Classifications

• • 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
  • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
  • G01N33/54326—Magnetic particles

Definitions

• the invention relates to the field of cell biology and single cell analysis.
• the invention relates to an improved method of handling cells in vitro.
• Various therapeutic and diagnostic procedures involve multiple steps of handling a population of cells.
• diagnostic and cell analysis methods of flow cytometry, fluorescence-activated cell sorting (FACS) and mass- spectrometry (CyTOF) require cell handling procedures including pipetting, centrifugation, resuspension and washes before applying a cell suspension to an instrument.
• Newer single-cell analysis techniques involve obtaining a cell suspension from a sample, and encapsulating of individual cells in droplets and fusing the cell- containing droplets with probe-containing droplets (Chro ium System, 10X Genomics, Pleasanton, Cal.).
• Patent 10,144,950 describes a novel process of single-cell analysis called Quantum Barcoding (QBC), where each cell is labeled with a unique combinatorial barcode.
• QBC Quantum Barcoding
• a cell suspension is subjected to multiple rounds of “split- pool” process comprising mixing the cells in a suspension, splitting the cell suspension into wells containing a barcode subunit and pooling the cells again for the next round of bar coding.
• Single cell analysis has applications in oncology and immunology where detection and study of multiple types of rare cells is needed.
• CyTOF flow cytometry and mass-spectrometry
• any cell loss during handling results in a loss of valuable data points derived from unique or rare cells in the sample.
• CAR-T cell therapy is an exciting pioneering treatment where patient’s own T cells are extracted and modified to specifically target and destroy the patient’s tumor cells.
• the multi-step process of preparing therapeutic T-cells includes isolating all leukocytes from the patients, specifically capturing T-cell subpopulations using an antibody conjugate or another targeting modality, activating the isolated T-cells with isolated activator molecules or whole antigen presenting cells (APCs), and transducing the activated T-cells with a chimeric T-cell receptor of choice prior to expanding the cells and returning them into the patient.
• the process includes several intermediate wash and purification steps. Any cell loss from handling reduced the yield of valuable material.
• the invention relates generally to the field of improved cell handling in diagnostic and therapeutic procedures in order to reduce or minimize cell loss.
• the invention is a method of decreasing cell loss from a suspension of cells during a cell handling procedure comprising: contacting a suspension of cells with magnetic particles wherein the magnetic particles do not have specific or non-specific affinity molecules capable of binding to the cells; performing the cell handling procedure; separating the magnetic particles from the cell suspension using a magnet.
• the magnetic particles range in size from about
• the magnetic particles range in relative size from about 0.5 to about 1 relative to the size of the cells in the cell suspension. In some embodiments, the magnetic particles are added at a ratio from about 0.3:1 to about 1:3 cell to particle.
• the magnetic particles comprise a magnetic core and a polymer coating, which may further comprise an epoxy coating. In some embodiments, the magnetic particles comprise a magnetic core and a glass coating. [0010] In some embodiments, the cell handling procedure involves mixing and dispensing the cell suspension. In some embodiments, the cell handling procedure comprises one or more rounds of split-pool process.
• the cell handling procedure comprises the steps of binding to the targets in a plurality of cells combined with magnetic particles a plurality of unique binding agents that are specific for one of the targets; adding multiple subcode oligonucleotides to each of the bound agents in the plurality of cells in an ordered manner during successive rounds of split pool synthesis wherein the subcode oligonucleotides in each round anneal adjacently to the subcode oligonucleotide from a previous round via an annealing region, and covalently linking the adjacently annealed subcode oligonucleotides to each other to create in each cell, a unique cell-originating nucleotide code.
• the unique binding agent may be an antibody or a nucleic acid probe.
• the unique cell-originating nucleotide code may be amplified prior to sequencing.
• the cell handling procedure comprises enzymatic tissue dissociation and one or more rounds of wash and centrifugation. In some embodiments, the cell handling procedure comprises lysis of red blood cells and one or more rounds of wash and centrifugation. [0013] In some embodiments, the cell handling procedure comprises the steps of: contacting the cell-magnetic particle mixture with a primary antibody, washing away the primary antibody, reconstituting the cell-magnetic particle mixture in a buffer and optionally also contacting the cell -magnetic particle mixture with a secondary antibody and washing away the primary antibody.
• the buffer is suitable for flow cytometry or mass-cytometry.
• the method further comprises the step of encapsulating the cells separated from the magnetic particles in oil-encapsulated droplets in a water-oil emulsion at about one cell per droplet.
