Methods of enumerating particles present in a cell composition

Abstract

Provided herein are methods of assessing or determining the presence or absence of particles, such as bead particles, present in a cell composition. Also provided are articles of manufacture and kits for use in the methods.

1 . A method for enumerating or detecting the presence or absence of particles in a cell composition, the method comprising the steps of:
 a) performing one or more incubations, thereby producing an output composition, wherein the one or more incubations comprise incubating a sample comprising at least a portion of a cell composition or a sample derived from the cell composition under a condition sufficient to induce osmotic lysis of one or more cells in the sample; and   b) determining the presence, absence, number and/or concentration of particles in the output composition, thereby enumerating or detecting the presence or absence of particles in the cell composition.   
     
     
         2 . The method of  claim 1 , wherein the condition sufficient to induce osmotic lysis comprises contacting the sample with a hypotonic solution. 
     
     
         3 . The method of  claim 1  or  claim 2 , wherein the incubating under a condition to induce osmotic lysis produces a lysed cell composition and the one or more incubations in step a) further comprises incubating the lysed cell composition or a composition derived from the lysed cell composition with a hypertonic solution. 
     
     
         4 . A method for enumerating or detecting the presence or absence of particles in a cell composition, the method comprising the steps of:
 a) performing one or more incubations, thereby producing an output composition, wherein the one or more incubations in step a) comprises:
 i) incubating a sample comprising at least a portion of a cell composition or a sample derived from the cell composition with a hypotonic solution or a hypertonic solution under a condition sufficient to induce lysis of one or more cells in the sample, thereby producing a lysed cell composition, and 
 ii) incubating at least a portion of the lysed cell composition or a sample derived from the lysed cell composition with the other of the hypotonic solution or the hypertonic solution; and 
   b) determining the presence, absence, number and/or concentration of particles in the output composition, thereby enumerating or detecting the presence or absence of particles in the cell composition.   
     
     
         5 . A method of enumerating or detecting the presence or absence of particles in a cell composition, the method comprising the steps of:
 a) performing one or more incubations, thereby producing an output composition, wherein the one or more incubations in step a) comprises
 i) incubating a sample comprising at least a portion of a cell composition or a sample derived from the cell composition with a hypotonic solution under a condition sufficient to induce lysis of one or more cells in the sample, thereby producing a lysed cell composition, and 
 ii) incubating at least a portion of the lysed cell composition or a sample derived from the lysed cell composition with a hypertonic solution; and 
   b) determining the presence, absence, number and/or concentration of particles in the output composition, thereby enumerating or detecting the presence or absence of particles in the cell composition.   
     
     
         6 . The method of any one of  claims 1 - 5 , wherein:
 the cell composition comprises or is suspected of comprising one or more of the particles bound to the surface of one or more cells in the cell composition;   the cell composition comprises or is suspected of comprising residual particles;   the cell composition is derived from a composition containing cells bound to one or more of the particles; and/or   the cell composition is derived from removal of particles from an input composition.   
     
     
         7 . The method of any one of  claims 1 - 6 , wherein the cell composition is produced by a method comprising:
 (1) mixing a population of cells with one or more of the particles thereby generating an input composition; and   (2) removing one or more of the particles from the cells in the input composition, thereby producing the cell composition.   
     
     
         8 . The method of  claim 7 , wherein the one or more of the particles are capable of binding one or more cells in the population. 
     
     
         9 . The method of any one of  claims 1 - 8 , wherein the particles are bead particles. 
     
     
         10 . The method of any one of  claims 1 - 9 , wherein the one or more incubations in step a) reduces or removes cell debris from the output composition. 
     
     
         11 . The method of any one of  claims 1 - 10 , wherein step a) further comprises rinsing or washing the output composition. 
     
     
         12 . The method of  claim 11 , wherein rinsing or washing the output composition comprises pelleting the particles and removing a volume or reducing a volume of the output composition. 
     
     
         13 . The method of  claim 12 , comprising reducing the volume of the output composition to about the same volume of the sample prior to the one or more incubations of step a). 
     
