • the multiple-coated surface may comprise cells attached to the surface.
  • the surface may be coated multiple times with the first coating layer and/or multiple times with the second coating layer. In other words, the coating process can be repeated numerous times until the desired order of coating layers is achieved.
  • the surface does not comprise any additional coating layers next to the first coating layer and the second coating layer.
  • the first coating comprises more than one carbohydrate-binding protein.
  • the second coating comprises more than one carbohydrate.
  • the first coating comprises more than one carbohydrate-binding protein and the second coating also comprises more than one carbohydrate.
  • this precoating layer may comprise more than one carbohydrate. With “more than one” is meant “a mixture of”.
  • the cells form a monolayer on the surface. In an embodiment the cells attach to the carbohydrate-binding protein. In an embodiment the cells attach to the carbohydrate or derivative thereof. In an embodiment the cells attach to the carbohydrate-binding protein and to the carbohydrate or derivative thereof.
  • the surface is a glass surface, a plastic surface, a metal surface, a ceramic surface or any combination thereof. In a preferred embodiment the surface is a glass surface, a plastic surface, a ceramic surface or any combination thereof.
  • the microfluidic device may be prepared from any suitable material including plastic, glass, ceramics, metal or any combination thereof. Preferably, the plastic is non-cytotoxic.
  • the plastic may include natural polymers and/or synthetic polymers.
  • the cell monolayer shows a high quality.
  • Quality can be measured for example by looking at the confluency of the cell monolayer.
  • the confluency should be 50% or higher, preferably 60% or higher, more preferably 70% or higher.
  • the advantage of the present invention is that the confluency can be manipulated to desired levels.
  • Quality can also be measured for example by looking at the dumpiness of the cell monolayer.
  • the cell monolayer does not show dumpiness.
  • Quality can also be measured for example by looking at the stability and/or robustness of the cell monolayer. Stability and/or robustness can be measured for example by measuring the resistance of the cell monolayer when subjected to shear flow force and/or acoustic force and/or centrifugal force.
  • the cells are cultured. Culturing of the cells may take place before, simultaneously with and/or after contacting the cells with the surface, e.g. the coated surface, and allowing the cells to attach to the surface to provide a microfluidic device comprising a surface comprising cells.
  • the carbohydrate is selected from the group consisting of mannose, fucose, xylose, arabinose, acetylglucosamine, fructose, sialic acid, glucuronic acid, galactose, N-acetylgalactosamine, any derivative or mixture thereof.
  • the carbohydrate comprised in the second coating and/or precoating is selected from the group consisting of mannose, fucose, xylose, arabinose, acetylglucosamine, fructose, sialic acid, glucuronic acid, galactose, N- acetylgalactosamine, any derivative or mixture thereof.
  • the carbohydrate is carbohydrate-R, wherein R is selected from the group consisting of a polyethylene glycol-NHS ester, a polyethylene glycol-antibody, a polyethylene-maleimide, a polyethylene-SPAC and a polyethylene-SPOCQ.
  • R is selected from the group consisting of a polyethylene glycol-NHS ester, a polyethylene glycol-antibody, a polyethylene-maleimide, a polyethylene-SPAC and a polyethylene-SPOCQ.
  • R is a chemical moiety capable of reacting with surface-bound OH groups such as phosphate or silane.
  • An example of a derivative of mannose and an example of a carbohydrate-R is mannose-6-R, wherein R is selected from the group consisting of a polyethylene glycol- NHS ester, a polyethylene glycol-antibody, a polyethylene-maleimide, a polyethylene- SPAC and a polyethylene-SPOCQ.
  • R is selected from the group consisting of a polyethylene glycol- NHS ester, a polyethylene glycol-antibody, a polyethylene-maleimide, a polyethylene- SPAC and a polyethylene-SPOCQ.
  • the carbohydrate comprised in the second coating is mannose-6-R, wherein R is selected from the group consisting of a polyethylene glycol-NHS ester, a polyethylene glycol-antibody, a polyethylene- maleimide, a polyethylene-SPAC and a polyethylene-SPOCQ.
  • the carbohydrate comprised in the second coating is a mix of mannose-6-R, wherein R is selected from the group consisting of a polyethylene glycol-NHS ester, a polyethylene glycol-antibody, a polyethylene-maleimide, a polyethylene-SPAC and a polyethylene- SPOCQ, and mannose/mannose-6-PEG.
  • the ratio of mannose-6-R, wherein R is selected from the group consisting of a polyethylene glycol-NHS ester, a polyethylene glycol-antibody, a polyethylene-maleimide, a polyethylene-SPAC and a polyethylene- SPOCQ, and mannose/ mannose-6-PEG can be selected based on the specific needs.
  • the carbohydrate in the precoating is mannose-6-R, wherein R is a chemical moiety capable of reacting with surface-bound OH groups such as phosphate or silane.
  • the carbohydrate comprised in the second coating differs from the carbohydrate comprised in the precoating. In an embodiment the carbohydrate comprised in the second coating is the same as the carbohydrate comprised in the precoating.
  • the carbohydrate-binding protein is a lectin.
  • the lectin is selected from the group consisting of concanavalin A (ConA), lentil lectins (LCH), snowdrop lectins (GNA), proteoglycans, type II transmembrane receptor lectins, collectins, selectins, natural killer lectins, macrophage mannose receptor (MMR) lectins, type I receptor lectins, tetranectins, polycystins, attractins, CTLD/acidic neck lectins, endosialins and any combination thereof.
  • the lectin is concanavalin A (ConA).
  • one or more active domains of a carbohydrate-binding protein may also be used in any of the processes for preparing a microfluidic device as described herein.
  • An "active domain” refers to an amino acid sequence found within the carbohydrate-binding protein that, in itself, provides function according to one or more properties of the carbohydrate-binding protein, such as providing structural support to cells and/or for attaching cells and/or for attaching a carbohydrate.
  • the peptide that includes an active domain of a carbohydrate-binding protein can have a "core sequence" of amino acid residues, and optionally one or more additional amino acid residues that flank (/.e., on the C-terminus, N-terminus, or both) the core sequence.
  • the one or more additional amino acids that flank the core sequence can correspond to the wild-type sequence in the relevant region of the protein, or can comprise one or more amino acid(s) that diverge from the wild-type sequence (e.g., a "variant amino acid sequence").
  • the variant amino sequence can be one that enhances properties of the peptide, such as providing enhanced ligand interaction.
  • a "peptide” is a short polymer of 25 or less amino acids linked by peptide bonds.
  • a "polypeptide” is a polymer of more than 25 amino acids linked by peptide bonds and which includes full length proteins.
  • a peptide having an active portion of a carbohydrate-binding protein can be synthesized by solid phase peptide synthesis (SPPS) techniques using standard techniques, such as Fmoc synthesis.
  • SPPS solid phase peptide synthesis
  • the first coating layer comprising the carbohydrate-binding protein may comprise other excipients and/or components as well.
  • the carbohydrate-binding protein is present in the first coating layer in an amount of at least 10% (w/w) (weight percentage of the carbohydrate-binding protein based on the total weight of the first coating layer comprising the carbohydrate-binding protein), at least 20% (w/w), at least 30% (w/w), at least 40% (w/w), at least 50% (w/w), at least 60% (w/w), at least 70% (w/w), at least 80% (w/w), at least 90% (w/w) or at least 100% (w/w).
  • the second coating layer comprising the carbohydrate and/or the precoating layer comprising a carbohydrate may comprise other excipients and/or components as well.
  • the carbohydrate is present in the second coating layer and/or the precoating layer in an amount of at least 10% (w/w) (weight percentage of the carbohydrate based on the total weight of the second coating layer comprising the carbohydrate), at least 20% (w/w), at least 30% (w/w), at least 40% (w/w), at least 50% (w/w), at least 60% (w/w), at least 70% (w/w), at least 80% (w/w), at least 90% (w/w) or at least 100% (w/w).
