CD34 stem cell mimetics

Abstract

Hydrogel beads with quantifiable attached biomolecules and their use as cellular mimetics in cytometric applications are described. The cellular mimetics described herein are selectively tunable to have at least one optical property substantially similar to at least one optical property of a target cell (e.g., CD34 + stem cell). The disclosure further relates to methods of using the disclosed cellular mimetics as enrichment controls in cytometric applications.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 535,233, filed August 29, 2023, the contents of which are incorporated herein by reference in their entirety.

[0003] Reference to the electronic sequence list

[0004] The contents of the electronic sequence list (SLIN_024_01WO_SeqList_ST26.xml; size: 17,366 bytes; and creation date: August 19, 2024) are incorporated herein by reference in their entirety. Technical Field

[0005] This disclosure relates to material compositions and methods that allow for the calibration and experimental control of stem cells. Background Technology

[0006] CD34-expressing stem cells are increasingly being used in cell therapy and other applications. CD34-expressing stem cells are typically enriched from donor-derived blood samples and phenotypically characterized using flow cytometry for purposes such as characterizing the starting material or for quality control before and / or after enrichment. Flow cytometry allows for the measurement of forward scattering (“FSC”) and side scattering (“SSC”), parameters relating to cell volume and the internal complexity of granules (e.g., nuclear shape, amount and type of cytoplasmic granules, or membrane roughness), as well as the analysis of cell surface markers indicating cell state. Using flow cytometry, CD34-expressing cells in heterogeneous cell populations can be sorted, counted, and / or characterized, allowing them to be used for desired applications.

[0007] Controls are used to calibrate flow cytometry parameters in order to distinguish the optical properties of CD34-expressing cells from other cells in a heterogeneous population. However, commercially available stem cell controls are primarily cell-based. Such controls are limited because they typically have a low percentage of CD34-expressing cells (leading to long sampling times or large control populations to collect sufficient events), suffer from supply inconsistencies (introducing cost and / or batch-to-batch variability), and / or are characterized by poor shelf life in sealed vials (leading to more frequent bridging studies in applications requiring comparative studies). Alternative stem cell controls require the use of mobilized peripheral blood to obtain a high percentage of CD34-expressing cells, which introduces significant costs to the process. Therefore, there is a need in the art for cell-free compositions that mimic CD34-expressing stem cells in order to calibrate devices (such as flow cytometers) used to analyze populations containing such cells. Summary of the Invention

[0008] In some aspects, this disclosure provides compositions comprising hydrogel beads of a first group, the hydrogel beads comprising: a) polymeric monomers and bifunctional monomers; and b) a cell surface biomarker spectrum comprising: i) extracellular domains of CD34 and CD45.

[0009] In some embodiments, the composition further comprises a second group of hydrogel beads containing: c) polymeric monomers and bifunctional monomers; and d) a cell surface biomarker profile containing: i) a CD45 extracellular domain but lacking a CD34 extracellular domain.

[0010] In some embodiments, the composition comprises a second group of hydrogel beads comprising: c) a polymeric monomer and a bifunctional monomer; and d) a cell surface biomarker profile comprising: i) a CD45 extracellular domain, wherein each hydrogel bead in the second group comprises no more than 10% of the median number of CD34 extracellular domains contained in the hydrogel beads of the first group.

[0011] In some embodiments, the composition comprises a second group of hydrogel beads comprising: c) polymeric monomers and bifunctional monomers; and d) a cell surface biomarker profile comprising: i) CD45 extracellular domains, wherein the hydrogel beads in the second group comprise no more than 10% of the median number of CD34 extracellular domains contained in the hydrogel beads of the first group.

[0012] In some embodiments, the hydrogel beads in the second population contain no more than 0.1%, no more than 0.2%, no more than 0.3%, no more than 0.5%, no more than 0.7%, no more than 1%, no more than 2%, no more than 3%, no more than 5%, or no more than 7% of the median number of CD34 extracellular domains contained in the hydrogel beads of the first population. In some embodiments, each hydrogel bead in the second population contains no more than 0.1%, no more than 0.2%, no more than 0.3%, no more than 0.5%, no more than 0.7%, no more than 1%, no more than 2%, no more than 3%, no more than 5%, or no more than 7% of the median number of CD34 extracellular domains contained in the hydrogel beads of the first population.

[0013] In some embodiments, the hydrogel beads in the first population contain about 10% to about 400% of the amount of the CD45 extracellular domain present on the cell surface of the target cells. In some embodiments, each hydrogel bead in the first population contains about 10% to about 400% of the amount of the CD45 extracellular domain present on the cell surface of the target cells.

[0014] In some embodiments, the hydrogel beads in the first group contain about 10% to about 300%, about 20% to about 400%, about 20% to about 300%, about 20% to about 200%, or about 50% to about 200% of the amount of the CD45 extracellular domain present on the cell surface of the target cells. In some embodiments, each hydrogel bead in the first group contains about 10% to about 300%, about 20% to about 400%, about 20% to about 300%, about 20% to about 200%, or about 50% to about 200% of the amount of the CD45 extracellular domain present on the cell surface of the target cells.

[0015] In some embodiments, the hydrogel beads in the first population contain about 10% to about 400% of the amount of the CD34 extracellular domain present on the cell surface of the target cells. In some embodiments, each hydrogel bead in the first population contains about 10% to about 400% of the amount of the CD34 extracellular domain present on the cell surface of the target cells.

[0016] In some embodiments, the hydrogel beads in the first group contain about 10% to about 300%, about 20% to about 400%, about 20% to about 300%, about 20% to about 200%, or about 50% to about 200% of the amount of CD34 extracellular domain present on the cell surface of the target cells. In some embodiments, each hydrogel bead in the first group contains about 10% to about 300%, about 20% to about 400%, about 20% to about 300%, about 20% to about 200%, or about 50% to about 200% of the amount of CD34 extracellular domain present on the cell surface of the target cells.

[0017] In some embodiments, the hydrogel beads in the second population contain about 10% to about 400% of the amount of the CD45 extracellular domain present on the cell surface of the target cells. In some embodiments, each hydrogel bead in the second population contains about 10% to about 400% of the amount of the CD45 extracellular domain present on the cell surface of the target cells.

[0018] In some embodiments, the hydrogel beads in the second population contain about 10% to about 300%, about 20% to about 400%, about 20% to about 300%, about 20% to about 200%, or about 50% to about 200% of the amount of the CD45 extracellular domain present on the cell surface of the target cells. In some embodiments, each hydrogel bead in the second population contains about 10% to about 300%, about 20% to about 400%, about 20% to about 300%, about 20% to about 200%, or about 50% to about 200% of the amount of the CD45 extracellular domain present on the cell surface of the target cells.

[0019] In some embodiments, the amount of CD34 extracellular domain present on the cell surface of the target cell in the hydrogel beads of the second population does not exceed 10%. In some embodiments, each hydrogel bead in the second population contains no more than 10% of the amount of CD34 extracellular domain present on the cell surface of the target cell.

[0020] In some embodiments, the amount of CD34 extracellular domains present on the cell surface of the target cells contained in the hydrogel beads of the second population is no more than 0.1%, no more than 0.2%, no more than 0.3%, no more than 0.5%, no more than 0.7%, no more than 1%, no more than 2%, no more than 3%, no more than 5%, or no more than 7%. In some embodiments, each hydrogel bead in the second population contains an amount of CD34 extracellular domains present on the cell surface of the target cells of no more than 0.1%, no more than 0.2%, no more than 0.3%, no more than 0.5%, no more than 0.7%, no more than 1%, no more than 2%, no more than 3%, no more than 5%, or no more than 7%.

[0021] In some implementations, the amount of CD34 and / or CD45 extracellular domains present on the cell surface of the target cells is the median amount of CD34 and / or CD45 extracellular domains present on the cell surface of cells in a leukopack of CD34+-rich cells treated with Protocol H.

[0022] In some implementations, the target cells are hematopoietic stem cells. In some implementations, the target cells are CD45dim-positive (CD45dim+) and CD34-positive (CD34+) stem cells.

[0023] In some implementations, the target cells are lymphocytes.

[0024] In some embodiments, flow cytometry is used to measure the amount of CD45 and / or CD34 extracellular domains present in the hydrogel and / or on the cell surface based on fluorescence intensity. In some embodiments, the fluorescence intensity of the CD45 extracellular domain is measured using a fluorophore-labeled CD45-specific binding molecule, and / or the fluorescence intensity of the CD34 extracellular domain is measured using a fluorophore-labeled CD34-specific binding molecule. In some embodiments, the binding molecule comprises a monoclonal antibody or an antigen-binding fragment thereof. In some embodiments, the CD34-specific binding molecule is selected from: phycoerythrin (PE)-labeled anti-CD34 antibody clone 8G12, phycoerythrin (PE)-labeled anti-CD34 antibody clone AC136, allophycocyanin (APC)-labeled anti-CD34 antibody clone 4H11, and Brilliant™ Violet 421 (BV421)-labeled anti-CD34 antibody clone 581. In some embodiments, the CD45-specific binding molecule is selected from: FITC-labeled anti-CD45 antibody clone 2D1, PerCP-Cyanine® 5.5 (PerCP-Cy5.5)-labeled anti-CD45 antibody clone 2D1, PE-labeled anti-CD45 antibody clone MEM-28, and BD Horizon™ V500-labeled anti-CD45 antibody clone HI30. In some embodiments, a combination of a CD34-specific binding molecule and a CD45-specific binding molecule is selected from one of the groups in Table 4; optionally, the combination is group 1 in Table 4.

[0025] In some embodiments, the biomarker is attached to the matrix of the hydrogel beads via a connector. In some embodiments, the biomarker is covalently attached to the matrix of the hydrogel beads via a connector. In some embodiments, the biomarker is non-covalently attached to the matrix of the hydrogel beads via a connector.

[0026] In some embodiments, the first and / or second group of hydrogel beads have an average diameter of about 1 μm to about 40 μm, about 20 μm to about 30 μm, about 3 μm to about 20 μm, or about 4-10 μm. In some embodiments, the first group of hydrogel beads has an average diameter of about 1 μm to about 40 μm, about 20 μm to about 30 μm, about 3 μm to about 20 μm, or about 4-10 μm. In some embodiments, the second group of hydrogel beads has an average diameter of about 1 μm to about 40 μm, about 20 μm to about 30 μm, about 3 μm to about 20 μm, or about 4-10 μm. In some embodiments, the first and second groups of hydrogel beads have an average diameter of about 1 μm to about 40 μm, about 20 μm to about 30 μm, about 3 μm to about 20 μm, or about 4-10 μm.

[0027] In some embodiments, the active portion of the composition comprises or consists of a first group of hydrogel beads and a second group of hydrogel beads.

[0028] In some embodiments, the first group comprises 1%-3% of the number of hydrogel beads in the active portion of the composition.

[0029] In some embodiments, the second group comprises 50%-99% of the number of hydrogel beads in the active portion of the composition; wherein any remaining portion of the active portion of the composition contains hydrogel beads lacking CD34 and CD45. In some embodiments, the second group comprises 97%-99% of the number of hydrogel beads in the active portion of the composition; wherein any remaining portion of the active portion of the composition contains hydrogel beads lacking CD34 and CD45.

[0030] In some embodiments, the first group comprises 1%-3% of the number of hydrogel beads in the active portion of the composition; and the second group comprises 97%-99% of the number of hydrogel beads in the active portion of the composition.

[0031] In some embodiments, the first group comprises 1.4%-1.8% of the number of hydrogel beads in the active portion of the composition; and the second group comprises 98.2%-98.6% of the number of hydrogel beads in the active portion of the composition.

[0032] In some embodiments, the first group of hydrogel beads and the second group of hydrogel beads together account for at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% of the number of hydrogel beads in the active portion of the composition.

[0033] In some embodiments, the ratio of the number of hydrogel beads in the first group to the number of hydrogel beads in the second group does not exceed 1, 0.5, 0.3, 0.2, 0.1, 0.07, 0.05, 0.03, 0.02, 0.018, 0.017, 0.016, 0.015, 0.01, 0.005, or 0.001. In some embodiments, the ratio of the number of hydrogel beads in the first group to the number of hydrogel beads in the second group is not greater than 0.0183.

[0034] In some embodiments, the ratio of the number of hydrogel beads in the first group to the number of hydrogel beads in the second group is about 1 to about 0.5, about 0.5 to about 0.2, about 0.2 to about 0.1, about 0.1 to about 0.05, about 0.05 to about 0.02, about 0.02 to about 0.015, about 0.015 to about 0.01, about 0.01 to about 0.005, about 0.005 to about 0.002, or about 0.002 to about 0.001, including all ranges and subranges therein. In some embodiments, the ratio of the number of hydrogel beads in the first group to the number of hydrogel beads in the second group is about 0.0183 to about 0.0142.

[0035] In some embodiments, the first group comprises 10%-95% of the number of hydrogel beads in the active portion of the composition.

[0036] In some embodiments, the first group comprises 80%-95% of the number of hydrogel beads in the active portion of the composition.

[0037] In some embodiments, the second group comprises 5%-90% of the number of hydrogel beads in the active portion of the composition. In some embodiments, any remaining portion of the active portion of the composition contains hydrogel beads lacking CD34 or CD45.

[0038] In some embodiments, the second group comprises 5%-20% of the number of hydrogel beads in the active portion of the composition.

[0039] In some embodiments, the first group comprises 80%-95% of the number of hydrogel beads in the active portion of the composition; and the second group comprises 5%-20% of the number of hydrogel beads in the active portion of the composition. In some embodiments, the first group comprises 90%-95% of the number of hydrogel beads in the active portion of the composition; and the second group comprises 5%-10% of the number of hydrogel beads in the active portion of the composition.

[0040] In some embodiments, the first group of hydrogel beads and the second group of hydrogel beads together account for at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the number of hydrogel beads in the active portion of the composition.