     
         14 . The method of  claim 12 , comprising reducing the volume of the output composition by less than 100% but greater than or greater than about 50%, 60%, 70%, 80%, 90% or 95%. 
     
     
         15 . The method of any one of  claims 1 - 14 , wherein one or more of the particles comprises a biomolecule capable of binding to a macromolecule on a surface of a cell in the cell composition. 
     
     
         16 . The method of  claim 15 , wherein the biomolecule is an antibody or antigen-binding fragment thereof. 
     
     
         17 . The method of any one of  claims 2 - 16 , wherein the hypotonic solution has an osmolarity less than 270 mOsm/L. 
     
     
         18 . The method of any one of  claims 2 - 17 , wherein:
 the hypotonic solution has an osmolarity between or between about 0 mOsm/L and 270 mOsm/L, 50 mOsm/L and 200 mOsm/L or 10 mOsm/L and 100 mOsm/L; or   the hypotonic solution has an osmolarity less than or less than about 250 mOsm/L, 200 mOsm/L, 150 mOsm/L, 100 mOsm/L, 50 mOsm/L, 10 mOsm/L or less.   
     
     
         19 . The method of any one of  claims 2 - 18 , wherein:
 the hypotonic solution comprises a solute concentration of between or between about 0 mM and 140 mM; or   the hypotonic solution comprises a solute concentration of less than or about less than 140 mM, less than or about less than 100 mM, less than or about less than 50 mM or less than or about less than 10 mM.   
     
     
         20 . The method of any one of  claims 2 - 19 , wherein:
 the hypotonic solution comprises a percentage weight for volume (% w/v) of solute of between or between about 0% and 0.8% or 0% and 0.5%; or   the hypotonic solution comprises a % w/v of solute of less than or about less than 0.8%, less than or about less than 0.6%, less than or about less than 0.4% or less than or about less than 0.2%.   
     
     
         21 . The method of any one of  claims 2 - 20 , wherein the hypotonic solution is solute-free. 
     
     
         22 . The method of any one of  claims 2 - 21 , wherein the hypotonic solution is sterile water for injection. 
     
     
         23 . The method of any one of  claims 3 - 22 , wherein the hypertonic solution has an osmolarity greater than 300 mOsm/L. 
     
     
         24 . The method of any one of  claims 3 - 23 , wherein:
 the hypertonic solution has an osmolarity of greater than or about 300 mOsm/L, greater than or about 400 mOsm/L, greater than or about 800 mOsm/L, greater than or about 1200 mOsm/L, greater than or about 1500 mOsm/L, greater than or about 2000 mOsm/L, greater than or about 2500 mOsm/L, greater than or about 3000 mOsm/L or greater than or about 4000 mOsm/L; or   the hypertonic solution has an osmolarity of between or about between 300 mOsm/L and 5000 mOsm/L, 1000 and 5000 mOsm/L or 1000 and 3000 mOsm/L.   
     
     
         25 . The method of any one of  claims 3 - 24 , wherein:
 the hypertonic solution has a solute concentration of greater than or about 200 mM, greater than or greater than about 400 mM, greater than or greater than about 600 mM, greater than or greater than about 800 mM, greater than or greater than about 1000 mM greater than or greater than about 2000 mM; or greater than or greater than about 5000 mM; or   the hypertonic solution has a solute concentration of between or between about 200 mM and 5000 mM, 500 mM and 2000 mM or 1000 mM and 2000 mM.   
     
     
         26 . The method of any one of  claims 3 - 25 , wherein:
 the hypertonic solution comprises a percentage weight for volume (% w/v) of solute of between or between about 1.5% and 15% or 2.5% and 12%; or   the hypertonic solution comprises a % w/v of solute of greater than or about greater than 1.5%, greater than or about greater than 3.0%, greater than or about greater than 6.0% or greater than or about greater than 8.0% or greater than or about greater than 10.0%.   
     
     
         27 . The method of any one of  claims 3 - 26 , wherein the hypertonic solution comprises a solute that is NaCl. 
     