  • the first (/.e. carbohydrate-binding protein comprising) and second (/.e. carbohydrate comprising) coating and any additional coating layer can be prepared to have a desired thickness.
  • the coated layers have a thickness in the range of about 2 nm to about 20 nm.
  • the first and second coating and any additional coating layer can be prepared to have a desired density and/or concentration.
  • the coating process involves placing the coating materials in contact with the device surface.
  • the device surface has been pretreated with a base coat.
  • the coating materials are applied to a surface and dried down or partially dried down.
  • the process of applying can be performed using any one of a variety of techniques including dipping, pouring, swabbing, siphoning, brushing, rolling, padding, ragging, painting, spraying, anodizing, electroplating, and/or laminating.
  • One exemplary method for applying a coating composition is by dip coating.
  • a typical dip coating procedure involves immersing the surface to be coated in a first coating composition, dwelling the object in the composition for a period of time, and then removing the surface from the composition. After the surface has been dip coated in the coating solution, it is removed and dried or partially dried. Drying can be carried out using any suitable method, including air drying the surface.
  • Another exemplary method for applying a coating composition is flowing the coating composition into a microfluidic device in order to coat the inner surface(s) of the microfluidic device.
  • the processes as described herein also comprise the step of contacting the microfluidic device comprising a surface comprising cells with a blocking agent.
  • the blocking agent may even further reduce background binding to the cells.
  • suitable blocking agents include, but are not limited to, N- hydroxysuccinimide (NHS) ester reagents.
  • An NHS ester reagent as defined herein is a compound comprising at least one succinimidyl group, which functional group is capable of forming an ester bond with a primary amine group on polypeptides, such as, for example, the amine groups as comprised in lysine residues, such that these amine groups are no longer available to attach to, for example, cells of interest.
  • the NHS ester reagent comprises a spacer, e.g. a polymer of a relative short length, up to about 100 Angstrom.
  • a polymer preferably is inert.
  • the polymer is polyethylene glycol (PEG). More preferably, the PEG length is up to 25 ethylene glycol units in length. Preferably, the number of units is selected from the range of 2 to 25, or from 3 to 10.
  • the NHS ester is a PEG-NHS ester.
  • Blocking may take place by providing a buffer suitable for the blocking reaction and sustaining cell viability of the cells present on the surface, e.g. phosphate, carbonatebicarbonate, HEPES or borate buffers at pH 7.2 to 8.5 for 0.5 to 4 h at room temperature or 4°C.
  • a buffer suitable for the blocking reaction e.g. phosphate, carbonatebicarbonate, HEPES or borate buffers at pH 7.2 to 8.5 for 0.5 to 4 h at room temperature or 4°C.
  • the NHS ester reagent is removed and the cells may, for example, be washed with serum containing medium or with a primary amine buffer such as Tris or glycine, which are not compatible because they compete for reaction, to quench (stop) the reaction.
  • NHS esters are known in the art, and it may be contemplated to utilize other compounds equally capable of reacting with amine groups as an alternative.
  • These compounds include, but are not limited to, isothiocyanates, isocyanates, acyl azides, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, carbodiimides, anhydrides, and fluorophenyl esters. Most of these compounds conjugate to amines by either acylation or alkylation. Without being bound by theory, as long as the compound is capable of reacting with the constituents responsible for the coating on the surface, it can be contemplated.
  • the cells are target cells or effector cells.
  • the cells are target cells.
  • the target cells are cells presenting an antigen.
  • a tumor antigen a viral antigen or a bacterial antigen.
  • Suitable target cells may include eukaryotic cells or prokaryotic cells.
  • Target cells that may be selected include fungal cells, animal cells, insect cells, mammalian cells, bacterial cells, yeasts, and protozoa.
  • the target cells are human cells.
  • the target cells are tumor cells such as human tumor cells.
  • the effector cells are human effector cells such as T cells, NK cells, B cells, dendritic cells, macrophages, or monocytes.
  • the effector cells may be genetically modified. When the genetic modification is with a CAR, cells such as CAR T cells can be obtained. Effector cells as used herein may thus also be CAR T cells.
  • extraneous DNA is removed from the cells before contacting the cells with the coated surface.
  • extraneous DNA is removed from the cells by treatment of the cells with a deoxyribonuclease (DNAse).
  • DNAse deoxyribonuclease
  • the present application also describes a process for determining cellular avidity comprising:
  • a microfluidic device comprising a surface comprising cells, wherein the surface comprises a coating comprising a carbohydrate-binding protein, a coating comprising a carbohydrate of derivative thereof and cells attached to the surface, and optionally a precoating comprising a carbohydrate or derivative thereof, wherein the precoating is located between the surface and the coating comprising a carbohydrate-binding protein,
  • the present application also describes a process for determining cellular avidity comprising:
  • a microfluidic device comprising a surface comprising cells, wherein the surface comprises a coating comprising a carbohydrate-binding protein, a coating comprising a carbohydrate of derivative thereof and cells attached to the surface, and a precoating comprising a carbohydrate or derivative thereof, wherein the precoating is located between the surface and the coating comprising a carbohydrate-binding protein,
  • the present application also describes a process for determining cellular avidity comprising:
  • cellular avidity is defined by the overall strength of interactions occurring in a cell-to-cell contact, involving a diversity of molecules at the surfaces of the cells that interact.
  • cellular avidity is a parameter that expresses the strength of binding between cells. It involves a multitude of interactions, including a diversity of receptor-ligand pairs, among which e.g. a specific receptor-ligand interaction, occurring at the membrane surface of a cell, working jointly forming a strong bond between cells. It may also involve active signalling and processes internal to the cells such as e.g. during immune synapse formation. It is understood that the cellular avidity of a cell of a certain type is defined relative to its target cell and conditions tested.
  • target cells and cells of interest relate to two different cells which are to interact specifically with each other.
  • the target cells or the cells of interest may express a ligand on their surface and the other cell may express a receptor for that ligand.
  • control cells the same cells as the cells of interest or target cells may be utilized, but these cells do not express such a ligand or receptor or express a variant thereof which is not functional.
  • the cells attached to the surface e.g. target cells
  • cells of interest e.g. effector cells
  • control cells thereof it is understood that this may involve cells that are to have a specific interaction, i.e. one cell carrying a receptor and the other cell having a ligand for the receptor.
  • ligand and receptor in this sense and in accordance with the invention may define their inter-relationship.
  • receptor may not be construed to be limiting in any way and is understood to mean a protein presented at the cell surface which can (specifically) interact with another protein (ligand) presented at another cell.
  • ligand and receptor are used to indicate a complementarity which is important for specific recognition between cells without restrictions on the complementary molecules that can be contemplated.
  • Receptors that may be of interest include, but are not limited to, a CAR, a TCR, a stimulatory or inhibitory co-receptor, or a receptor engaged via a bispecific antibody.
  • the cellular avidity may be determined. This is for example of interest when it is desirable to provide for a candidate CAR.
  • candidates can be selected and subsequently assessed with regard to functionality, e.g. by in vivo experiments.
  • cellular avidity is an important parameter for function, this way highly efficiently, suitable candidates can be selected.
  • the cellular avidity is determined.
  • the microfluidic device comprising a surface comprising cells is prepared by performing a process for preparing a microfluidic device comprising a surface comprising cells as described herein.
  • the cells are target cells or effector cells.
  • the cells of interest are target cells or effector cells.
  • the cells attached to the surface are target cells and the cells of interest are effector cells.
  • the target cells are cells presenting an antigen.
  • a tumor antigen a viral antigen or a bacterial antigen.
  • Suitable target cells may include eukaryotic cells or prokaryotic cells.
  • Target cells that may be selected include fungal cells, animal cells, insect cells, mammalian cells, bacterial cells, yeasts, and protozoa.