• magnetic particle refers to a micro particle having magnetic or paramagnetic properties.
• the particles may be covered in a coating not affecting its magnetic or paramagnetic properties.
• the instant invention does not require any specific affinity reagents or moieties on the particle although such reagents or moieties do not interfere with the method of the invention.
• nucleic acid refers to a nucleotide polymer, and unless otherwise limited, includes known analogs of natural nucleotides that can function in a similar manner (e.g., hybridize) to naturally occurring nucleotides.
• polynucleotide refers to a nucleotide polymer, and unless otherwise limited, includes known analogs of natural nucleotides that can function in a similar manner (e.g., hybridize) to naturally occurring nucleotides.
• nucleic acid and “oligonucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three dimensional structure, and may perform any function, known or unknown.
• polynucleotides coding or non-coding regions of a gene or gene fragment, intergenic DNA, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), small nucleolar RNA, ribozymes, complementary DNA (cDNA), which is a DNA representation of mRNA, usually obtained by reverse transcription of messenger RNA (mRNA) or by amplification; DNA molecules produced synthetically or by amplification, genomic DNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
• mRNA messenger RNA
• transfer RNA transfer RNA
• ribosomal RNA short interfering RNA
• shRNA short-hairpin
• a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. Polynucleotide sequences, when provided, are listed in the 5' to 3' direction, unless stated otherwise.
• a nucleic acid "probe” is an oligonucleotide capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, generally through complementary base pairing, usually through hydrogen bond formation, thus forming a duplex structure.
• the probe binds or hybridizes to a "probe binding site.”
• the probe can be labeled with a detectable label to permit facile detection of the probe, particularly once the probe has hybridized to its complementary target. Alternatively, however, the probe may be unlabeled, but may be detectable by specific binding with a ligand that is labeled, either directly or indirectly.
• the term "epitope” and "target molecule” are used interchangeably herein to refer to the molecule of interest (parts of it or the whole molecule) being detected and/or quantified by the methods described herein.
• the invention relates to an improved method of cell handling that minimizes cell loss. Many therapeutic and diagnostic procedures involve multiple steps of handling cells and cell loss occurs during such steps. For example, flow cytometry, fluorescence-activated cell sorting (FACS) and mass-spectrometry (CyTOF) require a cell handling procedures including pipetting, centrifugation, resuspension and washes before applying a cell suspension to an instrument. Each of these steps is associated with cell loss.
• FACS fluorescence-activated cell sorting
• CyTOF mass-spectrometry
• Patent 10,144,950 describes a novel process of single-cell analysis called Quantum Barcoding (QBC), where each cell in a cell suspension is labeled with a unique combinatorial barcode.
• QBC Quantum Barcoding
• the barcode is assembled via a split-pool process where a cell suspension is subjected to multiple rounds of mixing, splitting and pooling the cells performed using a microfluidic device or robotic or manual pipetting. Assembling a unique combination barcode required a sufficient nu ber of split-pool rounds, each associated with some cell loss.
• some therapeutic procedures involve multi-step process of handling a cell population. For example, chimeric antigen receptor T cell therapy (CAR-T cell therapy) involves ex-vivo handling of patient’s T cells. Cell loss associated with the multiple handling steps reduced the yield of CAR-T cells material.
• CAR-T cell therapy chimeric antigen receptor T cell therapy
• the present invention is based on a surprising discovery that magnetic particles (magnetic beads) when added as “ballast” to the cell handling procedures, significantly reduce cell loss.
• the magnetic property of the particles allows for easy separation of the particles from cells at the end of the cell-handling protocol.
• the invention is an improved method of handling cells comprising contacting a cell suspension with magnetic particles prior to at least some of the steps of a cell-handling protocol or prior to the very first step of the cell-handling protocol.
• cells can be prokaryotic or eukaryotic including animal, fungal and plant cells.
• organelles including mitochondria and plastids (chromoplasts and chloroplasts) and intra- cellular stages of the life cycle of the bacterial genus Mycoplasmae and Chlamydiae.
• a cell ranges in size between 1-5 micrometers for prokaryotes and 10-100 micrometers for eukaryotes.
• a typical human blood cell, e.g., a lymphocyte is 7-20 micrometers in diameter.
• the instant invention is not limited to any particular type of cells and does not rely on any intra-or extra- cellular marker or molecule to be present in or on the cell.