     
         28 . The method of any one of  claims 1 - 27 , wherein the concentration of the cell composition is at least or at least about 2×10 5  cells/mL, at least or at least about 5×10 5  cells/mL, at least or at least about 1×10 6  cells/mL, at least or at least about 5×10 6  cells/mL, or at least or at least about 1×10 7  cells/mL. 
     
     
         29 . The method of any one of  claims 1 - 28 , wherein:
 the volume of the cell composition is from or from about 0.2 mL to 50 mL, 0.2 mL to 20 mL, 0.5 mL to 10 mL, 0.5 mL to 5 mL, or 0.75 mL to 1.5 mL; or   the volume of the cell composition is at least or at least about 0.2 mL, 0.5 mL, 1.0 mL, 2.0 mL, 5.0 mL, 10.0 mL or 20 mL, 50 mL or more.   
     
     
         30 . The method of any one of  claims 1 - 29 , wherein one or more of the particles have or comprise particles having a diameter of greater than 0.001 μm, greater than 0.01 μm, greater than 0.1 μm, greater than 1.0 μm, greater than 10 μm, greater than 50 μm, greater than 100 μm or greater than 1000 μm. 
     
     
         31 . The method of any one of  claims 1 - 30 , wherein one or more of the particles have or comprise particles having a diameter of 1.0 μm to 500 μm, 1.0 μm to 150 μm, 1.0 μm to 30 μm, 1.0 μm to 10 μm or 1.0 μm to 5.0 μm. 
     
     
         32 . The method of any one of  claims 1 - 31 , wherein one or more of the particles have or comprise particles having a diameter that is substantially the same as the average diameter of a cell in the cell composition or is within 1.5-fold greater or less than the average diameter of a cell in the cell composition. 
     
     
         33 . The method of any one of  claims 1 - 32 , wherein one or more of the particles is magnetic and/or one or more of the particles comprise a magnetic core, a paramagnetic core or a superparamagnetic core. 
     
     
         34 . The method of  claim 33 , wherein the magnetic core is selected from among metal oxides, ferrites, metals, hematite, metal alloys, and combinations thereof. 
     
     
         35 . The method of any one of  claims 1 - 34 , wherein one or more of the particles comprise an iron oxide core. 
     
     
         36 . The method of any one of  claims 33 - 35 , wherein the magnetic core comprises a coat. 
     
     
         37 . The method of  claim 36 , wherein the coat protects, reduces or prevents the magnetic core from oxidation. 
     
     
         38 . The method of  claim 36  or  claim 37 , wherein the coat comprises a polymer, a polysaccharide, a silica, a fatty acid, a carbon or a combination thereof. 
     
     
         39 . The method of any one of  claims 36 - 37 , wherein the polymer, the polysaccharide, the silica, the fatty acid, the carbon or a combination thereof is biodegradable. 
     
     
         40 . The method of  claim 38  or  claim 39 , wherein the polysaccharide is chitosan, agarose, starch, dextran, a dextran derivative or combinations thereof. 
     
     
         41 . The method of  claim 38  or  claim 39 , wherein the polymer is polyethylene glycol, poly(lactic-co-glycolic acid), polyglutaraldehyde, polyurethane, polystyrene, and polyvinyl alcohol or combinations thereof. 
     
     
         42 . The method of any one of  claims 1 - 41 , wherein the cell has a diameter of between or about between 10 μm and 30 μm. 
     
     
         43 . The method of any one of  claims 1 - 42 , wherein the cell is an animal cell or the cell composition comprises animal cells. 
     
     
         44 . The method of any one of  claims 1 - 43 , wherein the cell is a human cell or the cell composition comprises human cells. 
     
     
         45 . The method of any one of  claims 1 - 44 , wherein the cell is a stem cell or the cell composition comprises stem cells. 
     
     
         46 . The method of  claim 45 , wherein the stem cell is an induced pluripotent stem cell (iPSC). 
     
     
         47 . The method of any one of  claims 1 - 46 , wherein the cell is an immune cell or the cell composition comprises immune cells. 
     
     
         48 . The method of  claim 47 , wherein the immune cell is a T cell, B cell, macrophage, neutrophil, natural killer (NK) cell or dendritic cell. 
     