  • the target cells are human cells.
  • the target cells are tumor cells such as human tumor cells.
  • the effector cells are human effector cells such as T cells, NK cells, B cells, dendritic cells, macrophages, or monocytes.
  • the effector cells may be genetically modified. When the genetic modification is with a CAR, cells such as CAR T cells can be obtained. Effector cells as used herein may thus also be CAR T cells.
  • the cells that are attached to the surface are preferably attached as a monolayer.
  • the subsequent cells of interest (and control cells thereof) that are to interact with the cells attached to the surface are preferably provided in a relatively low cell density as compared with those cells, such that substantially all cells of interest (and control cells thereof) can interact with those cells. In other words, there are more cells per cell of interest. Such provides for advantageous controllable conditions when applying the force on the cells of interest or the control cells thereof.
  • control cells of the target cells or the cells of interest are provided and the cellular avidity of the control cells is determined as well.
  • control cells of the cells that are attached to the surface and/or control cells of the cells of interest are provided and the cellular avidity of the control cells is determined as well.
  • the process for determining cellular avidity as described herein may be performed separately with cells of interest and their control cells and/or with cells that are attached to the surface and their control cells.
  • the process for determining cellular avidity as described herein may comprise the step of incubating the cells attached to the surface with the cells of interest and/or their control cells.
  • the process for determining cellular avidity as described herein may comprise the step of contacting cells of interest and/or their control cells with target cells. Control cells and/or cells of interest that have detached and/or remained attached are determined and cellular avidity scores are provided for the control cells and/or cells of interest with the cells that are attached to the surface.
  • control cells of the cells of interest instead of providing control cells of the cells of interest, alternatively control cells of the cells that are attached to the surface may be provided attached to a surface.
  • cells of interest are contacted with the cells that are attached to the surface and before or thereafter cells of interest are contacted with the control cells of the cells that are attached to the surface, and in each case cells of interest that have detached and/or remained attached (after force application as described herein) are determined and cellular avidity scores are provided for the cells of interest with the control cells of the cells that are attached to the surface and with the cells that are attached to the surface as such.
  • the cellular avidity scores obtained for cells and their respective controls can be compared and are of interest. Measuring the cellular avidity with and without control cells allows to better separate specific from non-specific binding.
  • control cells when cells of interest are used that express a specific ligand or receptor, control cells are used that are the same cells as the cells of interest but do not express the specific ligand or receptor or express a variant of the specific ligand or receptor which is not functional or not specific for the counterpart present on the cells that are attached to the surface, e.g. the target cells. Obviously, the same is true vice versa.
  • the cellular avidity is determined by exerting a force on the cells of interest.
  • a force can be applied and controlled on cells of interest that bind/interact/attach with cells attached to a surface, e.g. target cells, such a force can be contemplated in accordance with the invention.
  • the force is an acoustic force, a shear flow force or a centrifugal force.
  • cellular avidity is measured using the z-Movi® device as available from the company LUMICKS.
  • the device makes use of an acoustic force.
  • a microfluidic device as described herein can be used in the z-Movi® device. It may even be used repeatedly.
  • cellular avidity it is to be understood that this is to express the strength of binding of cells of interest (or control cells thereof) to cells attached to a surface (or control cells thereof). It is to be understood that where it is referred to specific forces applied to cells, this may refer to average forces, e.g. such forces may not be fully homogeneous, for example over the contact surface.
  • the cellular binding avidity is determined by exerting a force on the cells of interest, e.g. effector cells, away from the cells that are attached to the surface, e.g. target cells.
  • the force applied may be perpendicular (in the direction of z-axis) to the surface (x,y) to which the cells that are attached to the surface are attached, for example when a centrifugal force or acoustic force is applied.
  • the force may also be lateral (x-axis or y-axis), for example when a shear force is applied.
  • the force is applied and is controlled such that a defined force is exerted on the cells of interest that interact with the cells that are attached to the surface.
  • the force that is exerted on the cells of interest attached to the cells that are attached to the surface is to be substantially equal. This can be achieved when using for example a flat surface.
  • Other suitable surface shapes may be used (e.g. a tube with exerted concentrical force or laminar flow force in the direction of the length of the tube), as long as the force exerted can be substantially equal at a defined surface area, such a surface shape may be contemplated.
  • the force required to move a cell of interest away from the cells that are attached to the surface can be detected optically, e.g. via microscopy or other means. Cell detachment events can be monitored and counted.
  • the process for determining cellular avidity may further comprise the step of collection of cells, for example, collection of cells that experienced a defined force.
  • the cells of interest may be provided with a photoactivatable label which may subsequently be activated by illumination with light of a suitable wavelength only in a well-defined interaction region of the device (e.g. in a center region under an (acoustic) force transducer) to photoactivate and/or switch on the dye.
  • the cells can be sorted, for example, using fluorescence activated cell sorting (FACS) and only those cells which are activated are collected thereby obtaining the cells on which defined forces have been exerted.
  • FACS fluorescence activated cell sorting
  • the target cells and cells of interest bound thereto may be trypsinized thereby obtaining both the target cells and cells of interest that remained bound thereto in a suspension.
  • attached cells of interest can also be simply collected with physical means (e.g. by scraping) from the area of interest, i.e. the surface area with a well-defined nominal force.
  • the exact forces experienced by cells may also depend on cell size and or other cell properties such as density and compressibility.
  • the force may thus be a nominal force and not the true force experienced by the cells. Since it may be hard to precisely predict the average cell size, density, compressibility, etc. of the cells, the force may have been calculated based on theory alone or may have been calibrated using test particles with specific properties (see Kamsma, D. et al. (2016). Tuning the Music: Acoustic Force Spectroscopy (AFS) 2.0. Methods, 105, 26-33).
  • the force may be a calculated or calibrated force expressed with units of N (e.g.
  • Vpp input power
  • a piezo element see Sitters, G. et al. (2014). Acoustic force spectroscopy. Nature Methods, 12(1), 47-50), as angular velocity squared (&j 2 ) in the case of centrifugal forces or as flow speed v and or as shear stress (Pa) in case of shear forces.
  • &j 2 angular velocity squared
  • Pa shear stress
  • the percentage of cells of interest that remains bound at a certain applied force is indicative of cellular avidity, i.e. the larger the percentage of cells of interest that is bound, the higher the cellular avidity, it is useful to refer to the percentage of cells.
  • a different measure which relates to cellular avidity may be used, for example the percentage of detached cells, wherein conversely a low number is indicative of a higher cellular avidity.
  • percentage the ratio of cells that remain attached or are detached divided by the total number of cells that interacted may be provided. In case of a cellular avidity plot, the area under (or above) the curve may be determined.
  • a unit that is representative of the number of cells of interest that have detached or cells of interest that have remained attached, relative to the total number of cells that have interacted.
  • Such a unit allows for ranking cellular avidities, when comparing e.g. different cells of interest.
  • Providing such a unit may be referred to as providing a cellular avidity score.
  • a preferred cellular avidity score may be the percentage of cells of interest that remains attached relative to the cells of interest that have interacted, the latter being set at 100%.
  • a cellular avidity score is preferably provided.
  • any suitable force application method may be contemplated in processes for determining cellular avidity as described herein.
  • increasing the force is well controlled.
  • the applied force is a force ramp, preferably a linear force ramp. It is understood that when a force is selected to be applied it can be a constant force applied for a defined period.
  • the forces applied may be in various forms as a function of time.
  • the applied force is an increasing force. That is, after the incubation step, an increasing force is applied for a defined period until a defined end force is reached.
  • a linear force ramp may be applied for 150 seconds resulting in a defined end force of 1000 pN.
  • the ratio or difference between the cellular avidity of cells of interest and control cells is determined.
  • the ratio of the cellular avidity between cells of interest and control cells is preferably as large as possible.