[0041] In some embodiments, the ratio of the number of hydrogel beads in the first group to the number of hydrogel beads in the second group is at least 1, at least 2, at least 5, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 50, at least 70, or at least 100. In some embodiments, the ratio of the number of hydrogel beads in the first group to the number of hydrogel beads in the second group is at least 9.

[0042] In some embodiments, the ratio of the number of hydrogel beads in the first group to the number of hydrogel beads in the second group is about 1 to about 2, about 2 to about 5, about 5 to about 8, about 8 to about 10, about 10 to about 12, about 12 to about 15, about 15 to about 20, about 20 to about 50, about 50 to about 100, including all ranges and subranges therein. In some embodiments, the ratio of the number of hydrogel beads in the first group to the number of hydrogel beads in the second group is about 9 to about 19.

[0043] In some aspects, this disclosure provides a kit comprising two containers containing different compositions of this disclosure. In some embodiments, the first container contains a composition having more hydrogel beads in a second group than in the first group, and the second container contains a composition having more hydrogel beads in the first group than in the second group. In some embodiments, the first container contains at least 2, 3, 4, 5, 6, 7, 8, or more times more hydrogel beads than the second container.

[0044] In some implementations, the first container contains approximately 5 × 10 5 Approximately 2×10 6 10 beads, and the second container contains approximately 1×10 5 Approximately 4×10 5 10 beads. In some embodiments, the first container contains approximately 1 × 10⁻⁶ beads. 6 The second container contains approximately 2.5 × 10⁶ beads. 5 Each bead.

[0045] In some embodiments, the ratio of (a) the total number of hydrogel beads of the first group and the second group in the first container to (b) the total number of hydrogel beads of the first group and the second group in the second container is about 1 to about 10, about 2 to about 8, about 3 to about 6, or about 4, including all ranges and subranges therein.

[0046] In some implementations, the CD34 and CD45 cell surface markers each contain a fluorophore.

[0047] In some implementations, the CD34 and CD45 cell surface markers each contain different fluorophores.

[0048] In some embodiments, each fluorophore is independently selected from any of the following: polydinophyte chlorophyll protein-cyanine 5.5 dye (PerCP-Cy5.5); phycoerythrin-cyanine 7 (PE Cy7); allophycocyanin-cyanine 7 (APC-Cy7); fluorescein isothiocyanate (FITC); phycoerythrin (PE); allophycocyanin (APC); 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein succinimide; 5-(and-6)-carboxyeosin; 5-carboxyfluorescein; 6-carboxyfluorescein; 5-(and-6)-carboxyfluorescein; S-carboxyfluorescein-bis-(5-carboxymethoxy-2-nitrobenzyl) ether, -alanine-formamide, or succinimide; 5-carboxyfluorescein succinimide; 6 -Carboxyfluorescein succinimide; 5-(and-6)-carboxyfluorescein succinimide; 5-(4,6-dichlorotriazinyl)aminofluorescein; 2',7'-difluorofluorescein; Eosin-5-isothiocyanate; Erythrosin-5-isothiocyanate; 6-(fluorescein-5-formylamino)hexanoic acid or succinimide; 6-(fluorescein-5-(and-6)-formylamino)hexanoic acid or succinimide; fluorescein-S-EX succinimide; fluorescein-5-isothiocyanate; fluorescein-6-isothiocyanate; OregonGreen® 488 carboxylic acid or succinimide ester; Oregon Green® 488 isothiocyanate; Oregon Green® 488-X succinimide ester; Oregon Green® 500 carboxylic acid; Oregon Green® 500 carboxylic acid, succinimide ester, or triethylammonium salt; Oregon Green® 514 carboxylic acid; Oregon Green® 514 carboxylic acid or succinimide ester; RhodamineGreen™ carboxylic acid, succinimide ester, or hydrochloride; Rhodamine Green™ carboxylic acid, trifluoroacetamide, or succinimide ester; RhodamineGreen™-X succinimide ester or hydrochloride; RhodolGreen™ carboxylic acid, N,O-bis-(trifluoroacetyl) or succinimide ester; bis-(4-carboxypiperidinyl)sulfonylrhodamine or bis(succinimide ester); 5-(and-6)carboxynaphthalene fluorescein, 5-(and-6)carboxynaphthalene fluorescein succinimide ester; 5-carboxyrhodamine 6G hydrochloride; 6-carboxyrhodamine 6G hydrochloride, 5-carboxyrhodamine 6G succinimide ester; 6-carboxyrhodamine 6G succinimide ester; 5-(and-6)-carboxyrhodamine 6G succinimide ester; 5-carboxy-2',4',5',7'-tetrabromosulfone fluorescein succinimide ester or bis-(diisopropylethylammonium) salt; 5-carboxytetramethylrhodamine; 6-carboxytetramethylrhodamine; 5-(and-6)-carboxytetramethylrhodamine;5-Carboxytetramethylrhodamine succinimide; 6-Carboxytetramethylrhodamine succinimide; 5-(and-6)-Carboxytetramethylrhodamine succinimide; 6-Carboxy-X-rhodamine; 5-Carboxy-X-rhodamine succinimide; 6-Carboxy-X-rhodamine succinimide; 5-(and-6)-Carboxy-X-rhodamine succinimide; 5-Carboxy-X-rhodamine triethylammonium salt; Lissamine; TMRhodamine B sulfonyl chloride; Malachite green; Isothiocyanate; NANOGOLD® mono(sulfosuccinimide); QSY® 21 carboxylic acid or succinimide; QSY® 7 carboxylic acid or succinimide; Rhodamine Red™-X succinimide; 6-(tetramethylrhodamine-5-(and-6)-formamide)hexanoic acid; succinimide; tetramethylrhodamine-5-isothiocyanate; tetramethylrhodamine-6-isothiocyanate; tetramethylrhodamine-5-(and-6)-isothiocyanate; Texas Red® sulfonyl; Texas Red® sulfonyl chloride; Texas Red®-X STP ester or sodium salt; Texas Red®-X succinimide; Texas Red®-X succinimide; X-rhodamine-5-(and-6)isothiocyanate, BODIPY® FL; BODIPY® TMR STP ester; BODIPY® TR-X STP ester; BODIPY® 630 / 650-X STP ester; BODIPY® 650 / 665-X STP ester; 6-Dibromo-4,4-difluoro-5,7-dimethyl-4-boron-3a,4a-diaza-s-indaminone-3-propionate succinimide ester; 4,4-difluoro-4-boron-3a,4a-diaza-s-indaminone-3,5-dipropionic acid; 4,4-difluoro-5,7-dimethyl-4-boron-3a,4a-diaza-s-indaminone-3-pentanoic acid; 4,4-difluoro-5,7-dimethyl-4-boron-3a,4a-diazon-s-indaminone-3-pentanoic acid; 4,4-Diaza-S-indaminozide-3-pentanoic acid succinimide; 4,4-Difluoro-5,7-dimethyl-4-boron-3a,4a-diaza-S-indaminozide-3-propionic acid; 4,4-Difluoro-5,7-dimethyl-4-boron-3a,4a-diaza-S-indaminozide-3-propionic acid succinimide; 4,4-Difluoro-5,7-dimethyl-4-boron-3a,4a-diaza-S-indaminozide-3-propionic acid; sulfosuccinate Imide esters or sodium salts; 6-((4,4-difluoro-5,7-dimethyl-4-boron-3a,4a-diaza-s-indane-3-propionyl)amino)hexanoic acid; 6-((4,4-difluoro-5,7-dimethyl-4-boron-3a,4a-diaza-s-indane-3-propionyl)amino)hexanoic acid or succinimide esters; N-(4,4-difluoro-5,7-dimethyl-4-boron-3a,4a-diaza-s-indane) (3-propionyl)cysteine, succinimide ester or triethylammonium salt; 6-4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-boron-3a,4a-4,4-difluoro-5,7-diphenyl-4-boron-3a,4a-diaza-s-indaminone-3-propionic acid; 4,4-difluoro-5,7-diphenyl-4-boron-3a,4a-diaza-s-indaminone-3-propionic acid succinimide ester;4,4-Difluoro-5-phenyl-4-boron-3a,4a-diaza-s-indaminone-3-propionic acid; succinimide ester; 6-((4,4-difluoro-5-phenyl-4-boron-3a,4a-diaza-s-indaminone-3-propionyl)amino)hexanoic acid or succinimide ester; 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-boron-3a,4a-diaza-s-indaminone-3-propionic acid succinimide ester; 4,4-difluoro 5-(2-pyrrolidinyl)-4-boron-3a,4a-diaza-s-indane-3-propionic acid succinimide ester; 6-(((4,4-difluoro-5-(2-pyrrolidinyl)-4-boron-3a,4a-diaza-s-indane-3-yl)styryloxy)acetyl)aminohexanoic acid or succinimide ester; 4,4-difluoro-5-styryl-4-boron-3a,4a-diaza-s-indane-3-propionic acid; 4,4-difluoro-5-styrene 4,4-Boron-3a,4a-diaza-sindenene-3-propionic acid; succinimide ester; 4,4-difluoro-1,3,5,7-tetramethyl-4-boron-3a,4a-diaza-sindenene-8-propionic acid; 4,4-difluoro-1,3,5,7-tetramethyl-4-boron-3a,4a-diaza-sindenene-8-propionic acid succinimide ester; 4,4-difluoro-5-(2-thienyl)-4-boron-3a,4a-diaza-sindenene -3-propanoic acid succinimide ester; 6-(((4-(4,4-difluoro-5-(2-thienyl)-4-boron-3a,4a-diaza-s-indane-3-yl)phenoxy)acetyl)amino)hexanoic acid or succinimide ester; and 6-(((4,4-difluoro-5-(2-thienyl)-4-boron-3a,4a-diaza-s-indane-3-yl)styryloxy)acetyl)aminohexanoic acid or succinimide ester, Alexa Fluor® Alexa Fluor® 350 carboxylic acid; Alexa Fluor® 430 carboxylic acid; Alexa Fluor® 488 carboxylic acid; Alexa Fluor® 532 carboxylic acid; Alexa Fluor® 546 carboxylic acid; Alexa Fluor® 555 carboxylic acid; Alexa Fluor® 568 carboxylic acid; Alexa Fluor® 594 carboxylic acid; Alexa Fluor® 633 carboxylic acid; Alexa Fluor® 647 carboxylic acid; Alexa Fluor® 660 carboxylic acid; Alexa Fluor® 680 carboxylic acid; Cy3 NHS ester; Cy5 NHS ester; Cy5.5 NHS ester; and Cy7 NHS ester.

[0049] In some embodiments, the fluorophore is conjugated to an antibody or a fragment thereof, which binds to an epitope within the polymer bead.

[0050] In some embodiments, CD34 is derived from Homo sapiens. In some embodiments, the extracellular domain of CD34 comprises Ser32-Thr290 of uniprot P28906, or a sequence containing at least 70%, 75%, 80%, 85%, 90%, 95%, or 97.5% identity with SEQ ID NO: 1.

[0051] In some embodiments, CD45 is derived from Homo sapiens. In some embodiments, the extracellular domain of CD45 comprises Gln26-Lys577 of uniprot P08575, or a sequence containing at least 70%, 75%, 80%, 85%, 90%, 95%, or 97.5% identity with any of SEQ ID NO: 2-9. In some embodiments, the extracellular domain of CD45 comprises Gln26-Lys577 of uniprot P08575, or a sequence containing at least 70%, 75%, 80%, 85%, 90%, 95%, or 97.5% identity with SEQ ID NO: 10.

[0052] In some embodiments, the hydrogel beads exhibit at least one optical property substantially similar to the corresponding optical properties of the target cells. In some embodiments, the at least one optical property includes lateral scattering. In some embodiments, the at least one optical property includes forward scattering. In some embodiments, the at least one optical property includes both lateral scattering and forward scattering.

[0053] In some implementations, the target cells are hematopoietic stem cells. In some implementations, the target cells are lymphocytes.

[0054] In some aspects, this disclosure provides methods for calibrating a cell analyzer for detecting cells expressing CD34 in a cell population. In some aspects, this disclosure provides methods for calibrating a cell analyzer for detecting cells expressing CD45 in a cell population. In some aspects, this disclosure provides methods for calibrating a cell analyzer for detecting cells expressing both CD34 and CD45 in a cell population. In some embodiments, the method includes sampling a composition or kit of this disclosure and calibrating the cell analyzer based on at least one optical property of the hydrogel beads of the composition. In some embodiments, the method further includes sampling a cell population and obtaining cells containing at least one optical property. In some embodiments, the method includes forming a gating scheme based on at least one optical property.

[0055] In some aspects, this disclosure provides a method for enriching cells expressing CD34, comprising sampling a composition or kit of the disclosure and forming a gating scheme based on at least one optical property of the hydrogel beads of the composition, and selecting cells expressing CD34 from a cell population based on the gating scheme.

[0056] In some implementations, cells expressing CD34 express reduced amounts of CD45.

[0057] In some implementations, the cells expressing CD34 are CD34-expressing stem cells.

[0058] In some embodiments, at least one optical property includes FSC, SSC, median fluorescence intensity (MFI) of CD45, MFI of CD34, or any combination thereof.

[0059] In some aspects, this disclosure provides a first composition comprising a first group and a second group, wherein the hydrogel beads of the first group and the hydrogel beads of the second group together comprise the active portion of the composition. In some embodiments, the first group comprises 1%-3% of the number of hydrogel beads in the active portion of the composition. In some embodiments, the second group comprises 50%-99% of the number of hydrogel beads in the active portion of the composition; wherein any remaining portion of the active portion of the composition comprises hydrogel beads lacking CD34 or CD45. In some embodiments, the second group comprises 97%-99% of the number of hydrogel beads in the active portion of the composition. In some embodiments, the first group comprises 1%-3% of the number of hydrogel beads in the active portion of the composition; and the second group comprises 97%-99% of the number of hydrogel beads in the active portion of the composition. In some embodiments, the first group comprises 1.4%-1.8% of the number of hydrogel beads in the active portion of the composition; and the second group comprises 98.2%-98.6% of the number of hydrogel beads in the active portion of the composition.