     
         49 . The method of any one of  claims 1 - 48 , wherein the cell composition has been mixed with one or more of the particles, wherein the particle comprises a stimulating agent to effect stimulation and/or activation of a cell in the cell composition prior to the one or more incubations in step a). 
     
     
         50 . The method of  claim 49 , wherein the cell is a T cell and the stimulating agent is an anti-CD3 antibody and/or anti-CD28 antibody or an antigen-binding fragment thereof. 
     
     
         51 . The method of  claim 50 , wherein the cell is an antigen presenting cell and the stimulating agent is an anti-CD80 antibody and/or anti-CD86 antibody or an antigen-binding fragment thereof. 
     
     
         52 . The method of any one of  claims 1 - 51 , wherein the cell composition has been mixed with one or more of the particles, wherein the particle comprises an affinity reagent to effect isolation or enrichment of a cell in the cell composition prior to the one or more incubations in step a). 
     
     
         53 . The method of  claim 52 , wherein the affinity reagent comprises an antibody or antigen-binding fragment thereof that specifically binds to a cell surface protein on one or more cells in the cell composition. 
     
     
         54 . The method of  claim 53 , wherein the cell surface protein is selected from among CD2, CD3, CD4, CD5, CD8, CD25, CD27, CD28, CD29, CD31, CD44, CD45RA, CD45RO, CD54 (ICAM-1), CD127, MHCI, MHCII, CTLA-4, ICOS, PD-1, OX40, CD27L (CD70), 4-1BB (CD137), 4-1BBL, CD30L, LIGHT, IL-2R, IL-12R, IL-1R, IL-15R; IFN-gammaR, TNF-alphaR, IL-4R, IL 10R, CD18/CD11a (LFA-1), CD62L (L-selectin), CD29/CD49d (VLA-4), Notch ligand (e.g. Delta-like 1/4, Jagged 1/2, etc.), CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, and CXCR3. 
     
     
         55 . The method of any one of  claims 1 - 54 , wherein:
 the one or more incubation is for at least or at least about 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 20 minutes or 30 minutes; or   the one or more incubation is from or from about 30 seconds to 30 minutes, 1 minute to 20 minutes, 1 minute to 10 minutes or 1 minute to 5 minutes.   
     
     
         56 . The method of any one of  claims 2 - 55 , wherein:
 the volume of the hypotonic and/or hypertonic solution is at least or at least about 1 mL, 3 mL, 9 mL, 12 mL, 15 mL, 18 mL, 20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 45 mL, 50 mL or more; or   the volume of the hypotonic and/or hypertonic solution is from or from about 1 mL to 50 mL, 2 mL to 30 mL, 5 mL to 25 mL or 10 mL to 20 mL.   
     
     
         57 . The method of any one of  claims 1 - 56 , wherein the method does not destroy the surface of the particle or the coat on the surface of the particle or does remove the biomolecule attached to the surface of the particle. 
     
     
         58 . The method of any one of  claims 1 - 57 , wherein the determining in step b) comprises manual counting, electronic particle counting, affinity-based detection, microscopy, flow cytometry, or magnetic cell sorting. 
     
     
         59 . The method of any one of  claims 1 - 58 , wherein the determining in step b) comprises detecting one or more materials or biomolecules present, associated with or attached to the surface of the particles, optionally using a binding agent that specifically binds to the material or biomolecule. 
     
     
         60 . The method of  claim 59 , wherein the particles comprising a coat and the coat comprises the material. 
     
     
         61 . The method of  claim 59  or  claim 60 , wherein the material is a polysaccharide. 
     
     
         62 . The method of  claim 61 , wherein the material is dextran and/or the binding agent is an anti-dextran antibody. 
     
     
         63 . The method of  claim 59 , wherein the biomolecule is an antibody or antigen-binding fragment against a cell surface protein attached to the surface of the particle, which optionally is an anti-CD3 or anti-CD28 antibody. 
     