  • control cell background binding is about 15% or less and cells of interest binding is about 85% or more, for example from 85-95%.
  • the percentage difference between cellular avidity of control cells and cells of interest is at least 30%, at least 40%, at least 50%, at least 60% at least 70%, at least 80% and preferably at least 90%.
  • conditions are selected with the largest difference between the percentages. In an embodiment wherein e.g.
  • the present application also describes processes for identifying a candidate agent capable of modulating the cellular avidity between a cell of interest and a cell that is attached to the surface, wherein in the incubation step, and cellular avidity determination step of the processes as described herein agent(s) are provided and included in these steps. This way, when it is for instance of importance to block or enhance a particular interaction, agent(s) capable of doing so can be identified.
  • the cells that are attached to the surface can also be used in processes for screening of different cells of interest, by performing the processes as described herein, optionally in the presence of agent(s) as defined above, and comparing determined cellular avidities for the different cells of interest.
  • agent(s) as defined above
  • cellular avidities for the different cells of interest.
  • a candidate agent is provided for modulating the avidity between a cell of interest and a cell that is attached to the surface, and wherein said agent is present in the incubation step and step of determining cellular avidity.
  • the processes for determining cellular avidity as described herein may comprise the step of providing a cell engager.
  • Cell engagers include antibodies, or the like, which are capable of binding to a target cell and a cell of interest, e.g. an effector cell.
  • Such antibodies may include single chain antibodies comprising two binding domains such as scFv domains, and include BiTEs (/.e. bispecific T-cell engagers) or the like.
  • a conventional antibody design includes heavy and light chains, with one half of the antibody (one heavy chain and one light chain) engaging with a target cell, and the other half of the antibody (another heavy chain and another light chain) engaging with an effector cell, wherein preferably, the Fc domain is made inert.
  • suitable cell engagers are widely known in the art and the current invention allows to study and/or determine cellular avidity induced by a cell engager between a target cell and an effector cell.
  • a cell engager may be provided in addition and the cellular avidity score may be determined, induced by said cell engager, between a cell of interest (e.g. an effector cell) and a target cell.
  • said cell engager has a binding region capable of binding the cell of interest (e.g. an effector cell) and a binding region capable of binding the target cell. It is understood that the incubation step in the processes as described herein can thus be performed in the presence of a cell engager to allow the cells, i.e.
  • a cell engager is provided capable of binding an effector cell and a target cell, and the cell engager is included in the incubation step. Furthermore, such processes are highly useful for screening cell engagers, e.g. by identifying cell engagers that are particular capable of binding an effector cell and a target cell.
  • processes are provided for selecting and/or sorting cells of interest, comprising the steps of the processes as described herein, and optionally in the presence of agent(s) as defined above, comprising the further step of applying the selected force on the cells of interest and subsequently selecting and/or sorting cells of interest that have detached and/or that remain attached to the cells that are attached to the surface.
  • a process is provided comprising the steps of the processes of determining cellular avidity as described herein, wherein the process is for use in sorting and/or screening of cells of interest.
  • a microfluidic device comprising a surface comprising cells, wherein the surface comprises a coating comprising a carbohydrate-binding protein, a coating comprising a carbohydrate of derivative thereof and cells attached to the surface, and optionally a precoating comprising a carbohydrate or derivative thereof, wherein the precoating is located between the surface and the coating comprising a carbohydrate-binding protein,
  • a microfluidic device comprising a surface comprising cells, wherein the surface comprises a coating comprising a carbohydrate-binding protein, a coating comprising a carbohydrate of derivative thereof and cells attached to the surface, and a precoating comprising a carbohydrate or derivative thereof, wherein the precoating is located between the surface and the coating comprising a carbohydrate-binding protein,
  • the microfluidic device comprising a surface comprising cells is prepared by performing a process for preparing a microfluidic device comprising a surface comprising cells as described herein.
  • processes may be of use for the screening of candidate agents as well.
  • a further process is provided for screening candidate agents for modulating cellular avidity between of cells of interest and cells attached to a surface comprising the steps of:
  • a microfluidic device comprising a surface comprising cells, wherein the surface comprises a coating comprising a carbohydrate-binding protein, a coating comprising a carbohydrate of derivative thereof and cells attached to the surface, and optionally a precoating comprising a carbohydrate or derivative thereof, wherein the precoating is located between the surface and the coating comprising a carbohydrate-binding protein,
  • processes may be of use for the screening of candidate agents as well.
  • a further process is provided for screening candidate agents for modulating cellular avidity between of cells of interest and cells attached to a surface comprising the steps of:
  • a microfluidic device comprising a surface comprising cells, wherein the surface comprises a coating comprising a carbohydrate-binding protein, a coating comprising a carbohydrate of derivative thereof and cells attached to the surface, and a precoating comprising a carbohydrate or derivative thereof, wherein the precoating is located between the surface and the coating comprising a carbohydrate-binding protein,
  • the cells attached to the surface are incubated with the candidate agents followed by incubation with the cells of interest.
  • the candidate agents are incubated with the cells of interest followed by incubation of the cells attached to the surface with the candidate agents attached to the cells of interest.
  • the cells attached to the surface are incubated with the candidate agents and the cells of interest.
  • the cellular avidity score obtained in the presence of the candidate agents is compared to the cellular avidity score that is measured in the absence of the candidate agents. Based on the comparison, candidate agents that increase or decrease cellular avidity between the cells and the cells of interest can be identified and/or selected. Said candidate agents may subsequently in a further step be admixed with a pharmaceutically acceptable buffer or otherwise pharmaceutical acceptably formulated.
  • the microfluidic device comprising a surface comprising cells is prepared by performing a process for preparing a microfluidic device comprising a surface comprising cells as described herein.
  • Cells of interest which can be selected and/or sorted and thus be obtained accordingly with processes as described herein may subsequently in a further step be admixed with a pharmaceutically acceptable buffer or otherwise pharmaceutical acceptably formulated.
  • the present application also describes a microfluidic device obtainable by a process for preparing a microfluidic device comprising a surface comprising cells as described herein.
  • the present application also describes a microfluidic device obtained by a process for preparing a microfluidic device comprising a surface comprising cells as described herein.
  • the present application also describes a microfluidic device comprising a surface, wherein the surface comprises a coating comprising a carbohydrate-binding protein.
  • the present application also describes a microfluidic device comprising a surface, wherein the surface comprises a coating comprising a lectin.
  • the present application also describes a microfluidic device comprising a surface, wherein the surface comprises a coating comprising a lectin, and a coating comprising a carbohydrate or derivative thereof.
  • the present application also describes a microfluidic device comprising a surface, wherein the surface comprises a coating comprising concanavalin A.
  • the present application also describes a microfluidic device comprising a surface, wherein the surface comprises a coating comprising concanavalin A, and a coating comprising mannose and/or mannose-6-R, wherein R is selected from the group consisting of a polyethylene glycol-NHS ester, a polyethylene glycol-antibody, a polyethylene-maleimide, a polyethylene-SPAC and a polyethylene-SPOCQ.
  • the present application also describes a microfluidic device comprising a surface, wherein the surface comprises a coating comprising a carbohydrate-binding protein, a coating comprising a carbohydrate or derivative thereof and cells.
  • the present application also describes a microfluidic device comprising a surface, wherein the surface comprises a first coating comprising a carbohydrate-binding protein, a second coating comprising a carbohydrate or derivative thereof and cells.
  • the present application also describes a microfluidic device comprising a surface, wherein the surface comprises a first coating comprising a carbohydrate-binding protein, a second coating comprising a carbohydrate or derivative thereof and cells attached to the surface.