• the method of the invention does not necessarily require any affinity reagent but merely the presence of magnetic particles in the cell suspension undergoing a handling process. If a commercially available magnetic particle comprises an affinity reagent, for convenience, such a particle can be used in the method of the invention.
• the present invention involves a method of handling cells from a sample.
• the sample is derived from a subject or a patient.
• the sample may comprise a fragment of a solid tissue or a solid tumor derived from the subject or the patient, e.g. , by biopsy.
• the sample may also comprise body fluids ⁇ e.g., urine, sputum, serum, plasma or lymph, saliva, sputum, sweat, tear, cerebrospinal fluid, amniotic fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, cystic fluid, bile, gastric fluid, intestinal fluid, or fecal samples) that may contain cells.
• the sample may comprise whole blood or blood fractions where normal or tumor cells may be present.
• the sample is a cultured sample, e.g, a tissue culture containing cells.
• the cells of interest in the sample are infectious agents such as bacteria, protozoa or fungi.
• Nucleic acids, proteins or other markers of interest may be present in the cells and are the target of the cell-handling procedure.
• Each nucleic acid target is characterized by its nucleic acid sequence.
• Each protein target is characterized by its amino acid sequence and its epitopes recognized by specific antibodies.
• the target nucleic acid contains a locus of a genetic variant, e.g., a polymorphism, including a single nucleotide polymorphism or variant (SNP of SNV), or a genetic rearrangement resulting e.g., in a gene fusion.
• a protein biomarker contains an amino-acid change resulting in the creation of a unique epitope.
• the target nucleic acid or target protein comprises a biomarker, i.e., a gene or protein antigen whose variants are associated with a disease or condition.
• a biomarker i.e., a gene or protein antigen whose variants are associated with a disease or condition.
• the target nucleic acids and proteins can be selected from panels of disease-relevant markers described in U.S. Patent Application Ser. No. 14/774,518 filed on September 10, 2015. Such panels are available as AVENIO ctDNA Analysis kits (Roche Sequencing Solutions, Pleasanton, Cal.)
• the target nucleic acids or proteins are characteristic of a particular organism and aids in identification of the organism or a characteristic of the pathogenic organism such as drug sensitivity or drug resistance.
• the target nucleic acid or protein is a unique characteristic of a human subject, e.g., a combination of HLA or KIR sequences defining the subject’s unique HLA or KIR genotype.
• the target nucleic acid is a somatic sequence such as a rearranged immune sequence representing an immunoglobulin (including IgG, IgM and IgA immunoglobulin) or a T-cell receptor sequence (TCR).
• the target is a fetal sequence present in maternal blood, including a fetal sequence characteristic of a fetal disease or condition or a maternal condition related to pregnancy.
• the target could be one or more of the autosomal or X-linked disorders described in Zhang et al. (2019) Non- invasive prenatal sequencing for multiple Mendelian monogenic disorders using circulating cell-free fetal DNA, Nature Med. 25(3):439.
• the target is a nucleic acid (including mRNA, microRNA, viral RNA, cellular DNA or cell-free DNA (cfDNA) including circulating tumor DNA (ctDNA)).
• the target is a protein expressed in the cell.
• the protein target may be cell-surface protein.
• the cell surface protein is a lymphocyte surface protein selected from inhibitory receptors (such as Pdcdl, Havrcr2, Lag3, CD244, Entpdl, CD38, CD101, Tigit, CTLA4), cell surface receptors (such as TNFRSF9, TNFRSF4, Klrgl, CD28, Icos, IL2Rb, IL7R) or chemokine receptors (such as CX3CR1, CCL5, CCL4, CCL3, CSFl, CXCR5, CCR7, XCL1 and CXCL10).
• the proteins are selected from CD4, CD8, CD11, CD16, CD19, CD20, CD45, CD56 and CD279.
• magnetic particles are of a kind described in
• WO20 19086517 These particles comprise a stabilizer, a superparamagnetic core, and a liquid glass coating.
• the magnetic particle of this kind is a spherical particle 300-500 nanometers (nm) in size with the core being 270-290 nm.
• the particles have saturation magnetization of 50-70 Am 2 /kg and magnetic remanence below 3 Am 2 /kg.
• the liquid glass coating is 10-20 nm thick and comprises a silicate, e.g., sodium silicate, potassium silicate, calcium silicate, lithium silicate, and magnesium silicate.