     
         64 . A method of enumerating or detecting the presence or absence of particles in a cell composition, the method comprising the steps of:
 a) performing one or more incubations, thereby producing an output composition, wherein the one or more incubations comprise incubating a sample comprising at least a portion of a cell composition or a sample derived from the cell composition, under a condition sufficient to induce lysis of one or more cells in the sample; and   b) determining the presence, absence, number and/or concentration of particles in the output composition using a binding agent that specifically binds to a material, moiety or biomolecule present on, associated with or attached to the particle, thereby enumerating or detecting the presence or absence of particles in the cell composition.   
     
     
         65 . The method of  claim 64 , wherein the binding agent is an antibody or antigen-binding fragment thereof. 
     
     
         66 . The method of  claim 64  or  claim 65 , wherein the particle comprises a coat comprising the material. 
     
     
         67 . The method of any of  claims 64 - 66 , wherein the material is a polysaccharide. 
     
     
         68 . The method of any of  claims 64 - 67 , wherein the material is dextran and/or the binding agent is an anti-dextran antibody. 
     
     
         69 . The method of  claim 64  or  claim 65 , wherein the biomolecule is an antibody or antigen-binding fragment against a cell surface protein attached to the surface of the particle, which optionally is an anti-CD3 or anti-CD28 antibody. 
     
     
         70 . The method of  claim 69 , wherein the binding agent is an anti-idiotypic or anti-isotypic antibody against the biomolecule. 
     
     
         71 . The method of any of  claims 64 - 70 , wherein the one or more incubations induces osmotic cell lysis of one or more cells in the sample. 
     
     
         72 . The method of any of  claims 62 - 71 , wherein the one or more incubations comprises incubating the sample with a hypotonic solution. 
     
     
         73 . The method of  claim 72 , wherein the one or more incubations further comprises incubating the sample with a hypertonic solution. 
     
     
         74 . The method of any of  claims 64 - 73 , wherein the determining comprises fluorescence-activated cell sorting (FACS) for detection of one or more of the particles comprising the coat. 
     
     
         75 . The method of any one of  claims 1 - 74 , wherein the one or more incubations in step a) is/are performed at a temperature that is about 15° C. to 30° C., 18° C. to 28° C. or 20° C. to 25° C. 
     
     
         76 . The method of any one of  claims 1 - 75 , wherein the one or more incubations in step a) is/are performed at a temperature that is about 23° C. 
     
     
         77 . An article of manufacture, comprising:
 a container comprising a solution for effecting osmotic cell lysis;   packaging material; and   a label or package insert comprising instructions for enumerating or detecting the presence or absence of particles in a cell composition.   
     
     
         78 . The article of manufacture of  claim 77 , wherein the solution for effecting osmotic cell lysis is a hypotonic solution. 
     
     
         79 . The article of manufacture of  claim 77  or  claim 78 , further comprising a container comprising a hypertonic solution. 
     
     
         80 . The article of manufacture of any of  claims 77 - 79 , further comprising an instrument or reagent for detecting or identifying particles. 
     
     
         81 . The article of manufacture of  claim 80 , wherein the instrument or reagent comprises a hemocytometer. 
     
     
         82 . The article of manufacture of  claim 80 , wherein the instrument or reagent comprises a binding agent specific for a material, moiety or biomolecule on the surface of the particle. 
     
     
         83 . The article of manufacture of  claim 82 , wherein the binding agent is an antibody or an antigen-binding fragment thereof. 
     
     
         84 . The article of manufacture of  claim 82  or  claim 83 , wherein the particle comprises a coat comprising the material. 
     
     
         85 . The article of manufacture of  claim 84 , wherein the material is a polysaccharide. 
     
     
         86 . The article of manufacture of any of  claims 82 - 85 , wherein the material is dextran and/or the binding agent is an anti-dextran antibody. 
     
     
         87 . The article of manufacture or  claim 82  or  claim 83 , wherein the biomolecule is an antibody or antigen-binding fragment against a cell surface protein attached to the surface of the particle, which optionally is an anti-CD3 or anti-CD28 antibody. 
     
     
         88 . The article of manufacture of  claim 87 , wherein the binding agent is an anti-idiotypic or anti-isotypic antibody against the biomolecule. 
     
     
         89 . The article of manufacture of any of  claims 82 - 88 , wherein the binding agent is fluorescently labeled.