  • the present application also describes a microfluidic device comprising a surface, wherein the surface comprises:
  • a precoating comprising a carbohydrate or derivative thereof
  • the present application also describes a microfluidic device comprising a surface, wherein the surface comprises:
  • microfluidic device comprising a surface, wherein the surface comprises: • optionally, a precoating comprising a carbohydrate or derivative thereof,
  • the present application also describes a microfluidic device comprising a surface, wherein the surface comprises:
  • a microfluidic device as described herein comprises one or more internal spaces including a vessel, typically in the form of a flow channel.
  • the flow channel may be configured to convey a flow of fluid.
  • the microfluidic device as described herein may also comprise one or more compartments configured to contain one or more fluids.
  • the microfluidic device as described herein may also comprise one or more connectors for connecting purposes.
  • the microfluidic device comprises a unitary body in which the one or more internal spaces are at least partly formed.
  • the present application also describes a cell culture device comprising a surface, wherein the surface comprises a coating comprising at least a carbohydrate-binding protein.
  • the present application also describes a cell culture device comprising a surface, wherein the surface comprises a first coating comprising a carbohydrate-binding protein and a second coating comprising a carbohydrate or derivative thereof.
  • the present application also describes a cell culture device comprising a surface, wherein the surface comprises a first coating comprising a carbohydrate-binding protein, a second coating comprising a carbohydrate or derivative thereof and cells attached to the surface.
  • the carbohydrate is selected from the group consisting of mannose, fucose, xylose, arabinose, acetylglucosamine, fructose, sialic acid, glucuronic acid, galactose, N-acetylgalactosamine, any derivative and mixture thereof.
  • the carbohydrate is carbohydrate-R, wherein R is selected from the group consisting of a polyethylene glycol-NHS ester, a polyethylene glycol-antibody, a polyethylene-maleimide, a polyethylene-SPAC and a polyethylene-SPOCQ.
  • An example of a derivative of mannose and an example of a carbohydrate-R is mannose-6-R, wherein R is selected from the group consisting of a polyethylene glycol-NHS ester, a polyethylene glycol-antibody, a polyethylene-maleimide, a polyethylene-SPAC and a polyethylene-SPOCQ.
  • R is selected from the group consisting of a polyethylene glycol-NHS ester, a polyethylene glycol-antibody, a polyethylene-maleimide, a polyethylene-SPAC and a polyethylene-SPOCQ.
  • the carbohydrate is a mix of mannose/mannose-6-PEG and mannose-6-R, wherein R is selected from the group consisting of a polyethylene glycol-NHS ester, a polyethylene glycol-antibody, a polyethylene-maleimide, a polyethylene-SPAC and a polyethylene-SPOCQ).
  • R is selected from the group consisting of a polyethylene glycol-NHS ester, a polyethylene glycol-antibody, a polyethylene-maleimide, a polyethylene-SPAC and a polyethylene-SPOCQ.
  • the carbohydrate-binding protein is a lectin.
  • the lectin is selected from the group consisting of concanavalin A (ConA), lentil lectins (LCH), snowdrop lectins (GNA), proteoglycans, type II transmembrane receptor lectins, collectins, selectins, natural killer lectins, macrophage mannose receptor (MMR) lectins, type I receptor lectins, tetranectins, polycystins, attractins, CTLD/acidic neck lectins, endosialins and any combination thereof.
  • the cell culture device also comprises a precoating comprising a carbohydrate.
  • a "cell culture device” as used herein means a receptacle that can contain media for culturing a cell or tissue.
  • the cell culture device may include glass, ceramics, metal, plastic or any combination thereof.
  • the plastic is non-cytotoxic.
  • the plastic may include natural polymers and/or synthetic polymers.
  • Exemplary cell culture devices include, but are not limited to, single and multi-well plates, including 6-well and 12-well culture plates, and smaller welled culture plates such as 96-, 384-, and 1536-well plates, culture jars, culture dishes, petri dishes, culture flasks, culture plates, culture roller bottles, culture slides, including chambered and multi-chambered culture slides, culture tubes, coverslips, cups, spinner bottles, perfusion chambers, bioreactors, and fermenters.
  • single and multi-well plates including 6-well and 12-well culture plates, and smaller welled culture plates such as 96-, 384-, and 1536-well plates, culture jars, culture dishes, petri dishes, culture flasks, culture plates, culture roller bottles, culture slides, including chambered and multi-chambered culture slides, culture tubes, coverslips, cups, spinner bottles, perfusion chambers, bioreactors, and fermenters.
  • a cell culture device as described herein may also be used in a process for determining cellular avidity as described herein.
  • a cell culture device as described herein may also be used in a process for sorting or screening cells of interest as described herein.
  • a cell culture device as described herein may also be used in a process for screening candidate agents for modulating cellular avidity between cells as described herein.
  • the carbohydrate is selected from the group consisting of mannose, fucose, xylose, arabinose, acetylglucosamine, fructose, sialic acid, glucuronic acid, galactose, N- acetylgalactosamine, any derivative and mixture thereof.
  • the carbohydrate is carbohydrate-R, wherein R is selected from the group consisting of a polyethylene glycol-NHS ester, a polyethylene glycol-antibody, a polyethylene- maleimide, a polyethylene-SPAC and a polyethylene-SPOCQ.
  • An example of a derivative of mannose and an example of a carbohydrate-R is mannose-6-R, wherein R is selected from the group consisting of a polyethylene glycol-NHS ester, a polyethylene glycol-antibody, a polyethylene-maleimide, a polyethylene-SPAC and a polyethylene- SPOCQ.
  • R is selected from the group consisting of a polyethylene glycol-NHS ester, a polyethylene glycol-antibody, a polyethylene-maleimide, a polyethylene-SPAC and a polyethylene- SPOCQ.
  • the carbohydrate is a mix of mannose/mannose-6-PEG and mannose-6-R, wherein R is selected from the group consisting of a polyethylene glycol- NHS ester, a polyethylene glycol-antibody, a polyethylene-maleimide, a polyethylene- SPAC and a polyethylene-SPOCQ.
  • the carbohydrate comprised in the second coating is a mix of mannose/mannose-6-PEG and mannose-6-R, wherein R is selected from the group consisting of a polyethylene glycol-NHS ester, a polyethylene glycol-antibody, a polyethylene-maleimide, a polyethylene-SPAC and a polyethylene- SPOCQ.
  • the carbohydrate in the precoating is carbohydrate-R, wherein R is a chemical moiety capable of reacting with surface-bound OH groups such as phosphate or silane.
  • the carbohydrate- binding protein is a lectin.
  • the lectin is selected from the group consisting of concanavalin A (ConA), lentil lectins (LCH), snowdrop lectins (GNA), proteoglycans, type II transmembrane receptor lectins, collectins, selectins, natural killer lectins, macrophage mannose receptor (MMR) lectins, type I receptor lectins, tetranectins, polycystins, attractins, CTLD/acidic neck lectins, endosialins and any combination thereof.
  • the microfluidic devices and/or cell culture devices as described herein can be used for cell avidity measurements and for sorting or selecting of cells of interest, they however may also be used to conduct one or more of a wide range of investigations of interactions between cells and one or more substances.
  • Such interactions to be investigated may include, but are not limited to, an effect of the substance(s) on one or more of: cell attachment, cell growth, cell survival, cell differentiation, cell apoptosis/death and multicellular structure.
  • the activity or interaction of substances on cell motility, cell migration, cell to cell interactions as well as cell-protein signal interactions are also important activities that can be studied using the microfluidic devices and/or cell culture devices as described herein.
  • Substances of interest in such investigations may include, but are not limited to, pharmaceutical compounds or other therapeutics, exosomes, nanomicelles, nanoparticles, toxins, small molecules, nucleic acids (including nucleic acid vectors), oligonucleotides (including antisense oligonucleotides), oligopeptides, proteins, ribozymes, small interfering RNAs, microRNAs, short hairpin RNAs, aptamers, viruses, and antibodies or antigen binding parts thereof.