[0060] In some aspects, this disclosure provides a second composition comprising a first group and a second group, wherein the hydrogel beads of the first group and the hydrogel beads of the second group together comprise the active portion of the composition. In some embodiments, the first group comprises 10%-95% of the number of hydrogel beads in the active portion of the composition. In some embodiments, the first group comprises 80%-95% of the number of hydrogel beads in the active portion of the composition. In some embodiments, the second group comprises 5%-90% of the number of hydrogel beads in the active portion of the composition; wherein any remaining portion of the active portion of the composition comprises hydrogel beads lacking CD34 or CD45. In some embodiments, the second group comprises 5%-20% of the number of hydrogel beads in the active portion of the composition. In some embodiments, the first group comprises 80%-95% of the number of hydrogel beads in the active portion of the composition; and the second group comprises 5%-20% of the number of hydrogel beads in the active portion of the composition. In some embodiments, the first group comprises 90%-95% of the number of hydrogel beads in the active portion of the composition; and the second group comprises 5%-10% of the number of hydrogel beads in the active portion of the composition.

[0061] In some embodiments of any of the foregoing or related aspects, CD34 is derived from Homo sapiens. In some embodiments, CD45 is derived from Homo sapiens. In some embodiments, the extracellular domain of CD34 comprises Ser32-Thr290 of uniprot P28906 or SEQ ID NO: 11, or a sequence containing at least 70%, 75%, 80%, 85%, 90%, 95%, or 97.5% identity with Ser32-Thr290 of uniprot P28906 or SEQ ID NO: 11. In some embodiments, the extracellular domain of CD34 comprises SEQ ID NO: 1, or a sequence containing at least 70%, 75%, 80%, 85%, 90%, 95%, or 97.5% identity with SEQ ID NO: 1. In some embodiments, the CD45 extracellular domain comprises Gln26-Lys577 of uniprot P08575 or SEQ ID NO: 12, or a sequence containing at least 70%, 75%, 80%, 85%, 90%, 95%, or 97.5% identity with Gln26-Lys577 of uniprot P08575 or SEQ ID NO: 12. In some embodiments, the CD45 extracellular domain comprises SEQ ID NO: 2, or a sequence containing at least 70%, 75%, 80%, 85%, 90%, 95%, or 97.5% identity with SEQ ID NO: 2.

[0062] In some embodiments of any of the foregoing or related aspects, the hydrogel beads exhibit at least one optical property substantially similar to the corresponding optical properties of the target cells. In some embodiments, the at least one optical property is lateral scattering. In some embodiments, the at least one optical property is forward scattering. In some embodiments, the at least one optical property comprises both lateral scattering and forward scattering. In some embodiments, the target cells are hematopoietic stem cells.

[0063] In some aspects, this disclosure provides a method for calibrating a cytometer for detecting cells expressing CD34 in a cell population, comprising sampling a composition described herein and calibrating the cytometer based on at least one optical property of the hydrogel beads of the composition. In some embodiments, the method further comprises sampling a cell population and obtaining cells containing at least one optical property. In some embodiments, the cells are cells expressing CD34. Attached Figure Description

[0064] Figures 1A to 1C Bright-field and fluorescence images of the labeled hydrogel beads disclosed herein are provided.

[0065] Figures 2A to 2C The use of the hydrogel beads of this disclosure as calibrators for displaying various optical scattering properties of cell types is shown.

[0066] Figure 3A Flow cytometry gating protocols are provided for generating an exemplary mixture of hydrogel beads described herein, using a control prepared as an enrichment of CD34+ cell populations. The exemplary mixture contains CD45dim-positive (CD45dim+) hydrogel beads in a ratio of approximately 99:1 to CD45dim+ / CD34-positive (CD34+) hydrogel beads (referred to herein as "CD34-low" or "CD34-Lo" cell mimics).

[0067] Figure 3B Flow cytometry gating protocols are provided for generating exemplary mixtures of the hydrogel beads described herein, which are prepared as controls for enriched CD34+ cell populations. The exemplary mixture contains CD45dim+ hydrogel beads in a ratio of approximately 1:9 to CD45dim+ / CD34+ hydrogel beads (referred to herein as "CD34 High" or "CD34 Hi" cell mimics).

[0068] Figure 4ARepresentative analyses of the resulting immobilized leukopak cells were provided on the Cytek Aurora using a gating scheme prepared with CD34 Lo cell mimics. Plots of singlet cells for CD45dim+ population analysis and plots of CD45dim+ cells for percentage analysis of CD34+ cells are shown.

[0069] Figure 4B Representative analyses of stable CD34+ cell lines on Cytek Aurora using a gating scheme prepared with CD34 Hi cell mimics are provided. Plots of singlet cells for CD45dim+ population analysis and plots of CD45dim+ cells for percentage analysis of CD34+ cells are shown. Detailed Implementation

[0070] definition

[0071] The indefinite article “a / an” and the definite article “the” are intended to include both the singular and plural forms unless the context in which they are used explicitly indicates otherwise.

[0072] The terms “at least one” and “one or more” are used interchangeably to indicate that an article may include one or more of the listed elements.

[0073] As used in this article, “substantially similar” means at least 40% similar, at least 50% similar, at least 60% similar, at least 70% similar, at least 80% similar, at least 90% similar, at least 95% similar, at least 96% similar, at least 97% similar, at least 98% similar, or at least 99% similar.

[0074] As used herein, the term "MFI" refers to median fluorescence intensity. The median, as the 50th percentile of a population, indicates that half of the population is above this value while the other half is below it. MFI is separate from and distinct from "gMFI," which refers to geometric mean fluorescence intensity. Both MFI and gMFI measure changes in fluorescence intensity within a cell population.

[0075] Unless otherwise indicated, it should be understood that the quantities, ratios, and numerical characteristics of expressed ingredients, reaction conditions, etc., as used in the specification and claims are all intended to be modified by the term "about" in all cases.

[0076] Throughout this application, the term "about" is used to indicate that a value includes inherent error variations with respect to the apparatus or method used to determine the value, or variations that exist between the samples being measured. Unless otherwise stated or apparent from the context, the term "about" means within 10% above or below the reported value (unless such value would exceed 100% of the possible value or be below 0%). When used in conjunction with a range or series of values, unless otherwise stated, the term "about" applies to the endpoints of the range or each of the values ​​listed in the series. As used herein, the terms "about" and "approximately" are used as equivalents.

[0077] As mentioned in this article, the term "hydrogel" refers to macromolecular materials, whether dehydrated or hydrated.

[0078] When referring to compositions containing hydrogel beads of this disclosure, the term "active portion" refers to a portion of the hydrogel beads containing a spectrum of cell surface biomarkers. In some embodiments, such hydrogel beads can be used as a positive control for the presence of corresponding biomarkers in flow cytometry. In contrast, the inactive portion of the hydrogel beads may include, for example, hydrogel beads formed from polymeric monomers but containing no cell surface biomarkers.

[0079] The term "inclusive of all ranges and subranges" or its equivalents are used herein to mean that the disclosure of any range or series of possible values ​​inherently also discloses all ranges and subranges covered by the highest and lowest disclosed values. This term includes the entire range from the highest to the lowest disclosed value, as well as subranges from any two or more disclosed points. This term is also intended to disclose any subranges covered anywhere within the highest and lowest disclosed values, including between two points explicitly listed in the document, up to a decimal point. Therefore, the disclosure of the values ​​0, 5, 10, 15, 20 (inclusive of all ranges and subranges) should be interpreted as also covering the range 0-20, the range 0-5 or 5-15, and the range 2-16 or 3.1 to 19.8, etc.

[0080] Unless otherwise stated, it should be understood that all figures used in the specification to indicate the quantity, ratio, and numerical properties of components, reaction conditions, etc., are considered to be modifiable in all cases by the term "including all ranges and subranges thereof".

[0081] As used herein, “and / or” means and covers any and all possible combinations of one or more of the relevant listed items.

[0082] Overview

[0083] This disclosure relates to the use of quantitative biomolecular cell mimics to enhance the characterization of CD34-expressing cells in heterogeneous cell populations. These cell mimics serve as reliable quantitative controls for biomolecules with known antigen densities (e.g., CD45 and CD34 molecules). Incorporating cell mimics as reference materials enables standardization, thereby increasing comparability and data consistency between laboratories. These mimics play a crucial role in assessing transfer, thus facilitating reliable data exchange between different research environments. Furthermore, cell mimics with quantitative biomolecules aid in the development and validation of receptor occupancy assessment methods, optimizing staining conditions, antibody concentrations, and assay sensitivity and specificity.

[0084] These cell mimics are designed using hydrogel bead fabrication and biomolecule incorporation techniques. This innovative approach enables the design of cell mimics that can be independently tuned along optical and biochemical parameters, as well as optional physical parameters such as size. By precisely controlling these parameters, this synthetic cell printing technique provides a versatile and customizable tool for immunophenotyping applications.

[0085] These cell mimics are non-biohazardous and storage-stable alternatives to primary cells. They require no maintenance prior to staining, enabling resource-constrained laboratories to obtain reliable results. In summary, the use of quantitative cell mimics enhances stem cell characterization practices by providing quality control, standardization, method validation, and cost-effectiveness. By incorporating such quantitative cell mimics, clinical laboratories can achieve consistent and reliable results while optimizing resource utilization and advancing characterization techniques.

[0086] These cell mimics are suitable for evaluating starting materials (e.g., donor-derived blood samples) used to generate CD34-expressing stem cell populations, as well as for quality control of these populations after downstream processing steps (e.g., before and after enrichment).

[0087] This disclosure can be used on any suitable detection or analysis platform, including but not limited to imaging (e.g., microscopy, scanner, etc.), flow cytometry or other immunoassay methods (e.g., ELISA assays), electrophoresis, omics analysis (genomics, glycomics, proteomics, lipidomics analysis), molecular analysis (q-PCR, etc.). Analysis can be performed in fluorescence, bright-field, dark-field, or immunohistochemistry (e.g., chromogenic staining), such as imaging or detection.

[0088] hydrogel beads

[0089] Hydrogels are materials comprising a three-dimensional network of macromolecules that allows them to swell in the presence of water and shrink without dissolving in water in its absence (or by reducing the amount of water). Swelling (i.e., water absorption) results from the presence of hydrophilic functional groups connected to or dispersed within the macromolecular network. Crosslinking between adjacent macromolecules causes the water insolubility of these hydrogels. Crosslinking can be due to chemical (i.e., covalent) or physical (i.e., van der Waals forces, hydrogen bonds, ionic forces, etc.) bonds. Synthetically prepared hydrogels can be made by polymerizing monomeric materials to form a backbone and then crosslinking that backbone with a crosslinking agent. A characteristic of hydrogels with specific values ​​is that the material retains its general shape, whether dehydrated or hydrated. Therefore, if a hydrogel has a generally spherical shape in its dehydrated state, it will be spherical in its hydrated state.

[0090] The hydrogels in bead form provided herein are synthesized by polymerizing one or more monomers provided herein. Synthesis is carried out to form individual hydrogel beads. In one embodiment, monomeric materials (monomers) are polymerized to form homopolymers. However, in some embodiments, copolymers of different monomeric units (i.e., comonomers) are synthesized and used in the methods provided herein. In some embodiments, the monomers or comonomers used in the methods and compositions described herein are bifunctional monomers or include bifunctional monomers (wherein the comonomer is employed). In some embodiments, the hydrogels are synthesized in the presence of a crosslinking agent. In other embodiments, the hydrogels are synthesized in the presence of a polymerization initiator.

[0091] The amount of monomer can be varied by the user of this disclosure, for example, to obtain specific properties substantially similar to those of the target cells. In one embodiment, the monomer component (i.e., monomers, comonomers, bifunctional monomers, or combinations thereof, such as bis / acrylamides, allylamines, or other comonomers providing chemical functional groups for secondary labeling / conjugation, or alginates) is present in the hydrogel at about 10% to about 95% by weight. In another embodiment, the monomer component is present in the hydrogel at about 15% to about 90% by weight, or about 20% to about 90% by weight.

[0092] Examples of various monomers and crosslinking chemicals used in conjunction with this disclosure are provided in the Thermo Scientific Crosslinking Technical Handbook entitled “Easymolecular bonding crosslinking technology” (available at tools.lifetechnologies.com / content / sfs / brochures / 1602163-Crosslinking-Reagents-Handbook.pdf, the disclosure of which is incorporated herein by reference in its entirety for all purposes). For example, hydrazine (e.g., with NHS ester compounds) or EDC coupling reactions (e.g., with maleimide compounds) can be used to construct the hydrogels of this disclosure.

[0093] In some embodiments, the monomers used with the hydrogels provided herein are lactic acid, glycolic acid, acrylic acid, 1-hydroxyethyl methacrylate, ethyl methacrylate, 2-hydroxyethyl methacrylate (HEMA), propylene glycol methacrylate, acrylamide, N-vinylpyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glyceryl methacrylate (GMA), ethylene glycol methacrylate, ethylene glycol, fumaric acid, their derivatives, or combinations thereof.

[0094] In some embodiments, one or more of the following monomers are used herein to form the hydrogel of this disclosure: 2-hydroxyethyl methacrylate, hydroxyethoxyethyl methacrylate, hydroxydiethoxyethyl methacrylate, methoxyethyl methacrylate, methoxyethoxyethyl methacrylate, poly(ethylene glycol) methacrylate, methoxy-poly(ethylene glycol) methacrylate, methacrylic acid, sodium methacrylate, glyceryl methacrylate, hydroxypropyl methacrylate, hydroxybutyl methacrylate, or combinations thereof.