• the magnetic core is a defined aggregate of magnetic nanoparticles ⁇ 30 nm in size combined with the stabilizer such as citrate, histidine, cetyltrimethylammonium bromide (CTAB), cetyltrimethylammonium chloride (CTAC), sodium oleate or polyacrylic acid.
• the stabilizer such as citrate, histidine, cetyltrimethylammonium bromide (CTAB), cetyltrimethylammonium chloride (CTAC), sodium oleate or polyacrylic acid.
• the magnetic core is Fe 3 0 4 , alphaFe 2 0 3 , gammaFe 2 0 3 , MnFe x O y , CoFe x O y , NiFe x O y , CuFe x O y , ZnFe x O y , CdFe x O y , BaFe x O and SrFe x O, wherein x is 1 to 3 and y is 3 or 4.
• the core of the particle is FesOi.
• magnetic particles are of a kind described in
• These magnetic particles are slightly larger spherical particles 0.5-15 micrometers (pm) in size.
• the particles have a magnetic core made of a magnetic metal, and a glass coating comprising one or more of Si0 2 , B 2 0 3 , K 2 0, CaO, Al 2 0 3 and ZnO.
• magnetic particles are of a kind described in
• These magnetic particles are larger spherical superparamagnetic particles 5-40 micrometers (pm) in size and comprise a hypercrosslinked polymer matrix covering a magnetic core.
• the magnetic core is comprised of 1-20 magnetic nanoparticles and have a saturation magnetization between 10 Am 2 /kg to 20 Am 2 /kg. These particles may have a pore under 100 nm in size.
• the polymer coating may comprise tensides, silica, silicates, silanes, phosphates, phosphonates, phosphonic acids and mixtures of two or more thereof.
• the polymer coating of such particles may comprise polyacrylic acid derivatives, tricarboxylic acids, tricarboxylic acid salts, tricarboxylic acid derivatives, amino acids, amino acid salts, amino acid derivatives, surfactants, salts of surfactants, fatty acids, fatty acid salts and fatty acid derivatives.
• the particles are magnetic polymer- coated particles with magnetite embedded into coating during polymerization.
• the polymer is formed by polymerization of divinylbenzene and vinylbenzylchloride.
• Some commercially available magnetic particles comprise an affinity molecule for capture of targets labeled with a ligand for the affinity molecule.
• F or example, many commercially available particles comprise streptavidin for capture of biotinylated targets. While streptavidin is not needed for practicing the method of the invention, its presence is not detrimental to the performance of the method. For convenience, commercially available streptavidin-coated magnetic particles may be used in the method of the invention to effectively reduce or minimize cell loss.
• magnetic particles are of a kind described in
• the particles are 0.8 to 10 pm in size.
• the particles are magnetic polymer particles composed of a matrix polymer with pores and having superparamagnetic crystals on a surface or in the pores of the polymer and further having a polymer coating.
• the polymer coated is composed of two compounds selected from epoxides are selected from epichlorohydrin, epibromohydrin, isopropylglycidyl ether, butyl glycidyl ether, allylglycidyl ether, 1,4-butanediol diglycidyl ether (1,4-bis (2,3-epoxypropoxy) butane), ethylhexylglycidylether, methyl glycidylether neopentylglycol diglycidyl ether, ethylene glycol diglycidyl ether, glycerol diglycidyl ether, glycidol, and glycidyl methacrylate.
• epoxides are selected from epichlorohydrin, epibromohydrin, isopropylglycidyl ether, butyl glycidyl ether, allylglycidyl ether,
• magnetic particles are of a kind described in
• spherical particles are 5-8 pm in size and are composed of monodisperse epoxy coated porous matrix polymer having superparamagnetic crystals located mostly within the pores.
• the epoxy is selected from epichlorohydrin, epibromohydrin, isopropylglycidyl ether, butyl glycidyl ether, allylglycidyl ether, 1,4- butanediol diglycidyl ether (l,4-bis(2,3-epoxypropoxy) butane), neopentylglycol diglycidyl ether, ethylene glycol diglycidyl ether, glycerol diglycidyl ether, glycidol, glycidyl methacrylate, ethyl hexyl glycidylether, methyl glycidylether, glycerol propoxylate triglycidylether, poly(propylene glycol) dic
• the invention is an improved method of performing flow cytometry including fluorescence-activated cell-sorting (FACS) comprising preparing a cell suspension in the presence of magnetic particles and removing magnetic particles prior to applying the cell suspension to a flow cytometry instrument.