  • nucleic acids including nucleic acid vectors
  • oligonucleotides including antisense oligonucleotides
  • oligopeptides proteins
  • ribozymes small interfering RNAs
  • microRNAs microRNAs
  • short hairpin RNAs aptamers
  • viruses and antibodies or antigen binding parts thereof.
  • the present application also describes a kit of parts comprising a microfluidic device comprising a surface, a carbohydrate or derivative thereof, and a carbohydrate- binding protein.
  • the present application also describes a kit of parts comprising a microfluidic device comprising a surface comprising a coating comprising a carbohydrate-binding protein, and a carbohydrate or derivative thereof.
  • the present application also describes a kit of parts comprising:
  • a microfluidic device comprising a surface
  • a precoating comprising a carbohydrate or derivative thereof
  • the present application also describes a kit of parts comprising:
  • a microfluidic device comprising a surface comprising a coating comprising a carbohydrate-binding protein, and optionally a precoating comprising a carbohydrate or derivative thereof, and
  • a microfluidic device present in the kit of parts refers to a device having the required structural configuration of a microfluidic devices as described herein, but in which a first coating comprising a carbohydrate-binding protein and/or a second coating comprising a carbohydrate or derivative thereof is yet to be formed.
  • the carbohydrate or derivative thereof is a mix, for example a mix comprising mannose/mannose-6-PEG and mannose-6-R, wherein R is selected from the group consisting of a polyethylene glycol-NHS ester, a polyethylene glycol-antibody, a polyethylene-maleimide, a polyethylene-SPAC and a polyethylene-SPOCQ
  • the kit may additionally comprise all carbohydrates and derivatives thereof present in the mix. Said carbohydrates and derivatives thereof may be present in a single container or alternatively in separate containers.
  • the present application also describes a kit of parts comprising a microfluidic device comprising a surface, a mannose/mannose-6-PEG and/or mannose-6-R, wherein R is selected from the group consisting of a polyethylene glycol- NHS ester, a polyethylene glycol-antibody, a polyethylene-maleimide, a polyethylene- SPAC and a polyethylene-SPOCQ, and a lectin.
  • the kit may additionally comprise the carbohydrate present in the precoating.
  • kits of parts as described herein may be stored in one or more containers prior to their application and/or use.
  • kits of parts as described herein may be multi-pack kits wherein different components are stored in a plurality of containers.
  • a kit of parts as described herein may further comprise instructions for coating the surface with the carbohydrate and/or the carbohydrate-binding protein and, optionally, instructions for contacting and attaching cells with a surface to provide a microfluidic device comprising a surface comprising cells.
  • the present application describes a process for preparing a microfluidic device comprising a coated surface, the process comprising the steps of:
  • the present application also describes a process for preparing a microfluidic device comprising a coated surface, the process comprising the steps of:
  • a microfluidic device comprising a carbohydrate-binding protein coated surface, wherein the surface optionally comprises a coating comprising a carbohydrate or derivative thereof between the surface and the coating comprising the carbohydrate-binding protein,
  • the present application also describes a process for preparing a microfluidic device comprising a surface comprising cells, the process comprising the steps of:
  • the present application also describes a process in accordance with any one of the previously described processes, wherein the carbohydrate is selected from the group consisting of mannose, fucose, xylose, arabinose, acetylglucosamine, fructose, sialic acid, glucuronic acid, galactose, N-acetylgalactosamine, any derivative or mixture thereof.
  • the present application also describes a process in accordance with any one of the previously described processes, wherein the carbohydrate is mannose-6-R, wherein R is selected from the group consisting of a polyethylene glycol-NHS ester, a polyethylene glycol-antibody, a polyethylene-maleimide, a polyethylene-SPAC and a polyethylene-SPOCQ.
  • the present application also describes a process in accordance with any one of the previously described processes, wherein the carbohydrate-binding protein is a lectin.
  • the present application also describes a process in accordance with the previously described process, wherein the lectin is selected from the group consisting of concanavalin A (ConA), lentil lectins (LCH), snowdrop lectins (GNA), proteoglycans, type II transmembrane receptor lectins, collectins, selectins, natural killer lectins, macrophage mannose receptor (MMR) lectins, type I receptor lectins, tetranectins, polycystins, attractins, CTLD/acidic neck lectins, endosialins and any combination thereof.
  • ConA concanavalin A
  • LCH lentil lectins
  • GDA snowdrop lectins
  • proteoglycans type II transmembrane receptor lectins
  • collectins collectins, selectins, natural killer lectins, macrophage mannose receptor (MMR) lectins, type I receptor lectins,
  • the present application also describes a process for determining cellular avidity comprising:
  • a microfluidic device comprising a surface comprising cells, wherein the surface comprises a coating comprising a carbohydrate-binding protein, a coating comprising a carbohydrate of derivative thereof and cells attached to the surface, and optionally a precoating comprising a carbohydrate or derivative thereof, wherein the precoating is located between the surface and the coating comprising a carbohydrate-binding protein,
  • the present application also describes a process in accordance with the previously described process, wherein the cellular avidity is determined by exerting a force on the cells of interest.
  • the present application also describes a process in accordance with the previously described process, wherein the force is an acoustic force, a shear flow force or a centrifugal force.
  • the present application also describes a process in accordance with any one of the three previously described processes, wherein the microfluidic device comprising a surface comprising cells is prepared by performing a process in accordance with any one of the previously described processes 3-8.
  • the present application also describes a microfluidic device comprising a surface, wherein the surface comprises:
  • a precoating comprising a carbohydrate or derivative thereof
  • the present application also describes a microfluidic device comprising a surface, wherein the surface comprises:
  • a precoating comprising a carbohydrate or derivative thereof
  • the present application also describes a kit of parts comprising:
  • a microfluidic device comprising a surface
  • a precoating comprising a carbohydrate or derivative thereof
  • the present application also describes a kit of parts comprising:
  • a microfluidic device comprising a surface comprising a coating comprising a carbohydrate-binding protein, and optionally a precoating comprising a carbohydrate or derivative thereof, and a carbohydrate or derivative thereof.
  • Two glass surfaces were activated by contacting the surfaces for 10 minutes at room temperature with a 1 M aqueous sodium hydroxide solution, leading to exposure of silanol groups.
  • one glass surface was contacted for 30 minutes at room temperature with a 20 mM aqueous solution of a carbohydrate (mannose-6-phosphate) yielding a carbohydrate-coated surface, while the other glass surface was kept untreated.
  • a carbohydrate mannose-6-phosphate
  • one glass surface was contacted for 30 minutes at room temperature with a 20 mM aqueous solution of a carbohydrate (mannose-6-phosphate) yielding a carbohydrate-coated surface and stored for 16 hours at room temperature.
  • the carbohydrate-coated surface was coated with a carbohydrate-binding protein (concanavalin A) for 10 minutes at room temperature using an aqueous solution of concanavalin A with a concentration of 5 mg/ml, yielding a surface coated with a first coating comprising a carbohydrate and a second coating comprising a carbohydrate- binding protein.
  • a carbohydrate mannose-6-phosphate
  • the other glass surface was coated with a carbohydrate-binding protein (concanavalin A) for 16 hours at room temperature using an aqueous solution of concanavalin A with a concentration of 5 mg/ml, yielding a coated surface with a coating comprising a carbohydrate-binding protein.
  • a carbohydrate-binding protein concanavalin A
  • a microfluidic device was coated with a first coating comprising a carbohydrate and a second coating comprising a carbohydrate- binding protein using an identical process and conditions as described above.
  • the microfluidic device used in the experiment was a z-Movi® chip (obtained and as available from LUMICKS).
  • the cell monolayer quality was analyzed by analyzing the confluency of the cell monolayer after force application.