[0095] In other embodiments, one or more of the following monomers are used herein to form tunable hydrogels: phenyl acrylate, phenyl methacrylate, benzyl acrylate, benzyl methacrylate, 2-phenylethyl acrylate, 2-phenylethyl methacrylate, 2-phenoxyethyl acrylate, 2-phenoxyethyl methacrylate, phenyl thioethyl acrylate, phenyl thioethyl methacrylate, 2,4,6-tribromophenyl acrylate, 2,4,6-tribromophenyl methacrylate, pentabromophenyl acrylate, pentabromophenyl methacrylate, pentachlorophenyl acrylate, methacrylate Pentachlorophenyl acrylate, 2,3-dibromopropyl acrylate, 2,3-dibromopropyl methacrylate, 2-naphthyl acrylate, 2-naphthyl methacrylate, 4-methoxybenzyl acrylate, 4-methoxybenzyl methacrylate, 2-benzyloxyethyl acrylate, 2-benzyloxyethyl methacrylate, 4-chlorophenoxyethyl acrylate, 4-chlorophenoxyethyl methacrylate, 2-phenoxyethoxyethyl acrylate, 2-phenoxyethoxyethyl methacrylate, N-phenylacrylamide, N-phenylmethylacrylamide, N-benzylacrylamide Amine, N-benzylmethacrylamide, N,N-dibenzylmethacrylamide, N,N-dibenzylmethacrylamide, N-diphenylmethacrylamide, N-(4-methylphenyl)methacrylamide, N-1-naphthylacrylamide, N-4-nitrophenylacrylamide, N-(2-phenylethyl)acrylamide, N-triphenylmethylacrylamide, N-(4-hydroxyphenyl)acrylamide, N,N-methylphenylacrylamide, N,N-phenylphenylethylacrylamide, N-diphenylmethylmethacrylamide, N-(4-methylphenyl)acrylamide N-(2-phenylethyl)methacrylamide, N-1-naphthylmethacrylamide, N-4-nitrophenylmethacrylamide, N-(2-phenylethyl)methacrylamide, N-triphenylmethylmethacrylamide, N-(4-hydroxyphenyl)methacrylamide, N,N-methylphenylmethacrylamide, N,N′-phenylphenylethylmethacrylamide, N,N-vinylcarbazole, 4-vinylpyridine, and 2-vinylpyridine, as described in U.S. Patent No. 6,657,030, are incorporated herein by reference in their entirety for all purposes.

[0096] Both synthetic and biological monomers can be used in the hydrogels described herein to form synthetic, biohydrogels, or hybrid hydrogels comprising synthetic and biological components (e.g., peptides, proteins, monosaccharides, disaccharides, polysaccharides, primary amine thiol groups, carbonyl groups, carbohydrates, and carboxylic acids present on biomolecules). For example, proteins, peptides, or carbohydrates can be used as individual monomers to form hydrogels that may or may not include synthetic monomers (or polymers) and are combined with chemically compatible comonomers and crosslinking chemicals (see, for example, Thermo Scientific Crosslinking Technical Handbook entitled “Easy molecular bonding → crosslinking → technology,” available at tools.lifetechnologies.com / content / sfs / brochures / 1602163-Crosslinking-Reagents-Handbook.pdf, the entire contents of which are incorporated herein by reference for all purposes). Compatible crosslinking chemicals include, but are not limited to, amines, carboxyl groups, and other reactive chemical side groups. Representative reactive groups suitable for the hydrogels and monomers described herein are provided in Table 1 below.

[0097] Table 1. Reactive groups of cross-linking agents suitable for biomonomer conjugation

[0098] Reactivity Category Target functional groups reactive chemical groups Amine reactivity -NH2 NHS ester imine ester pentafluorophenyl ester hydroxymethylphosphine Carboxyl-amine reactivity -COOH Carbodiimide (e.g., EDC) Thiol reactivity -SH Maleimide haloacetyl (bromo- or iodo-)pyridyl sulfonate thiosulfonate vinyl sulfonate Aldehyde reactivity (oxidation of sugars, carbonyl groups) -CHO hydrazine alkoxyamine Photoreactivity, i.e., non-selective, random insertion. random Bispropidine aryl azide Hydroxyl (non-aqueous) reactivity -OH Isocyanates azide reactivity -N3 phosphine

[0099] Generally, polymers can be formed using any form of polymerization chemistry / method commonly known to those skilled in the art. Polymerization can be catalyzed by UV-induced group formation and reaction progression. The hydrogel beads of this disclosure can be produced by polymerization of acrylamide or acrylate. For example, in one embodiment, the acrylamide is a polymerizable carbohydrate-derived acrylamide as described in U.S. Patent No. 6,107,365, the disclosure of which is incorporated herein by reference in its entirety for all purposes. As described herein and known to those skilled in the art, the specific linkage of acrylamide groups to sugars readily adapts to a range of monosaccharides and higher polysaccharides, such as synthetic polysaccharides or polysaccharides derived from natural sources, such as glycoproteins found in serum or tissues.

[0100] In some embodiments, acrylate-functionalized polyethylene glycol monomers are used as hydrogel monomers. For example, in one embodiment, the PEG is an acrylate or acrylamide-functionalized PEG.

[0101] In some embodiments, the hydrogel beads comprise a monofunctional monomer polymerized with at least one bifunctional monomer. One example includes, but is not limited to, the formation of a polyacrylamide polymer using acrylamide and bisacrylamide (bifunctional monomers). In another embodiment, the hydrogel beads provided herein comprise a bifunctional monomer polymerized with a second bifunctional monomer. One example includes, but is not limited to, the formation of polymers having a mixed composition comprising compatible chemicals such as acrylamide, bisacrylamide, and bisacrylamide structural homologues comprising a wide range of additional chemicals. The range of chemically compatible monomers, bifunctional monomers, and mixed compositions will be apparent to those skilled in the art and follows principles of chemical reactivity known to those skilled in the art. (Refer to Thermo Handbook and Acrylamide Polymerization Handbook). See, for example, the Thermo Scientific Crosslinking Technical Handbook entitled “Easy molecular bonding crosslinking technology” (available at tools.lifetechnologies.com / content / sfs / brochures / 1602163-Crosslinking-Reagents-Handbook.pdf) and the Polyacrylamide Emulsions Handbook (SNF Floerger, available at snf.com.au / downloads / Emulsion_Handbook_E.pdf), the contents of which are incorporated herein by reference in their entirety for all purposes.

[0102] In some embodiments, the hydrogel beads provided herein comprise a polymerizable monofunctional monomer and are monofunctional acrylic monomers. Non-limiting examples of monofunctional acrylic monomers used herein are acrylamide; methacrylamide; N-alkylacrylamides, such as N-ethylacrylamide, N-isopropylacrylamide, or N-tert-butylacrylamide; N-alkylmethylacrylamides, such as N-ethylmethylacrylamide or N-isopropylmethylacrylamide; N,N-dialkylacrylamides, such as N,N-dimethylacrylamide and N,N-diethylacrylamide; N-[(dialkylamino)alkyl]acrylamides, such as N-[3-dimethylamino)propyl]acrylamide. Or N-[3-(diethylamino)propyl]acrylamide; N-[(dialkylamino)alkyl]methacrylamide, such as N-[3-dimethylamino)propyl]methacrylamide or N-[3-(diethylamino)propyl]methacrylamide; alkyl acrylates, such as 2-(dimethylamino)ethyl acrylate, 2-(dimethylamino)propyl acrylate or 2-(diethylamino)ethyl acrylate; and alkyl methacrylates, such as 2-(dimethylamino)ethyl methacrylate.

[0103] A bifunctional monomer is any monomer that can be polymerized with a monofunctional monomer of the present disclosure to form a hydrogel as described herein, the monomer containing a second functional group that can participate in a second reaction (e.g., conjugation of a fluorophore or a cell surface receptor (or its domain)).

[0104] In some embodiments, the bifunctional monomer is selected from the group consisting of allylamine, allyl alcohol, allyl isothiocyanate, allyl chloride, and allyl maleimide.

[0105] The bifunctional monomer can be a bifunctional acrylic monomer. Non-limiting examples of bifunctional acrylic monomers are N,N'-methylenebisacrylamide, N,N'-methylenebismethylacrylamide, N,N'-ethylenebismethylacrylamide, N,N'-propylenebisacrylamide, and N,N'-(1,2-dihydroxyethylene)bisacrylamide.

[0106] Higher-order branched and linear comonomers can be substituted in polymer blends to adjust the refractive index while maintaining polymer density, as described in U.S. Patent No. 6,657,030, which is incorporated herein by reference in its entirety for all purposes.

[0107] In some implementations, the hydrogel contains molecules that modulate the properties of the hydrogel. These molecules, capable of altering the properties of the hydrogel, are discussed further below.

[0108] In some embodiments, individual hydrogel beads or groups thereof comprise biodegradable polymeric monomers. In one embodiment, the biodegradable polymeric monomer is a poly(ester) based on polylactide (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), poly(lactic acid-copoly-glycolic acid) (PLGA), and copolymers thereof. In some embodiments, the biodegradable polymeric monomer is a carbohydrate or protein, or a combination thereof. For example, in one embodiment, monosaccharides, disaccharides, or polysaccharides (e.g., glucose, sucrose, or maltodextrin), peptides, proteins (or their domains) are used as hydrogel monomers. Other biodegradable polymers include poly(hydroxyalkanoates) of the PHB-PHV class, additional poly(esters), and natural polymers such as modified poly(saccharides), such as starch, cellulose, and chitosan. In other embodiments, the biocompatible polymer is an adhesion protein, cellulose, carbohydrate, starch (e.g., maltodextrin, 2-hydroxyethyl starch, alginate), dextran, lignin, polyamino acids, amino acids, or chitin. Such biodegradable polymers are commercially available, for example, from Sigma Aldrich (St. Louis, MO).

[0109] In some embodiments, the biodegradable monomers are selected from the group consisting of: agar, agarose, alginic acid, alginate, α-glucan, amylopectin, amylose, arabinoxylan, β-glucan, callose, capsullan, carrageenan, cellulose, animal cellulose, cellulose, chitin, chitosan, leucobacterium, gelatin, cyclodextrin, α-cyclodextrin, dextrin, sucrose, fructan, fucoidan, galactomannan, galactomannan, galactosamine galactomannan, gellan gum, dextran, glucomannan, glucuronic acid xylan, glycocalyx, glycogen, hemicellulose, and cyclodextrin. Sugars, hydroxypropyl methylcellulose, icodextrin, inulin, kefiran, kelp polysaccharides, lentinan, polysaccharides, polysaccharides, lichen polysaccharides, mannan, mixed-linked glucans, euglena starch, pectic acid, pectin, pentameric starch, plant glycogen, pleuran, polydextrose, polysaccharide peptides, laver polysaccharides, pullulan, schizofiran, sinistrin, sizofiran, vegan gum, xanthan gum, xylan, xyloglucan, yeast polysaccharides, and combinations thereof.

[0110] In some embodiments, the hydrogel beads of this group comprise agar, agarose, alginic acid, alginate, α-glucan, amylopectin, amylose, arabinoxylan, β-glucan, callose, capsullan, carrageenan, cellulose dextrin, animal cellulose, cellulose, chitin, chitosan, leucobacterium, gelatin, cyclodextrin, α-cyclodextrin, dextrin, sucrose, fructan, fucoidan, galactomannan, galactomannan, galactosamine galactomannan, gellan gum, dextran, glucomannan, glucuronic acid xylan, glycocalyx, glycogen, hemicellulose, and other similar components. Polysaccharides, hydroxypropyl methylcellulose, icodextrin, inulin, kefiran, kelp polysaccharides, lentinan, polysaccharides, lichen polysaccharides, mannan, mixed-linked glucans, euglena starch, pectic acid, pectin, pentameric starch, plant glycogen, pleuran, polydextrose, polysaccharide peptides, laver polysaccharides, pullulan, schizofiran, sinistrin, sizofiran, vegan gum, xanthan gum, xylan, xyglucan, yeast polysaccharides, or combinations thereof.

[0111] In some embodiments, the protein contains only natural amino acids. However, this disclosure is not limited thereto. For example, self-assembled artificial proteins and proteins having non-natural amino acids (e.g., proteins incorporated into non-ribosomal peptides or synthetically introduced by synthetic methods, see, for example, Zhang et al. (2013). Current Opinion in Structural Biology 23, pp. 581-587, the disclosure of which is incorporated herein by reference in its entirety for all purposes) or their protein domains can also be used as hydrogel monomers. The range of non-natural / unnatural amino acids that can be incorporated into such compositions is well known to those skilled in the art (Zhang et al. (2013). Current Opinion in Structural Biology 23, pp. 581-587; incorporated herein by reference in its entirety for all purposes). In one embodiment, a biodegradable polymer is used as a comonomer, i.e., in a mixture of monomers. In one embodiment, the biodegradable polymer is a bifunctional monomer.

[0112] In some embodiments, the biomonomer is functionalized with acrylamide or acrylate. For example, in one embodiment, the polymerizable acrylamide-functionalized biomolecule is an acrylamide or acrylate-functionalized protein (e.g., acrylamide-functionalized collagen or functionalized collagen domain), an acrylamide or acrylate-functionalized peptide, or an acrylamide or acrylate-functionalized monosaccharide, disaccharide, or polysaccharide.

[0113] Any monosaccharide, disaccharide, or polysaccharide (functionalized or otherwise) can be used as a hydrogel monomer. In some embodiments, acrylamide or acrylate-functionalized monosaccharides, disaccharides, or polysaccharides are used as polymerizable hydrogel monomers. In some embodiments, structural polysaccharides are used as polymerizable hydrogel monomers. In other embodiments, the structural polysaccharide is arabinoylxylan, cellulose, chitin, or pectin. In other embodiments, alginate is used as a polymerizable hydrogel monomer. In other embodiments, glycosaminoglycans (GAGs) are used as polymerizable monomers in the hydrogels provided herein. In other embodiments, GAGs are chondroitin sulfate, dermatan sulfate, keratin sulfate, heparin, heparin sulfate, or hyaluronic acid (also known in the art as hyaluronan or hyaluronic acid salts) used as polymerizable hydrogel monomers. Further scope of compatible biomonomers and their reactive chemicals is known to those skilled in the art and follows general principles of chemical reactivity.