• FACS fluorescence-activated cell-sorting
• the improved flow- cytometry method starts with obtaining a cell suspension containing approximately between 10 5 and 10 6 cells.
• the cells are contacted with magnetic particles at a ratio of about 1:2 to 1:3 cell to particle.
• the invention is not limited to a particular cell-particle ratio. Rather, the optimal ratio may be determined experimentally for each particular size and type of cell wherein a range of celhparticle ratios is satisfactory to reduce or minimize cell loss.
• the cell-magnetic particle mixture is optionally washed one or more times by centrifugation and resuspension of the cell-particle mixture in a suitable buffer.
• the cell-magnetic particle mixture is contacted with a suitable primary and secondary antibody.
• a centrifugation step pellets cells and magnetic particles from the staining suspension.
• the antibody-containing supernatant is removed.
• the traces of the antibody are removed by optional one or more washes including centrifugation of the cell-magnetic particle mixture, removal of the supernatant, and resuspension of the cell-magnetic particle mixture in a fresh buffer.
• the cell-magnetic particle mixture prior to the final centrifugation, the cell-magnetic particle mixture is thoroughly mixed (vortexed) in a fresh buffer.
• the first (primary) antibody is conjugated to a fluorochrome.
• the primary antibody must be bound to a secondary antibody, which is fluorochrome-conjugated.
• the cell-magnetic particle mixture is contacted with a secondary antibody and a wash procedure described above for the first (primary) antibody is repeated.
• the cell- magnetic particle mixture is further contacted with a blocking reagent comprising a high concentration of immunoglobulin that will bind to the Fc-receptors on cells like monocytes, thereby blocking the non-specific binding of the staining antibody reagents to these receptors.
• the blocking reagent is a species- matched immunoglobulin, e.g., human g-globulin for human or humanized primary and secondary antibody.
• staining of the cell-magnetic particle mixture with antibodies proceeds for 30 minutes at 4°C or on ice in the optional presence of sodium azide.
• the cell-magnetic particle mixture is fixed with formaldehyde.
• the magnetic particles are separated from the cell- magnetic particle mixture using a magnet, and cells are loaded onto the flow cytometry or FACS instrument.
• 500,000 cells are mixed with an equal number of magnetic particles, centrifuged, separated from the supernatant and resuspended in a final volume of 100 m ⁇ of 1 percent BSA (Bovine serum albumin)- PBS (phosphate-buffered saline).
• BSA Bovine serum albumin
• PBS phosphate-buffered saline
• the resuspension is contacted with 2 to 3 pg of primary antibody per 1 to 200,000 cells and incubated on ice for 45 minutes.
• the stained cell-magnetic particle mixture is washed three times with 1% BSA-PBS and resuspended in 100 m ⁇ of 1% BSA-PBS.
• the washed cell-magnetic particle mixture is contacted with 2 to 3 pg of FITC labeled second antibody and incubated on ice for 45 minutes.
• the stained cell-magnetic particle mixture is washed three times with PBS without BSA and fixed with 1% paraformaldehyde for 2 minutes.
• the cell- magnetic particle mixture is washed with PBS, re-suspend in -300 pi of PBS and magnetic particles are removed.
• the invention is an improved method of performing time-of-flight mass cytometry (CyTOF) comprising preparing a cell suspension in the presence of magnetic particles and removing magnetic particles prior to applying the cell suspension to a mass cytometry instrument.
• CyTOF time-of-flight mass cytometry
• the improved mass cytometry method begins with isolating cells from a sample, e.g., isolating peripheral blood mononuclear cells (PBMCs) from blood by Ficoll-plaque density gradient centrifugation.
• PBMCs peripheral blood mononuclear cells
• the cells were prepared from dissociated tissue using commercially available reagents and instruments (e.g., gentleMACS Dissociator and Human Tumor Dissociation kit, Miltenyi Biotec, Waltham, Mass.)
• the isolated cells are contacted with magnetic particles at a ratio between 1:2 to 1:3 cell to particle.
• the invention is not limited to a particular cell-particle ratio. Rather, the optimal ratio may be determined experimentally for each particular size and type of cell wherein a range of celhparticle ratios is satisfactory to reduce or minimize cell loss.
• the cell-magnetic particle mixture is directly stained with antibodies labeled with a metal-tag.
• the antibody-containing supernatant is removed.
• the traces of the antibody are removed by optional one or more washes including centrifugation of the cell -magnetic particle mixture, removal of the supernatant and resuspension of the cell-magnetic particle mixture in a fresh buffer.