  • the chip was placed on the z-Movi® cell avidity analyzer (obtained and as available from LUMICKS) and the monolayer was observed using the software of the z-Movi® cell avidity analyzer, The chip was subjected to a 1000 pN force in the z-Movi® cell avidity analyzer resulting in a cell-coated surface with more than 80% confluency after application of the force.
  • Chip A The glass surface of a z-Movi® chip was activated by contacting the surface for 10 minutes at room temperature with a 1 M aqueous sodium hydroxide solution leading to exposure of silanol groups. Next, the glass surface of the chip was coated with a carbohydrate-binding protein (concanavalin A) for 16 hours at room temperature using an aqueous solution of concanavalin A with a concentration of 5 mg/ml yielding a coated surface with a coating comprising a carbohydrate-binding protein.
  • a carbohydrate-binding protein concanavalin A
  • Raji cells 100 million cells/ml cultured in RPMI + Glutamax supplemented with 10% heat inactivated fetal bovine serum and penicillin-streptomycin was made.
  • the Raji cells i.e. target cells
  • the Raji cells were contacted for 30 minutes at 37°C with the coated surface of the chip allowing the cells to attach to the coated surface in a monolayer to provide a microfluidic device comprising a surface comprising cells.
  • untransduced cells of interest i.e. effector cells, i.e. primary T cells
  • effector cells i.e. primary T cells
  • the effector cells were introduced in the microfluidic device (i.e. z- Movi® chip) comprising a surface comprising cells, incubated for 5 minutes, and then the chip was subjected during 2.5 minutes to a 1 to 1000 pN force ramp in a z-Movi® cell avidity analyzer operated with the Ocean V1.4 software.
  • the monolayer was stained with a Trypan Blue solution to qualitatively assess the target cell viability.
  • Chip B The glass surface of a z-Movi® chip was activated by contacting the surface for 10 minutes at room temperature with a 1 M aqueous sodium hydroxide solution leading to exposure of silanol groups. Next, the glass surface of the chip was coated with a carbohydrate-binding protein (concanavalin A) for 16 hours at room temperature using an aqueous solution of concanavalin A with a concentration of 5 mg/ml yielding a coated surface with a coating comprising a carbohydrate-binding protein. Next, a suspension of Raji cells (100 million cells/ml) cultured in RPMI + Glutamax supplemented with 10% heat inactivated fetal bovine serum and penicillin-streptomycin was made. The Raji cells (/.e. target cells) were contacted for 30 minutes at 37°C with the coated surface of the chip allowing the cells to attach to the coated surface in a monolayer to provide a microfluidic device comprising a surface comprising cells.
  • CAR transduced cells of interest /.e. effector cells, i.e. CAR transduced primary T cells
  • the cells of interest were stained with CellTraceTM Far Red dye (Thermo Fisher Scientific) at 1 pM for 15 minutes in PBS at 37°C, then resuspended at 10 million/mL in complete medium and used for the avidity experiments.
  • the effector cells were introduced in the microfluidic device (i.e. z-Movi® chip) comprising a surface comprising cells, incubated for 5 minutes, and then the chip was subjected during 2.5 minutes to a 1 to 1000 pN force ramp in a z-Movi® cell avidity analyzer operated with the Ocean V1.4 software.
  • the monolayer was stained with a Trypan Blue solution to qualitatively assess the target cell viability.
  • Chip C The glass surface of a z-Movi® chip was activated by contacting the surface for 10 minutes at room temperature with a 1 M aqueous sodium hydroxide solution leading to exposure of silanol groups. Next, the glass surface of the chip was contacted for 30 minutes at room temperature with a 20 mM aqueous solution of a carbohydrate (mannose-6-phosphate) yielding a carbohydrate-coated surface and stored at room temperature for 16 hours in the dark.
  • a carbohydrate mannose-6-phosphate
  • carbohydrate-coated surface was coated with a carbohydrate-binding protein (concanavalin A) for 10 minutes at room temperature using an aqueous solution of concanavalin A with a concentration of 5 mg/ml, yielding a surface coated with a first coating comprising a carbohydrate and a second coating comprising a carbohydrate-binding protein.
  • a carbohydrate-binding protein concanavalin A
  • Raji cells 100 million cells/ml cultured in RPMI + Glutamax supplemented with 10% heat inactivated fetal bovine serum and penicillin-streptomycin was made.
  • the Raji cells i.e. target cells
  • the Raji cells were contacted for 30 minutes at 37°C with the coated surface of the chip allowing the cells to attach to the coated surface in a monolayer to provide a microfluidic device comprising a surface comprising cells.
  • untransduced cells of interest i.e. effector cells, i.e. primary T cells
  • effector cells i.e. primary T cells
  • the effector cells were introduced in the microfluidic device (i.e. z- Movi® chip) comprising a surface comprising cells, incubated for 5 minutes, and then the chip was subjected during 2.5 minutes to a 1 to 1000 pN force ramp in a z-Movi® cell avidity analyzer operated with the Ocean V1.4 software.
  • the monolayer was stained with a Trypan Blue solution to qualitatively assess the target cell viability.
  • Chip D The glass surface of a z-Movi® chip was activated by contacting the surface for 10 minutes at room temperature with a 1 M aqueous sodium hydroxide solution leading to exposure of silanol groups. Next, the glass surface of the chip was contacted for 30 minutes at room temperature with a 20 mM aqueous solution of a carbohydrate (mannose-6-phosphate) yielding a carbohydrate-coated surface and stored at room temperature for 16 hours in the dark.
  • a carbohydrate mannose-6-phosphate
  • carbohydrate-coated surface was coated with a carbohydrate-binding protein (concanavalin A) for 10 minutes at room temperature using an aqueous solution of concanavalin A with a concentration of 5 mg/ml, yielding a surface coated with a first coating comprising a carbohydrate and a second coating comprising a carbohydrate-binding protein.
  • a carbohydrate-binding protein concanavalin A
  • a suspension of Raji cells (100 million cells/ml) cultured in RPMI + Glutamax supplemented with 10% heat inactivated fetal bovine serum and penicillin-streptomycin was made.
  • the Raji cells (/.e. target cells) were contacted for 30 minutes at 37°C with the coated surface of the chip allowing the cells to attach to the coated surface in a monolayer to provide a microfluidic device comprising a surface comprising cells.
  • CAR transduced cells of interest /.e. effector cells, i.e. CAR transduced primary T cells
  • the cells of interest were stained with CellTraceTM Far Red dye (Thermo Fisher Scientific) at 1 pM for 15 minutes in PBS at 37°C, then resuspended at 10 million/mL in complete medium and used for the avidity experiments.
  • the effector cells were introduced in the microfluidic device (i.e. z-Movi® chip) comprising a surface comprising cells, incubated for 5 minutes, and then the chip was subjected during 2.5 minutes to a 1 to 1000 pN force ramp in a z-Movi® cell avidity analyzer operated with the Ocean V1.4 software.
  • the monolayer was stained with a Trypan Blue solution to qualitatively assess the target cell viability.
  • Chip A The glass surface of a z-Movi® chip was activated by contacting the surface for 10 minutes at room temperature with a 1 M aqueous sodium hydroxide solution leading to exposure of silanol groups. Next, the glass surface of the chip was coated with a carbohydrate-binding protein (concanavalin A) for 16 hours at room temperature using an aqueous solution of concanavalin A with a concentration of 5 mg/ml yielding a coated surface with a first coating comprising a carbohydrate-binding protein.
  • a carbohydrate-binding protein concanavalin A
  • a suspension of Raji cells (100 million cells/ml) cultured in RPMI + Glutamax supplemented with 10% heat inactivated fetal bovine serum and penicillin-streptomycin was made.
  • the Raji cells (/.e. target cells) were contacted for 30 minutes at 37°C with the coated surface of the chip allowing the cells to attach to the coated surface in a monolayer to provide a microfluidic device comprising a surface comprising cells.