[0114] Another range of biocompatible monomers that can be incorporated is known in the art, see, for example, the non-degradable biocompatible monomers disclosed in the following literature: Shastri (2003). Current Pharmaceutical Biotechnology 4, pp. 331-337, which is incorporated herein by reference in its entirety for all purposes. Other monomers are provided in the following references: de Moraes Porto (2012). Polymer Biocompatibility, Polymerization, Dr. Ailton De Souza Gomes (ed.), ISBN: 978-953-51-0745-3; InTech, DOI:10.5772 / 47786; Heller et al. (2010). Journal of Polymer Science Part A: PolymerChemistry 49, pp. 650-661; Final Report for Biocompatible Materials (2004), The Board of the Biocompatible Materials and the Molecular Engineering in Polymer Science programmes, ISBN 91-631-4985-0. The contents of each of these references are hereby incorporated in their entirety by reference.

[0115] In one embodiment, the biocompatible monomers used with the hydrogel described herein include ethylene glycol dimethacrylate (EGDMA), 2-hydroxyethyl methacrylate (HEMA), methyl methacrylate (MMA), methacryloxymethyltrimethylsilane (TMS-MA), N-vinyl-2-pyrrolidone (N-VP), styrene, or combinations thereof.

[0116] Naturally occurring hydrogels that can be used in this disclosure include a variety of polysaccharides available from natural sources, such as plants, algae, fungi, yeasts, marine invertebrates, and arthropods. Non-limiting examples include agarose, dextran, chitin, cellulose-based compounds, starch, derived starches, etc. These typically have repeating glucose units as the main component of the polysaccharide backbone. Crosslinking chemicals for such polysaccharides are known in the art, see, for example, the Thermo Scientific Crosslinking Technical Handbook entitled “Easymolecular bonding crosslinking technology” (available at tools.lifetechnologies.com / content / sfs / brochures / 1602163-Crosslinking-Reagents-Handbook.pdf).

[0117] In one embodiment, hyaluronic acid is used as a hydrogel monomer (as a single monomer or as a comonomer). In one embodiment, hyaluronic acid is functionalized, for example, with acrylate or acrylamide. Hyaluronic acid is a high molecular weight GAG composed of disaccharide repeating units of N-acetylglucosamine and glucuronic acid linked together by alternating β-1,4 and β-1,3 glycosidic bonds. In the human body, hyaluronic acid is present in several soft connective tissues, including skin, umbilical cord, synovial fluid, and vitreous humor. Therefore, in one embodiment, where it is desired to mimic one or more properties of target cells, hyaluronic acid is used as a hydrogel monomer. Methods for manufacturing hydrogel beads are described in Xu et al. (2012). Soft Matter. 8, pp. 3280-3294, the disclosure of which is incorporated herein by reference in its entirety for all purposes. As described herein, hyaluronic acid can be derived using a variety of reactive handles, depending on the desired crosslinking chemistry and other monomers used to form hydrogel beads.

[0118] In yet another embodiment, a linear polysaccharide chitosan composed of randomly distributed β-(1-4)-linked D-glucosamine (deacetylation unit) and N-acetyl-D-glucosamine (acetylation unit) is used as a hydrogel monomer (as a single monomer or as a comonomer).

[0119] In some embodiments, individual hydrogel beads or multiple hydrogel beads comprise peptides, proteins, protein domains, or combinations thereof as hydrogel monomers or multiple hydrogel monomers. In other embodiments, the protein is a structural protein or its domain, such as filament, elastin, titanin, or collagen or its domains. In some embodiments, the protein is an extracellular matrix (ECM) component (e.g., collagen, elastin, proteoglycans, fibronectin, lysine, fibronectin). In other embodiments, the structural protein is collagen. In other embodiments, the collagen is type I collagen, type II collagen, or type III collagen or a combination thereof. In other embodiments, the hydrogel monomer comprises proteoglycans. In other embodiments, the proteoglycan is a core proteoglycan, a disaccharide, a testosterone, a bikunizidine, fibrinolytic proteoglycan, a photoproteoglycan, or its domains.

[0120] In other embodiments, acrylate-functionalized structural protein hydrogel monomers are used as components of the hydrogels provided herein (e.g., acrylate-functionalized proteins or protein domains, such as filaments, elastin, titin, collagen, proteoglycans, or their functionalized domains). In further embodiments, the acrylate-functionalized structural protein hydrogel monomers comprise proteoglycans, such as core proteoglycans, disaccharides, testosterone, bisKunitz inhibitors, fibrinolytic proteoglycans, photoproteoglycans, or their domains).

[0121] In some embodiments, the PEG monomer and oligopeptide may be mimicking extracellular matrix proteins, such as those used in the hydrogels provided herein with vinyl sulfone-functionalized multi-arm PEG, integrin-binding peptides, and biscysteine ​​matrix metalloproteinase peptides, as described by Lutolf et al. (2003). Proc. Natl. Acad. Sci. USA 100, 5413-5418, which is incorporated herein by reference in its entirety for all purposes. In this particular embodiment, the hydrogel is formed via a Michael-type addition reaction on PEG between a dithiolized oligopeptide and a vinyl sulfone group. A range of other compatible chemicals that may be incorporated herein will be apparent to those skilled in the art and follow general principles of chemical reactivity, see, for example, the Thermo Scientific Crosslinking Technical Handbook entitled “Easy molecular bonding crosslinking technology” (available at tools.lifetechnologies.com / content / sfs / brochures / 1602163-Crosslinking-Reagents-Handbook.pdf).

[0122] Other bioactive domains in natural proteins can also be used as hydrogel monomers or portions thereof. For example, cell adhesion integrin-binding domains, controlled-release affinity-binding domains, or transglutaminase crosslinking domains can be used in the hydrogels presented herein. Details on the production of such hydrogels can be found in the following references: Martino et al. (2009). Biomaterials 30, 1089; Martino et al. (2011). Sci. Trans. Med. 3, 100ra89; Hu and Messersmith (2003). J. Am. Chem. Soc. 125, 14298, each of which is incorporated in its entirety by reference for all purposes.

[0123] In some implementations, recombinant DNA methods are used to produce proteins that are designed to gel in response to changes in pH or temperature, for example, by the method described by Petk et al. (1998). Science 281, pp. 389-392, which is incorporated herein by reference in its entirety for all purposes. In short, the protein consists of terminal leucine zipper domains flanked by water-soluble polyelectrolyte segments. In near-neutral aqueous solutions, the coiled-helical aggregates of the terminal domains form a three-dimensional hydrogel polymer network.

[0124] Common crosslinking agents that can be used to crosslink the hydrogels presented herein include, but are not limited to, ethylene glycol dimethacrylate (EGDMA), tetraethylene glycol dimethacrylate, and N,N'-15-methylenebisacrylamide. A range of other crosslinking chemicals that can be used will be apparent to those skilled in the art and follow general principles of chemical reactivity, see, for example, the Thermo Scientific Crosslinking Technical Handbook entitled "Easy molecular bonding crosslinking technology" (available at tools.lifetechnologies.com / content / sfs / brochures / 1602163-Crosslinking-Reagents-Handbook.pdf).

[0125] In some embodiments, the polymerization of the hydrogel is initiated by an equivalent initiator formed from a persulfate or a catalytic group. The range of compatible initiators is known to those skilled in the art and follows general principles of chemical reactivity; see, for example, the Thermo Scientific Crosslinking Technical Handbook entitled “Easy molecular bonding crosslinking technology” (available at tools.lifetechnologies.com / content / sfs / brochures / 1602163-Crosslinking-Reagents-Handbook.pdf). The persulfate can be any water-soluble persulfate. Non-limiting examples of water-soluble persulfates are ammonium persulfate and alkali metal persulfates. Alkali metals include lithium, sodium, and potassium. In some embodiments, the persulfate is ammonium persulfate or potassium persulfate. In other embodiments, the polymerization of the hydrogel provided herein is initiated by ammonium persulfate.

[0126] The polymerization of hydrogels can be accelerated by an accelerator that catalyzes the formation of polymerization-unstable chemical side groups. A range of possible accelerators is known to those skilled in the art and follows general principles of chemical reactivity, see, for example, the Thermo Scientific Crosslinking Technical Handbook entitled “Easy molecular bonding crosslinking technology” (available at tools.lifetechnologies.com / content / sfs / brochures / 1602163-Crosslinking-Reagents-Handbook.pdf). In one embodiment, the accelerator is a tertiary amine. The tertiary amine can be any water-soluble tertiary amine. In one embodiment, the accelerator is used in the polymerization reaction and is N,N,N',N'-tetramethylethylenediamine, 3-dimethylamino)propionitrile, or N,N,N',N'-tetramethylethylenediamine (TEMED). In another embodiment, the accelerator is used in the polymerization reaction and is azobis(isobutyronitrile) (AIBN).

[0127] As discussed above, the hydrogels used in the compositions and methods described herein may comprise any of the monomer units and crosslinking agents as described herein, and in one aspect, are generated as hydrogel beads by polymerizing droplets. Microfluidic methods for generating multiple droplets comprising fluid droplets and rigid droplets are known to those skilled in the art and are described in U.S. Patent Publication No. 2011 / 0218123 and U.S. Patent No. 7,294,503, each of which is incorporated herein by reference in its entirety for all purposes. Such methods provide multiple droplets comprising a first fluid and substantially surrounded by a second fluid, wherein the first and second fluids are substantially immiscible (e.g., droplets comprising an aqueous liquid substantially surrounded by an oil-based liquid).

[0128] Multiple fluid droplets (e.g., prepared using a microfluidic device) may be polydisperse (e.g., having a range of different sizes), or in some cases, the fluid droplets may be monodisperse or substantially monodisperse, for example, having a uniform diameter distribution, such that no more than about 10%, about 5%, about 3%, about 1%, about 0.03%, or about 0.01% of the droplets have an average diameter greater than about 10%, about 5%, about 3%, about 1%, about 0.03%, or about 0.01% of the average diameter. As used herein, the average diameter of a droplet population refers to the arithmetic mean of the diameters of the droplets. The average diameter of the beads can be measured, for example, by light scattering techniques. In one embodiment, the average diameter of the hydrogel beads is customized, for example, by varying the flow rates of the first and second fluids within the channels of the microfluidic device or by varying the volume of the channels of the microfluidic device.

[0129] In some embodiments, the average diameter of the hydrogel beads is about 0.1 μm to about 100 μm, about 0.5 μm to about 60 μm, or about 1 μm to about 40 μm. In other embodiments, the average diameter of the hydrogel beads is about 1-2 μm, about 2-3 μm, about 3-5 μm, about 5-8 μm, about 8-12 μm, about 12-20 μm, or about 20-40 μm. In other embodiments, the average diameter of the hydrogel beads is about 1-4 μm, about 2-8 μm, about 3-12 μm, about 4-16 μm, about 5-20 μm, or about 10-40 μm, including all ranges and subranges therein. In some embodiments, the average diameter of the hydrogel beads is approximately the same as the diameter of CD34+ human stem cells. In some embodiments, the average diameter of the hydrogel beads is approximately the same as the diameter of human cells with reduced CD34+ and CD45.

[0130] In some embodiments, the hydrogel particles are carried by suspension polymerization, also known in the art as bead polymerization, granular polymerization, or particulate polymerization (see Elbert (2011). Acta Biomater. 7, pp. 31-56, which is incorporated herein by reference in its entirety for all purposes). In suspension polymerization, the monomer is insoluble in a continuous phase, such as an aqueous monomer solution in a continuous oil phase. In suspension polymerization, polymerization initiation occurs within monomer-rich droplets, and each droplet has more than one group at any given time. In one embodiment, the monomer phase comprises monomers, which may be bifunctional monomers or multiple monomer species (comonomers, which may be multiple bifunctional monomers). In one embodiment, the monomer phase comprises an initiator and / or a crosslinking agent.

[0131] Emulsion polymerization can also be used to form the hydrogel particles described herein. In emulsion polymerization, the monomers have poor solubility in the continuous phase, similar to suspension polymerization; however, polymerization initiation occurs outside the monomer droplets (see Elbert (2011). Acta Biomater. 7, pp. 31–56, which is incorporated herein by reference in its entirety for all purposes). In emulsion polymerization embodiments, if a surfactant is present, the initiator causes chain growth of the monomers (or comonomers) dissolved in the continuous phase or the monomers contained in the micelles.

[0132] In other embodiments, hydrogel particles are formed via precipitation polymerization, as described, for example, in Elbert (2011). Acta Biomater. 7, pp. 31-56, which is incorporated herein by reference in its entirety for all purposes. Precipitation polymerization is a technique that utilizes the difference in solubility between monomers and polymers to produce microparticles. Specifically, it is known that the solubility of larger polymer chains is generally lower than that of smaller polymer chains. Therefore, above a certain molecular weight, phase separation can be facilitated. Precipitation polymerization initially begins with solution polymerization in a single-phase homogeneous system. Shortly after the start of polymerization, in one embodiment, a relatively high concentration of polymer chains is present, which facilitates phase separation by nucleation. As polymerization proceeds, the concentration of polymer chains decreases, and existing particles capture chains before the nucleation of new particles can occur. Therefore, particle nucleation occurs only shortly after the start of the reaction and lasts for a short period of time, which in one embodiment results in a narrow particle size distribution. Other methods include, but are not limited to, lithographic grain formation (Helgeson et al. (2011). Curr. Opin. Colloid. Interface Sci. 16, pp. 106-117, incorporated herein by reference in its entirety for all purposes), membrane emulsification (e.g., microsieve emulsification techniques described by Nanomi BV (Netherlands)), microchannel emulsification (Sugiura et al. (2002). Languimir 18, pp. 5708-5712, incorporated herein by reference in its entirety), and bulk emulsification (SNFFloerger, available at snf.com.au / downloads / Emulsion_Handbook_E.pdf, incorporated herein by reference in its entirety).