• the magnetic particles are separated from the cell-magnetic particle mixture using a magnet, and cells are loaded onto the mass-cytometry instrument.
• the invention is an improved method of performing quantum barcoding (QBC) described in U.S. Patent 10,144,950 comprising preparing a cell suspension in the presence of magnetic particles and removing magnetic particles prior to isolating nucleic acid barcodes for sequencing.
• QBC quantum barcoding
• the magnetic particles are removed prior to cell counting.
• cells are isolated from a sample. The isolated cells are contacted with magnetic particles at a ratio of about 1:2 to 1:3 cell to particle. The invention is not limited to a particular cell-particle ratio.
• the optimal ratio may be determined experimentally for each particular size and type of cell and size and type of magnetic particle wherein a range of celhparticle ratios is satisfactory to reduce or minimize cell loss.
• the cell-magnetic particle mixture is contacted with a unique binding agent, e.g., DNA or RNA probe or an antibody.
• the unique binding agent comprises at least one part or element specifically interacting with a target and an element allowing for assembly of a combinatorial barcode.
• the assay is a multiplex assay whereby a plurality of target molecules is detected in a plurality of cells in a single reaction mixture using a plurality of different binding agents of same or different kind (e.g., a plurality of different nucleic acid probes, or a plurality of different antibodies, or a combination of nucleic acid probes and antibodies).
• the unique binding agent may be an aptamer, including a nucleic acid aptamer (i.e., single- stranded DNA molecules or single-stranded RNA molecules) and a peptide aptamer.
• An antibody may comprise a linker oligonucleotide that facilitates assembly of a barcode.
• the cell-magnetic particle mixture is subjected to a split-pool barcode assembly described in more detail in the U.S. Patent 10,144,950, which is incorporated herein by reference.
• the unique cell- originating code is assembled on each cell where a unique binding agent has bound.
• the unique barcode is a modular structure assembled from subunits.
• the unique code is assembled by stepwise addition of subunits.
• the subunits attach to each other or to a common backbone via attachment regions, e.g., complementary nucleic acid sequences.
• the attachment may comprise one or both of hybridization and ligation to the backbone or to the adjacent code subunit.
• the unique codes are separated from cells and sequenced to identify each code.
• the codes are amplified prior to sequencing.
• the cells are counted prior to sequencing the barcodes.
• the magnetic particles are separated from the cells in the cell- magnetic particle mixture prior to the cell counting step.
• the invention is an improved method of handling cells in a single cell analysis workflow involving distributing cells into one cell compartments (such as wells or oil-encapsulated droplets) and contacting each isolated cell with unique identifiers encapsulated in another set of oil droplets. See U.S. Patent Nos. 9,695,468 and 10,221,442.
• the improved method of single cell analysis comprises preparing a cell suspension in the presence of magnetic particles and removing the magnetic particles prior to distributing the cells of the cell suspension into compartments.
• the improved method includes steps of enzymatic tissue and extracellular matrix disruption, pipetting, washes, centrifugation and resuspension conducted in the presence of magnetic particles in the cell suspension.
• the cell suspension with an improved yield is subjected to the single cell analysis procedure such as single cell gene expression (whole transcriptome analysis) or single cell immune profiling described in U.S. Patent Nos. 9,695,468 and 10,221,442 and enabled by the instruments distributed by 10X Genomics (Pleasanton, Cal.)
• the improved cell handling method of the invention is not limited to the exemplary applications listed above but can be used in any diagnostic, prognostic, therapeutic, patient stratification, drug development, treatment selection, and screening process that involves handling of cells and where reducing or minimizing cell loss is desired.
• PanT cells (Stem Cell Technologies, Cambridge, Mass.) were stained with a 9 antibody panel. After staining, the cells were divided into two samples of 680,000 cells each. 2 million magnetic beads were added to one of the samples. After completing the QBC workflow (Nolan, G., et al., (2020) "Ultra-high throughput single-cell analysis of proteins and RNAs by split-pool synthesis," Communications Biology, In Press) on both samples, the magnetic beads were removed by magnetic separation from the cells that remained in the supernatant. The cells were counted in both samples and the sample without magnetic beads had a cell count of 257,000 cells while the sample that was processed with magnetic beads spiked in had a cell count of 363,000 cells. 1.41 x more cells were recovered by using magnetic beads. (Table 1).
• PBMC Peripheral Blood Mononuclear cells

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