  • untransduced cells of interest /.e. effector cells, i.e. primary T cells
  • effector cells i.e. primary T cells
  • the effector cells were introduced in the microfluidic device (i.e. z- Movi® chip) comprising a surface comprising cells, incubated for 5 minutes, and then the chip was subjected during 2.5 minutes to a 1 to 1000 pN force ramp in a z-Movi® cell avidity analyzer operated with the Ocean V1.4 software.
  • the monolayer was stained with a Trypan Blue solution to qualitatively assess the target cell viability.
  • Chip B The glass surface of a z-Movi® chip was activated by contacting the surface for 10 minutes at room temperature with a 1 M aqueous sodium hydroxide solution leading to exposure of silanol groups. Next, the glass surface of the chip was coated with a carbohydrate-binding protein (concanavalin A) for 16 hours at room temperature using an aqueous solution of concanavalin A with a concentration of 5 mg/ml yielding a coated surface with a first coating comprising a carbohydrate-binding protein.
  • a carbohydrate-binding protein concanavalin A
  • a suspension of Raji cells (100 million cells/ml) cultured in RPMI + Glutamax supplemented with 10% heat inactivated fetal bovine serum and penicillin-streptomycin was made.
  • the Raji cells (/.e. target cells) were contacted for 30 minutes at 37°C with the coated surface of the chip allowing the cells to attach to the coated surface in a monolayer to provide a microfluidic device comprising a surface comprising cells.
  • CAR transduced cells of interest /.e. effector cells, i.e. CAR transduced primary T cells
  • the cells of interest were stained with CellTraceTM Far Red dye (Thermo Fisher Scientific) at 1 pM for 15 minutes in PBS at 37°C, then resuspended at 10 million/mL in complete medium and used for the avidity experiments.
  • the effector cells were introduced in the microfluidic device (i.e. z-Movi® chip) comprising a surface comprising cells, incubated for 5 minutes, and then the chip was subjected during 2.5 minutes to a 1 to 1000 pN force ramp in a z-Movi® cell avidity analyzer operated with the Ocean V1.4 software.
  • the monolayer was stained with a Trypan Blue solution to qualitatively assess the target cell viability.
  • Chip C The glass surface of a z-Movi® chip was activated by contacting the surface for 10 minutes at room temperature with a 1 M aqueous sodium hydroxide solution leading to exposure of silanol groups. Next, the glass surface of the chip was contacted for 30 minutes at room temperature with a 20 mM aqueous solution of a carbohydrate (mannose-6-phosphate) yielding a carbohydrate-coated surface and stored at room temperature for 16 hours in the dark.
  • a carbohydrate mannose-6-phosphate
  • carbohydrate-coated surface was coated with a carbohydrate-binding protein (concanavalin A) for 10 minutes at room temperature using an aqueous solution of concanavalin A with a concentration of 5 mg/ml, yielding a surface coated with a first coating comprising a carbohydrate-binding protein.
  • a carbohydrate-binding protein concanavalin A
  • a suspension of Raji cells (100 million cells/ml) cultured in RPMI + Glutamax supplemented with 10% heat inactivated fetal bovine serum and penicillin-streptomycin was made.
  • the Raji cells i.e. target cells
  • the coated surface of the chip allowing the cells to attach to the coated surface in a monolayer to provide a microfluidic device comprising a surface comprising cells.
  • the surface was contacted for 10 minutes at room temperature with a 20 mM solution of mannose in PBS (1 M), yielding, a surface coated with a first coating comprising a carbohydrate-binding protein, cells, and a second coating comprising a carbohydrate.
  • the surface was subsequently rinsed with PBS and serum-free medium and used for further experimentation.
  • untransduced cells of interest /.e. effector cells, i.e. primary T cells
  • effector cells i.e. primary T cells
  • the effector cells were introduced in the microfluidic device (i.e. z- Movi® chip) comprising a surface comprising cells, incubated for5 minutes, and then the chip was subjected during 2.5 minutes to a 1 to 1000 pN force ramp in a z-Movi® cell avidity analyzer operated with the Ocean V1.4 software.
  • the monolayer was stained with a Trypan Blue solution to qualitatively assess the target cell viability.
  • Chip D The glass surface of a z-Movi® chip was activated by contacting the surface for 10 minutes at room temperature with a 1 M aqueous sodium hydroxide solution leading to exposure of silanol groups. Next, the glass surface of the chip was contacted for 30 minutes at room temperature with a 20 mM aqueous solution of a carbohydrate (mannose-6-phosphate) yielding a carbohydrate-coated surface and stored at room temperature for 16 hours in the dark.
  • a carbohydrate mannose-6-phosphate
  • carbohydrate-coated surface was coated with a carbohydrate-binding protein (concanavalin A) for 10 minutes at room temperature using an aqueous solution of concanavalin A with a concentration of 5 mg/ml, yielding a surface coated with a first coating comprising a carbohydrate and a second coating comprising a carbohydrate-binding protein.
  • a carbohydrate-binding protein concanavalin A
  • a suspension of Raji cells (100 million cells/ml) cultured in RPMI + Glutamax supplemented with 10% heat inactivated fetal bovine serum and penicillin-streptomycin was made.
  • the Raji cells i.e. target cells
  • the coated surface of the chip allowing the cells to attach to the coated surface in a monolayer to provide a microfluidic device comprising a surface comprising cells.
  • the surface was contacted for 10 minutes at room temperature with a 20 mM solution of mannose in PBS (1 M), yielding, a surface coated with a first coating comprising a carbohydrate-binding protein, cells, and a second coating comprising a carbohydrate.
  • the surface was subsequently rinsed with PBS and serum-free medium and used for further experimentation.
  • CAR transduced cells of interest i.e. effector cells, i.e. CAR transduced primary T cells
  • the cells of interest were stained with CellTraceTM Far Red dye (Thermo Fisher Scientific) at 1 pM for 15 minutes in PBS at 37°C, then resuspended at 10 million/mL in complete medium and used for the avidity experiments.
  • the effector cells were introduced in the microfluidic device (i.e. z-Movi® chip) comprising a surface comprising cells, incubated for 5 minutes, and then the chip was subjected during 2.5 minutes to a 1 to 1000 pN force ramp in a z-Movi® cell avidity analyzer operated with the Ocean V1.4 software.
  • the monolayer was stained with a Trypan Blue solution to qualitatively assess the target cell viability.
  • both glass surfaces were contacted for 30 minutes at room temperature with a 20 mM aqueous solution of mannose-6-phosphate and stored for 16 hours at room temperature.
  • the surface was coated with a carbohydrate-binding protein (concanavalin A) for 10 minutes at room temperature using an aqueous solution of concanavalin A with a concentration of 5 mg/ml, yielding a surface coated with a carbohydrate-binding protein.
  • a carbohydrate-binding protein (concanavalin A)
  • One of the glass surfaces (surface A) was kept as is, while the other glass surface (surface B) was coated for 10 minutes at room temperature with a 20 mM solution of a carbohydrate (mannose) in PBS (1 M), yielding a multiple-coated surface.
  • Raji cells 100 million cells/ml cultured in RPMI + Glutamax supplemented with 10% heat inactivated fetal bovine serum and penicillin-streptomycin was made.
  • the Raji cells (/.e. target cells) were contacted for 30 minutes at 37°C with surface A and surface B allowing the cells to attach to the coated surfaces in a monolayer.

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Abstract

L'invention concerne des dispositifs adaptés à la mesure de processus biologiques, par exemple à la mesure de l'avidité cellulaire. L'invention concerne également des procédés de fabrication et d'utilisation des dispositifs.
PCT/EP2023/081023 2022-11-08 2023-11-07 Dispositif microfluidique WO2024100056A1 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
EP22205995 2022-11-08
EP22205979 2022-11-08
EP22205979.2 2022-11-08
EP22205995.8 2022-11-08
EP22205993 2022-11-08
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