[0133] In some embodiments, hydrogel particles are formed within a microfluidic device having two oil channels focused on a central flow of an aqueous monomer solution. In this embodiment, droplets form at the interface between the two channels and the central flow to disrupt droplets in the water-in-oil emulsion. Once droplets form, in one embodiment, the droplets are stabilized prior to polymerization, for example, by adding a surfactant to the oil phase. However, in another embodiment, the droplets are unstable prior to polymerization. In one embodiment, polymerization of the monomer is triggered by adding a promoter (e.g., N,N,N',N' tetramethylethylenediamine) to one or both oil channels after the initial droplet formation.

[0134] Hydrogel beads as cell mimics

[0135] The three main deconvolution modes of flow cytometry are the two passive optical properties of the particles (forward scattering FSC, corresponding to the refractive index (or RI), and side scattering SSC) and the biomarkers present on the surface of a given cell type. Therefore, compositions that allow the hydrogel particles of this disclosure to mimic specific cell types (e.g., cells expressing CD34) in these three modes can be used to provide synthetic, robust calibrators for flow cytometry.

[0136] In one embodiment, the disclosed hydrogel particles have a refractive index (RI) greater than about 1.10, greater than about 1.15, greater than about 1.20, greater than about 1.25, greater than about 1.30, greater than about 1.35, greater than about 1.40, greater than about 1.45, greater than about 1.50, greater than about 1.55, greater than about 1.60, greater than about 1.65, greater than about 1.70, greater than about 1.75, greater than about 1.80, greater than about 1.85, greater than about 1.90, greater than about 1.95, greater than about 2.00, greater than about 2.10, greater than about 2.20, greater than about 2.30, greater than about 2.40, greater than about 2.50, greater than about 2.60, greater than about 2.70, greater than about 2.80, or greater than about 2.90.

[0137] In another embodiment, the refractive index (RI) of the disclosed hydrogel particles is about 1.10 to about 3.0, or about 1.15 to about 3.0, or about 1.20 to about 3.0, or about 1.25 to about 3.0, or about 1.30 to about 3.0, or about 1.35 to about 3.0, or about 1.4 to about 3.0, or about 1.45 to about 3.0, or about 1.50 to about 3.0, or about 1.6 to about 3.0, or about 1.7 to about 3.0, or about 1.8 to about 3.0, or about 1.9 to about 3.0, or about 2.0 to about 3.0.

[0138] In some embodiments, the disclosed hydrogel particles have a refractive index (RI) less than about 1.10, less than about 1.15, less than about 1.20, less than about 1.25, less than about 1.30, less than about 1.35, less than about 1.40, less than about 1.45, less than about 1.50, less than about 1.55, less than about 1.60, less than about 1.65, less than about 1.70, less than about 1.75, less than about 1.80, less than about 1.85, less than about 1.90, less than about 1.95, less than about 2.00, less than about 2.10, less than about 2.20, less than about 2.30, less than about 2.40, less than about 2.50, less than about 2.60, less than about 2.70, less than about 2.80, or less than about 2.90. The preceding paragraphs provide various minimum and maximum values ​​for the refractive index. This part of the disclosure is intended to be composable, such that the disclosure provides a range having both maximum and minimum values. For example, in some embodiments, the disclosed hydrogel particles exhibit a refractive index of about 1.10 to about 3.0.

[0139] The SSC of the disclosed hydrogel particles compared to the SSC of target cells (e.g., cells expressing CD34) is the most meaningful measurement. In some embodiments, the SSC of the disclosed hydrogel particles is within 30%, 25%, 20%, 15%, 10%, 5%, or 1% of the SSC of target cells (e.g., cells expressing CD34), as measured by a cytometry device.

[0140] In one embodiment, the SSC of the hydrogel particles is adjusted by incorporating high-refractive-index molecules (or more) into the hydrogel. In one embodiment, the high-refractive-index molecules are provided in the hydrogel particles, and in another embodiment, the high-refractive-index molecules are colloidal silica, alkyl acrylates, alkyl methacrylates, or combinations thereof. Thus, in some embodiments, the hydrogel particles of this disclosure comprise alkyl acrylates and / or alkyl methacrylates. In one embodiment, the concentration of the monomer is adjusted to further adjust the refractive index of the hydrogel particles.

[0141] Alkyl acrylates or alkyl methacrylates may contain 1 to 18, 1 to 8, or 2 to 8 carbon atoms in the alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, 2-ethylhexyl, heptyl or octyl. The alkyl group may be branched or straight-chain.

[0142] High refractive index molecules may also include vinyl aromatics, such as styrene and methylstyrene, which may optionally be substituted with alkyl groups, such as methyl, ethyl or tert-butyl, or with halogens, such as chlorostyrene.

[0143] In some embodiments, the forward scattering concentration (FSC) is adjusted by regulating the percentage of monomers present in the composition, thereby altering the water content present during hydrogel particle formation. In one embodiment, when using both monomers and comonomers, the ratio of monomers to comonomers is adjusted to modify the forward scattering properties of the hydrogel particles.

[0144] The FSC of the disclosed hydrogel particles is most meaningful when compared to the FSC of target cells (e.g., cells expressing CD34). In some embodiments, the FSC of the disclosed hydrogel particles is within 30%, 25%, 20%, 15%, 10%, 5%, or 1% of the FSC of target cells (e.g., cells expressing CD34), as measured by a cytometry device.

[0145] Forward scattering (FSC) is related to particle volume and can therefore be tuned by changing the particle diameter, as described herein. It has generally been observed that larger objects refract more light than smaller objects, resulting in a higher forward scattering signal (and vice versa). Therefore, in one embodiment, the particle diameter is varied to tune the FSC properties of the hydrogel particles. For example, in one embodiment, the hydrogel particle diameter is varied by utilizing larger microfluidic channels during particle formation.

[0146] SSCs can be engineered by encapsulating nanoparticles within a hydrogel to mimic organelles in target cells (e.g., cells expressing CD34). In some embodiments, the hydrogel particles of this disclosure comprise one or more types of nanoparticles selected from the group consisting of polymethyl methacrylate (PMMA) nanoparticles, polystyrene (PS) nanoparticles, and silica nanoparticles. Not wishing to be bound by theory, the ability to selectively tune both the forward and side scattering of the hydrogel particles, as described herein, allows for a robust platform to mimic a wide range of cell types (e.g., cells expressing CD34).

[0147] Although this invention is primarily described in relation to modifications of optical properties, it is not limited thereto. For example, hydrogel particles can be prepared and modulated to adjust the capacitance of the particles, for example, to calibrate a coulter counter. In one embodiment, the capacitance of the hydrogel particles is adjusted by changing the amount of hydrogel monomers in the composition. For example, the concentrations of polyaniline, polyacetylene, polyphenylene oxide, polypyrrole (X = NH) and polythiophene (X = S) comonomers, as well as polyaniline (X = NH / N) and polyphenylene sulfide (X = S) comonomers, can all be adjusted to change the capacitance. In one embodiment, increasing the concentration of one or more of these monomers increases the capacitance of the hydrogel particles.

[0148] In some embodiments, the hydrogel particles of this disclosure have material modulus properties (e.g., elasticity) more similar to those of target cells (e.g., cells expressing CD34) compared to polystyrene beads of the same diameter.

[0149] In some embodiments, the hydrogel beads of this disclosure contain artificial light scattering properties substantially similar to the corresponding light scattering properties of target cells (e.g., cells expressing CD34), said artificial light scattering properties being provided by: comonomers, chemical side groups, encapsulating materials, colloidal silica, or a ratio of acrylamide to bisacrylamide.

[0150] In some embodiments, the hydrogel beads of this disclosure contain scattering modifiers. In some embodiments, the light scattering property substantially similar to the corresponding light scattering property of the target cells (e.g., cells expressing CD34) is side scattering (SSC). In some embodiments, the light scattering property substantially similar to the corresponding light scattering property of the target cells (e.g., cells expressing CD34) is forward scattering (FSC).

[0151] In some embodiments, the scattering modifier comprises a comonomer. In some embodiments, the scattering modifier comprises a suspension of nanoparticles.

[0152] Target cells can be virtually any type of cell, including prokaryotic and eukaryotic cells. Suitable prokaryotic target cells include, but are not limited to, bacteria such as E. coli, various Bacillus species, and extremophiles such as thermophiles.

[0153] Suitable eukaryotic target cells include, but are not limited to, fungi such as yeast and filamentous fungi, including species of the genera *Saccharomyces*, *Aspergillus*, *Trichoderma*, and *Neurospora*; plant cells, including those of corn, sorghum, tobacco, rapeseed, soybean, cotton, tomato, potato, alfalfa, sunflower, etc.; and animal cells, including fish, birds, and mammals. Suitable fish cells include, but are not limited to, fish cells from species of salmon, trout, tilapia, tuna, carp, halibut, bighead carp, swordfish, cod, and zebrafish. Suitable bird cells include, but are not limited to, cells from chickens, ducks, quails, pheasants, turkeys, and other bushfowl or game birds. Suitable mammalian cells include, but are not limited to, cells from horses, cattle, buffalo, deer, sheep, rabbits, rodents (such as mice, rats, hamsters, and guinea pigs), goats, pigs, primates, marine mammals (including dolphins and whales), and cell lines such as human cell lines of any tissue or stem cell type, as well as stem cells (including pluripotent and non-pluripotent stem cells) and non-human fertilized eggs. In some implementations, the target cells are human cells.

[0154] In some embodiments, the target cells are hematopoietic stem cells. In some embodiments, the target cells are cells expressing CD34 (e.g., CD34-expressing stem cells). In some embodiments, the CD34-expressing cells are obtained from a donor (e.g., a human donor). In some embodiments, the CD34-expressing cells are present in blood obtained from a donor (e.g., a human donor). In some embodiments, the CD34-expressing cells are present in blood obtained from a donor (e.g., a human donor) who has been given a stem cell mobilizing agent.

[0155] In some implementations, cells expressing CD34 are engineered to express CD34.

[0156] In some implementation schemes, the target cells are primary cells, cultured cells, established cells, normal cells, transformed cells, infected cells, stably transfected cells, transiently transfected cells, proliferating cells, or terminally differentiated cells.

[0157] In some embodiments, the target cells are immune cells. Non-limiting examples of immune cells include B lymphocytes (also known as B cells), T lymphocytes (also known as T cells), natural killer (NK) cells, lymphokine-activated killer (LAK) cells, monocytes, macrophages, neutrophils, granulocytes, mast cells, platelets, Langerhans cells, stem cells, dendritic cells, peripheral blood monocytes, tumor-infiltrating (TIL) cells, genetically modified immune cells (including hybridomas), drug-modified immune cells, and derivatives, precursors, or progenitors of any of the cell types listed herein. In some embodiments, immune cells are plasma cells, lymphocytes, immune cells, biomolecule-presenting cells (e.g., dendritic cells, macrophages, B cells), naive B cells, memory B cells, naive T cells, memory T cells, chimeric antigen receptor T cells (CAR-T cells), regulatory T cells, cytotoxic T cells, NK cells, or any other suitable cells.

[0158] In some embodiments, the target cells are freshly collected from the subject. In some embodiments, the target cells are cultured cell lines. In some embodiments, the target cells are established cell lines. In some embodiments, the target cells are cultured from preserved or frozen samples.

[0159] In some embodiments, the target cell is a stem cell. In some embodiments, the stem cell is a pluripotent stem cell, totipotent stem cell, multipotent stem cell, oligopotent stem cell, or unipotent stem cell. In some embodiments, the pluripotent stem cell is an embryonic stem cell. In some embodiments, the stem cell is an undifferentiated pluripotent stem cell. In some embodiments, the totipotent stem cell is, but is not limited to, embryonic stem cells, neural stem cells, bone marrow stem cells, hematopoietic stem cells, cardiomyocytes, neurons, astrocytes, muscle cells, or connective tissue cells. In some embodiments, multipotent stem cells are, but are not limited to, myeloid progenitor cells or lymphoid progenitor cells. In some embodiments, the stem cell is an induced pluripotent stem cell (iSPC). In some embodiments, the stem cell is an adult stem cell. In some embodiments, the stem cell is an undifferentiated pluripotent stem cell. In some embodiments, the stem cell is a mammalian stem cell. In some embodiments, the stem cell is a primate stem cell. In some embodiments, the stem cell is a human stem cell.

[0160] In some embodiments, the stem cells are derived from any source within the animal. For example, stem cells can be harvested from an embryo or any primitive germ layer therein, from placental or chorionic tissue, or from more mature tissues such as adult stem cells, including but not limited to adipose, bone marrow, neural tissue, mammary gland tissue, liver tissue, pancreas, epithelial, respiratory, gonadal, and muscle tissue. In some embodiments, the stem cells are placental or chorionic stem cells.

[0161] In some embodiments, the target cells are blood cells. In some embodiments, the target cells are peripheral blood mononuclear cells (PMBCs). In some embodiments, the peripheral blood mononuclear cells are lymphocytes, monocytes, or dendritic cells. In some embodiments, the lymphocytes are T cells, B cells, or NK cells. In some embodiments, the target cells are natural killer (NK) cells.

[0162] In some implementations, the target cells are lymphocytes. In other implementations, the target cells are T cells, NK cells, or B cells.

[0163] In some embodiments, the target cells are selected from the exemplary target cells listed in Table 2 of this disclosure.

[0164] Biomolecules bound to hydrogel beads

[0165] After the hydrogel particles are formed, one or more particle surfaces can be functionalized, for example, to mimic one or more properties of target cells. Hydrogel beads may also contain biomolecules, such as antigens.

[0166] In some embodiments, the hydrogel beads contain a specific copy number of biomolecules, such as cell surface markers, like cell surface receptors or their domains, such as their epitope binding regions. For example, individual subpopulations of hydrogel particles can each be derived to have a unique copy number; for instance, one subpopulation would contain 100 copies of the cell surface marker, a second subpopulation would contain 1,000 copies of the same cell surface marker, a third subpopulation would contain 10,000 copies of the same cell surface marker, and so on. The hydrogel particle population is fluorescently stained against the corresponding cell surface marker, and the fluorescence of the hydrogel particles in each subpopulation is detected. In this respect, the hydrogel particle subpopulations can be used to generate a standard curve of fluorescence emission from target cells containing the corresponding cell marker. The cell surface marker can be any of the cell surface markers provided therein or their binding regions, or cell surface markers known to those skilled in the art.

[0167] The hydrogel particles of this disclosure behave similarly to target cells in procedures such as flow cytometry or FACS staining and analysis. For example, in one embodiment, the hydrogel particles have one or more optical properties substantially similar to any of the cell types described herein.

[0168] In one embodiment, the hydrogel particles are functionalized with one or more cell surface markers or fragments thereof (e.g., the extracellular portion in the case of transmembrane proteins), for example by linking one or more cell surface markers, extracellular portions, or ligand-binding regions thereof to the particles via free amines, free carboxyl groups, and / or free hydroxyl groups present on the surface of the hydrogel particles. Functionalization of the hydrogel particles with dyes or cell surface molecules can also occur via linkers, such as streptavidin / biotin conjugates.

[0169] Depending on the target cell, individual hydrogel particles can be derived using one or more cell surface markers or fragments thereof (e.g., the extracellular portion in the case of transmembrane proteins) to further mimic the structural properties of the target cell. The table below provides a non-limiting list of cell surface markers that can be used to derive hydrogel particles, depending on the target cell. Although cell surface markers are provided, it should be understood that a portion of a cell surface marker (e.g., the receptor-binding portion, ligand-binding portion, or extracellular portion of the marker) can be used to derive hydrogel particles (at free functional groups, as described above).

[0170] In some embodiments, this disclosure provides hydrogel beads functionalized with CD34 or a portion thereof (e.g., the CD34 extracellular domain). In some embodiments, this disclosure provides hydrogel beads functionalized with CD45 or a portion thereof (e.g., the CD45 extracellular domain). In some embodiments, this disclosure provides hydrogel beads functionalized with both CD34 or a portion thereof (e.g., the CD34 extracellular domain) and CD45 or a portion thereof (e.g., the CD45 extracellular domain).

[0171] In some embodiments, this disclosure provides a first population of hydrogel beads comprising a spectrum of cell surface biomarkers containing extracellular domains of CD34 and CD45. In some embodiments, the hydrogel beads comprise polymeric monomers and bifunctional monomers. In some embodiments, this disclosure provides hydrogel beads having various predetermined levels of CD34 and CD45 biomarkers, which can be used as controls for various assays (e.g., flow cytometry). In such embodiments, the populations of hydrogels are designed to mimic corresponding animal cell populations. Thus, in some embodiments, these populations of hydrogel beads are configured with appropriate CD34 and / or CD45 biomarkers (or their extracellular domains) at concentrations approximating those biomarkers in various animal cell populations.

[0172] In some embodiments, each hydrogel bead in the first group contains about 10% to about 400% of the amount of CD34 extracellular domain present on the cell surface of the target cell. In some embodiments, each hydrogel bead in the first group contains about 10% to about 400% of the amount of CD45 extracellular domain present on the cell surface of the target cell. In some embodiments, each hydrogel bead in the first group contains about 10% to about 50%, about 10% to about 70%, about 10% to about 100%, about 10% to about 120%, about 10% to about 150%, about 10% to about 200%, about 10% to about 250%, about 10% to about 300%, about 10% to about 400%, about 20% to about 50%, about 20% to about 70%, about 20% to about 100%, about 20% to about 120%, about 20% to about 150%, about... 20% to about 200%, about 20% to about 250%, about 20% to about 300%, about 20% to about 400%, about 30% to about 50%, about 30% to about 70%, about 30% to about 100%, about 30% to about 120%, about 30% to about 150%, about 30% to about 200%, about 30% to about 250%, about 30% to about 300%, about 30% to about 400%, about 40% to about 50%, about 40% to about 70%, about 40% to about 100%, about 40% to about 120%, about 40% to about 150%, about 40% to about 200%, about 40% to about 250%. Approximately 40% to approximately 300%, approximately 40% to approximately 400%, approximately 50% to approximately 70%, approximately 50% to approximately 100%, approximately 50% to approximately 120%, approximately 50% to approximately 150%, approximately 50% to approximately 200%, approximately 50% to approximately 250%, approximately 50% to approximately 300%, approximately 50% to approximately 400%, approximately 60% to approximately 70%, approximately 60% to approximately 100%, approximately 60% to approximately 120%, approximately 60% to approximately 150%, approximately 60% to approximately 200%, approximately 60% to approximately 250%, approximately 60% to approximately 300%, approximately 60% to approximately 400%, approximately 70% to approximately 100%, approximately 70% to approximately 120%, about 70% to about 150%, about 70% to about 200%, about 70% to about 250%, about 70% to about 300%, about 70% to about 400%, about 80% to about 100%, about 80% to about 120%, about 80% to about 150%, about 80% to about 200%, about 80% to about 250%, about 80% to about 300%, about 80% to about 400%, about 90% to about 100%, about 90% to about 120%, about 90% to about 150%, about 90% to about 200%, about 90% to about 250%, about 90% to about 300%, about 90% to about 400%.About 100% to about 120%, about 100% to about 150%, about 100% to about 200%, about 100% to about 250%, about 100% to about 300%, about 100% to about 400%, about 120% to about 150%, about 120% to about 200%, about 120% to about 250%, about 120% to about 300%, about 120% to about 400%, about 150% to about 200%, about 150% to about 250%, about 150% to about 300%, or about 150% to about 400%, including all ranges and subranges therein.

[0173] In some embodiments, this disclosure provides a second group of hydrogel beads, each containing a cell surface biomarker profile that includes a CD45 extracellular domain but at a lower level than that of the first group of hydrogel beads. In some embodiments, each hydrogel bead in the second group contains no more than 10% of the median number of CD34 extracellular domains contained in the first group of hydrogel beads. In some embodiments, each hydrogel bead in the second group contains no more than 0.1%, no more than 0.2%, no more than 0.3%, no more than 0.5%, no more than 0.7%, no more than 1%, no more than 2%, no more than 3%, no more than 5%, or no more than 7% of the median number of CD34 extracellular domains contained in the first group of hydrogel beads. In some embodiments, the cell surface biomarker profile of each hydrogel bead in the second group lacks a CD34 extracellular domain. In some embodiments, the hydrogel beads contain a polymeric monomer and a bifunctional monomer.

[0174] In some embodiments, each hydrogel bead in the second population contains about 10% to about 400% of the amount of the CD45 extracellular domain present on the cell surface of the target cell. In some embodiments, each hydrogel bead in the second population contains about 10% to about 50%, about 10% to about 70%, about 10% to about 100%, about 10% to about 120%, about 10% to about 150%, about 10% to about 200%, about 10% to about 250%, about 10% to about 300%, about 10% to about 400%, about 20% to about 50%, about 20% to about 70%, about 20% to about 100%, about 20% to about 120%, about 20% to about 150%, about 20% to about 200%, about 20% to about 250%, about 20% to about 250%, about 20% to about 50%, about 20% to about 70%, about 20% to about 100%, about 20% to about 120%, about 20% to about 150%, about 20% to about 20 ...150%, about 20% to about 200%, about 20% to about 250%, about 20% to about 50%, about 20% to about 100%, about 20% to about 120%, about 2 Approximately 300%, approximately 20% to approximately 400%, approximately 30% to approximately 50%, approximately 30% to approximately 70%, approximately 30% to approximately 100%, approximately 30% to approximately 120%, approximately 30% to approximately 150%, approximately 30% to approximately 200%, approximately 30% to approximately 250%, approximately 30% to approximately 300%, approximately 30% to approximately 400%, approximately 40% to approximately 50%, approximately 40% to approximately 70%, approximately 40% to approximately 100%, approximately 40% to approximately 120%, approximately 40% to approximately 150%, approximately 40% to approximately 200%, approximately 40% to approximately 250%, approximately 40% to approximately 300%, approximately 40% to approximately 400%, approximately 50% to approximately 70%, approximately 50% to approximately 100%, approximately 50% to approximately 120%, approximately 50% to approximately 150%, approximately 50% to approximately 200%, approximately 50% to approximately 250%, approximately 50% to approximately 300%, approximately 50% to approximately 400%, approximately 60% to approximately 70%, approximately 60% to approximately 100%, approximately 60% to approximately 120%, approximately 60% to approximately 150%, approximately 60% to approximately 200%, approximately 60% to approximately 250%, approximately 60% to approximately 300%, approximately 60% to approximately 400%, approximately 70% to approximately 100%, approximately 70% to approximately 120%, approximately 70% to approximately 150%, approximately 70% to approximately 200%, approximately 70% to approximately 250%, approximately 70% to approximately 300%, approximately 70% to approximately 400%, approximately 80% About 100%, about 80% to about 120%, about 80% to about 150%, about 80% to about 200%, about 80% to about 250%, about 80% to about 300%, about 80% to about 400%, about 90% to about 100%, about 90% to about 120%, about 90% to about 150%, about 90% to about 200%, about 90% to about 250%, about 90% to about 300%, about 90% to about 400%, about 100% to about 120%, about 100% to about 150%, about 100% to about 200%, about 100% to about 250%, about 100% to about 300%, about 100% to about 400%, about 120% to about 150%.Approximately 120% to approximately 200%, approximately 120% to approximately 250%, approximately 120% to approximately 300%, approximately 120% to approximately 400%, approximately 150% to approximately 200%, approximately 150% to approximately 250%, approximately 150% to approximately 300%, or approximately 150% to approximately 400%, inclusive of all ranges and subranges therein.

[0175] In some embodiments, the amount of CD34 extracellular domain in each hydrogel bead of the second population is lower than the amount of CD34 extracellular domain in each hydrogel bead of the first population. In some embodiments, each hydrogel bead of the second population contains no more than 10% of the amount of CD34 extracellular domain present on the cell surface of the target cell. In some embodiments, each hydrogel bead of the second population contains no more than 50%, no more than 40%, no more than 30%, no more than 20%, or no more than 10% of the amount of CD34 extracellular domain present on the cell surface of the target cell. In some embodiments, each hydrogel bead of the second population contains no more than 0.1%, no more than 0.2%, no more than 0.3%, no more than 0.5%, no more than 0.7%, no more than 1%, no more than 2%, no more than 3%, no more than 5%, or no more than 7% of the amount of CD34 extracellular domain present on the cell surface of the target cell.

[0176] In some embodiments, the hydrogel beads in the first group contain about 10% to about 400% of the amount of CD34 extracellular domain present on the cell surface of the target cells. In some embodiments, the hydrogel beads in the first group contain about 10% to about 400% of the amount of CD45 extracellular domain present on the cell surface of the target cells. In some embodiments, the hydrogel beads in the first group contain about 10% to about 50%, about 10% to about 70%, about 10% to about 100%, about 10% to about 120%, about 10% to about 150%, about 10% to about 200%, about 10% to about 250%, about 10% to about 300%, about 10% to about 400%, about 20% to about 50%, about 20% to about 70%, about 20% to about 100%, about 20% to about 120%, about 20% to about 150%, about 20% to about 2 ... Approximately 250%, approximately 20% to approximately 300%, approximately 20% to approximately 400%, approximately 30% to approximately 50%, approximately 30% to approximately 70%, approximately 30% to approximately 100%, approximately 30% to approximately 120%, approximately 30% to approximately 150%, approximately 30% to approximately 200%, approximately 30% to approximately 250%, approximately 30% to approximately 300%, approximately 30% to approximately 400%, approximately 40% to approximately 50%, approximately 40% to approximately 70%, approximately 40% to approximately 100%, approximately 40% to approximately 120%, approximately 40% to approximately 150%, approximately 40% to approximately 200%, approximately 40% to approximately 250%, approximately 40% to approximately 300%, approximately 40% to approximately 400%. Approximately 50% to approximately 70%, approximately 50% to approximately 100%, approximately 50% to approximately 120%, approximately 50% to approximately 150%, approximately 50% to approximately 200%, approximately 50% to approximately 250%, approximately 50% to approximately 300%, approximately 50% to approximately 400%, approximately 60% to approximately 70%, approximately 60% to approximately 100%, approximately 60% to approximately 120%, approximately 60% to approximately 150%, approximately 60% to approximately 200%, approximately 60% to approximately 250%, approximately 60% to approximately 300%, approximately 60% to approximately 400%, approximately 70% to approximately 100%, approximately 70% to approximately 120%, approximately 70% to approximately 150%, approximately 70% to approximately 200%, approximately 7 0% to about 250%, about 70% to about 300%, about 70% to about 400%, about 80% to about 100%, about 80% to about 120%, about 80% to about 150%, about 80% to about 200%, about 80% to about 250%, about 80% to about 300%, about 80% to about 400%, about 90% to about 100%, about 90% to about 120%, about 90% to about 150%, about 90% to about 200%, about 90% to about 250%, about 90% to about 300%, about 90% to about 400%, about 100% to about 120%, about 100% to about 150%, about 100% to about 200%.About 100% to about 250%, about 100% to about 300%, about 100% to about 400%, about 120% to about 150%, about 120% to about 200%, about 120% to about 250%, about 120% to about 300%, about 120% to about 400%, about 150% to about 200%, about 150% to about 250%, about 150% to about 300%, or about 150% to about 400%, including all ranges and subranges therein.

[0177] In some embodiments, this disclosure provides a second population of hydrogel beads comprising a cell surface biomarker profile containing a CD45 extracellular domain but at a lower level than that of the first population of hydrogel beads. In some embodiments, the hydrogel beads in the second population contain no more than 10% of the median number of CD34 extracellular domains contained in the hydrogel beads of the first population. In some embodiments, the hydrogel beads in the second population contain no more than 0.1%, no more than 0.2%, no more than 0.3%, no more than 0.5%, no more than 0.7%, no more than 1%, no more than 2%, no more than 3%, no more than 5%, or no more than 7% of the median number of CD34 extracellular domains contained in the hydrogel beads of the first population. In some embodiments, the cell surface biomarker profile of the hydrogel beads in the second population lacks a CD34 extracellular domain. In some embodiments, the hydrogel beads comprise a polymeric monomer and a bifunctional monomer.

[0178] In some embodiments, the hydrogel beads in the second population contain about 10% to about 400% of the amount of the CD45 extracellular domain present on the cell surface of the target cells. In some embodiments, the hydrogel beads in the second population contain about 10% to about 50%, about 10% to about 70%, about 10% to about 100%, about 10% to about 120%, about 10% to about 150%, about 10% to about 200%, about 10% to about 250%, about 10% to about 300%, about 10% to about 400%, about 20% to about 50%, about 20% to about 70%, about 20% to about 100%, about 20% to about 120%, about 20% to about 150%, about 20% to about 200%, about 20% to about 250%, about 20% to about 300%, or about 20%. About 400%, about 30% to about 50%, about 30% to about 70%, about 30% to about 100%, about 30% to about 120%, about 30% to about 150%, about 30% to about 200%, about 30% to about 250%, about 30% to about 300%, about 30% to about 400%, about 40% to about 50%, about 40% to about 70%, about 40% to about 100%, about 40% to about 120%, about 40% to about 150%, about 40% to about 200%, about 40% to about 250%, about 40% to about 300%, about 40% to about 400%, about 50% to about 70%, about 50% to about 100%, about 50% to about 120%, about 50% to about 150%, about 50% to about 200%, about 50% to about 250%, about 50% to about 300%, about 50% to about 400%, about 60% to about 70%, about 60% to about 100%, about 60% to about 120%, about 60% to about 150%, about 60% to about 200%, about 60% to about 250%, about 60% to about 300%, about 60% to about 400%, about 70% to about 100%, about 70% to about 120%, about 70% to about 150%, about 70% to about 200%, about 70% to about 250%, about 70% to about 300%, about 70% to about 400%, about 80% to about 100%, about 80% to About 120%, about 80% to about 150%, about 80% to about 200%, about 80% to about 250%, about 80% to about 300%, about 80% to about 400%, about 90% to about 100%, about 90% to about 120%, about 90% to about 150%, about 90% to about 200%, about 90% to about 250%, about 90% to about 300%, about 90% to about 400%, about 100% to about 120%, about 100% to about 150%, about 100% to about 200%, about 100% to about 250%, about 100% to about 300%, about 100% to about 400%, about 120% to about 150%, about 120% to about 200%.Approximately 120% to approximately 250%, approximately 120% to approximately 300%, approximately 120% to approximately 400%, approximately 150% to approximately 200%, approximately 150% to approximately 250%, approximately 150% to approximately 300%, or approximately 150% to approximately 400%, inclusive of all ranges and subranges therein.

[0179] In some embodiments, the amount of CD34 extracellular domain in the hydrogel beads of the second population is lower than the amount of CD34 extracellular domain in the hydrogel beads of the first population. In some embodiments, the amount of CD34 extracellular domain present on the cell surface of the target cells contained in the hydrogel beads of the second population does not exceed 10%. In some embodiments, the amount of CD34 extracellular domain present on the cell surface of the target cells contained in the hydrogel beads of the second population does not exceed 50%, 40%, 30%, 20%, or 10%. In some embodiments, the amount of CD34 extracellular domain present on the cell surface of the target cells contained in the hydrogel beads of the second population does not exceed 0.1%, 0.2%, 0.3%, 0.5%, 0.7%, 1%, 2%, 3%, 5%, or 7%.

[0180] In some embodiments, the amount of CD34 and / or CD45 extracellular domains present on the cell surface of the target cells is the median amount of CD34 and / or CD45 extracellular domains present on the cell surface of cells in a package of CD34+-rich leukocytes treated with Protocol H. In some embodiments, leukopak is used in accordance with an IRB-approved protocol using SpectraOptia®. ®The Apheresis System collects enriched leukopak products from healthy, consenting donors. In some embodiments, leukopak is collected from at least 10, at least 20, or at least 30 healthy, consenting donors. Leukopaks allow for scalability and reproducibility of experiments by limiting the impact of donor-to-donor variability. In some embodiments, the leukopak contains acid-citric acid-glucose solution A (ACDA) as an anticoagulant. In some embodiments, the leukopak (before CD34+ cell enrichment) contains up to about 50% T cells, about 20% monocytes, about 10% B cells, about 10% NK cells, about 3% granulocytes, and about 3% hematocrit. Protocol H is a mobilization protocol known in the art and is performed by AllCells® to collect mobilized peripheral blood. In this procedure, a healthy donor is given an FDA-approved drug to “mobilize” HSPCs from a bone marrow stem cell niche into the peripheral blood circulation. In some implementations, Protocol H treatment involves subcutaneous administration of Filgrastim (Neupogen®) 10 ug / kg / day for 5 days, followed by subcutaneous administration of Plexafor (Mozobil®) 240 ug / kg / day for 2 days on days 4 and 5 (evening), and removal on days 5 and 6. The cytokine G-CSF (Neupogen®) is the most commonly used mobilizing agent and has been the gold standard in clinical practice for over 20 years. The bicyclic imamidine molecule Plexafor (Mozobil®) has been shown to effectively salvage HSPC mobilization in individuals who cannot be mobilized with G-CSF. Further information on Protocol H treatment can be found, for example, at the web link “allcells.com / dual-mobilization-enhances-cd34-yield-across-all-donor-types / ”, the contents of which are incorporated herein by reference in their entirety. Although the amount of CD34 and / or CD45 extracellular domains can vary in cell populations within leukocyte packs, the median amount of CD34 and / or CD45 extracellular domains present on the surface of cells in leukocyte packs rich in CD34+ cells has the smallest batch-to-batch variation.

[0181] In some embodiments, multiple forms or variants of the CD34 and / or CD45 extracellular domains may be present in the hydrogel beads or on the cell surface of the target cells. For example, the CD45 extracellular domain has several naturally occurring isotypes (e.g., SEQ ID NO: 2-9), and multiple isotypes may be present on the surface of the same cell. In some embodiments, the CD34 and / or CD45 extracellular domains in the hydrogel beads may be present in different forms. For example, the hydrogel beads may have multiple CD34 extracellular domain variants, one variant containing a first tag and possible mutations (e.g., insertion, deletion, or substitution), and another variant containing a second tag and possible different mutations (e.g., insertion, deletion, or substitution). In this case, unless otherwise stated, the amount of the CD34 and / or CD45 extracellular domains is the total amount of all variants that can bind to CD34-specific binding molecules and / or CD45-specific binding molecules in a quantification assay. In some embodiments, the quantification assay is a fluorescence intensity measurement using flow cytometry. That is, in some embodiments, flow cytometry is used to measure the amount of CD45 and / or CD34 extracellular domains present in the hydrogel and / or on the cell surface based on fluorescence intensity. In some embodiments, the amount of CD34 and / or CD45 extracellular domains in the hydrogel is compared with the median amount of CD34 and / or CD45 extracellular domains present on the surface of cells in a leukocyte package rich in CD34+ cells treated with Protocol H, based on fluorescence intensity during flow cytometry (e.g., using a combination of CD34-specific binding molecules and CD45-specific binding molecules according to Group 1 in Table 4).

[0182] In some embodiments, the fluorescence intensity of the CD45 extracellular domain is measured using a CD45-specific binding molecule. In some embodiments, the fluorescence intensity of the CD34 extracellular domain is measured using a CD34-specific binding molecule. In some embodiments, the binding molecule comprises a monoclonal antibody or an antigen-binding fragment thereof. In some embodiments, the binding molecule comprises a peptide, protein, aptamer, or polymer. In some embodiments, the binding molecule is a complex (e.g., a complex of a primary antibody and a fluorophore-labeled secondary antibody). In some embodiments, the binding molecule binds to CD34 or CD45 with an equilibrium dissociation constant (Kd) of less than 1000 nM, less than 100 nM, less than 50 nM, less than 10 nM, less than 5 nM, less than 1 nM, less than 0.5 nM, or less than 0.1 nM. In some embodiments, the binding molecule is fluorophore-labeled or contains a fluorophore.

[0183] In some embodiments, the CD34-specific binding molecule is selected from: phycoerythrin (PE)-labeled anti-CD34 antibody clone 8G12, phycoerythrin (PE)-labeled anti-CD34 antibody clone AC136, allophycocyanin (APC)-labeled anti-CD34 antibody clone 4H11, and Brilliant™ Violet 421 (BV421)-labeled anti-CD34 antibody clone 581. In some embodiments, the CD45-specific binding molecule is selected from: FITC-labeled anti-CD45 antibody clone 2D1, PerCP-Cyanine® 5.5 (PerCP-Cy5.5)-labeled anti-CD45 antibody clone 2D1, phycoerythrin (PE)-labeled anti-CD45 antibody clone MEM-28, and BD Horizon™ V500-labeled anti-CD45 antibody clone HI30. In some embodiments, the combination of CD34-specific binding molecules and CD45-specific binding molecules is selected from one group in Table 4. In some implementations, the combination of the CD34-specific binding molecule and the CD45-specific binding molecule is a phycoerythrin (PE)-labeled anti-CD34 antibody clone 8G12 and a FITC-labeled anti-CD45 antibody clone 2D1.

[0184] In some embodiments, CD34 is derived from Homo sapiens. In some embodiments, the extracellular domain of CD34 comprises Ser32-Thr290 of uniprot P28906 or SEQ ID NO: 1, or contains at least 70%, 75%, 80%, 85%, 90%, 95%, or 97.5% identity with Ser32-Thr290 of uniprot P28906 or SEQ ID NO: 1. In some embodiments, the extracellular domain of CD34 comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 1. In some embodiments, the extracellular domain of CD34 comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a segment of at least 20 consecutive amino acids in SEQ ID NO: 1. In some embodiments, the CD34 extracellular domain comprises at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the amino acid sequence identical to at least 30 consecutive amino acids in SEQ ID NO: 1. In some embodiments, the CD34 extracellular domain comprises at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the amino acid sequence identical to at least 40 consecutive amino acids in SEQ ID NO: 1. In some embodiments, the CD34 extracellular domain comprises at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the amino acid sequence identical to at least 50 consecutive amino acids in SEQ ID NO: 1. In some embodiments, the CD34 extracellular domain comprises at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the amino acid sequence identical to at least 70 consecutive amino acids in SEQ ID NO: 1. In some embodiments, the CD34 extracellular domain comprises at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the amino acid sequence identical to at least 100 consecutive amino acids in SEQ ID NO: 1. In some embodiments, the CD34 extracellular domain comprises a fluorophore.

[0185] In some embodiments, CD45 is derived from Homo sapiens. In some embodiments, the extracellular domain of CD45 comprises Gln26-Lys577 of uniprot P08575 or SEQ ID NO: 2, or a sequence containing at least 70%, 75%, 80%, 85%, 90%, 95%, or 97.5% identity with Gln26-Lys577 of uniprot P08575 or SEQ ID NO: 2. In some embodiments, the extracellular domain of CD45 comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to any of SEQ ID NO: 2-9. In some embodiments, the CD45 extracellular domain comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to that of SEQ ID NO: 10. In some embodiments, the CD45 extracellular domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to that of at least 20 consecutive amino acid segments in SEQ ID NO: 10. In some embodiments, the CD45 extracellular domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to that of at least 30 consecutive amino acid segments in SEQ ID NO: 10. In some embodiments, the CD45 extracellular domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a segment of at least 40 consecutive amino acids in SEQ ID NO: 10. In some embodiments, the CD45 extracellular domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a segment of at least 50 consecutive amino acids in SEQ ID NO: 10. In some embodiments, the CD45 extracellular domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a segment of at least 70 consecutive amino acids in SEQ ID NO: 10. In some embodiments, the CD45 extracellular domain contains at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the same amino acid sequence as at least 100 consecutive amino acids in SEQ ID NO: 10.In some embodiments, the CD45 extracellular domain contains a fluorophore. In some embodiments, the fluorophore of the CD45 extracellular domain is different from the fluorophore of the CD34 extracellular domain.

[0186] Table 5: Non-restrictive exemplary sequences of the extracellular domains of CD34 and CD45

[0187] protein Non-limiting exemplary sequence SEQ ID NO CD34 extracellular domain SLDNNGTATPELPTQGTFSNVSTNVSYQETTTPSTLGSTSSLHPVSQHGNEATTNITETTVKFTSTSVITSVYGNTNSSVQSQTSVISTVFTTPANVSTPETTLKPSLSPGNVSDLSTTTSLATSPTKP YTSSSPILSDIKAEIKCSGIREVKLTQGICLEQNKTSSCAEFKKDRGEGLARVLCGEEQADADAGAQVCSLLLAQSEVRPQCLLLVLANRTEISSKLQLMKKHQSDLKKLGILDFTEQDVASHQSYSQKT 1 CD45 extracellular domain ABC isotype QSPTPSPTGLTTAKMPSVPLSSDPLPTHTTAFSPASTFERENDFSETTTSLSPDNTSTQVSPDSLDNASAFNTTGVSSVQTPHLPTHADSQTPSAGTDTQTFSGSAANAKLNPTPGSNAISDVPGERSTASTFPTDPVSPLTTTLSLAHHSSAALPARTSNTTITANTSDAYLNASETTTLSPSGSAVISTTTIATTPSKPTCDEKYANITVDYLYNKETKLFTAKLNVNENVECGNNTCTNNEVHNLTECKNASVSISHNSCTAPDKTLILDVPPGVEKFQLHDCTQVEKADTTICLKWKNIETFTCDTQNITYRFQCGNMIFDNKEIKLENLEPEHEYKCDSEILYNNHKFTNASKIIKTDFGSPGEPQIIFCRSEAAHQGVITWNPPQRSFHNFTLCYIKETEKDCLNLDKNLIKYDLQNLKPYTKYVLSLHAYIIAKVQRNGSAAMCHFTTKSAPPSQVWNMTVSMTSDNSMHVKCRPPRDRNGPHERYHLEVEAGNTLVRNESHKNCDFRVKDLQYSTDYTFKAYFHNGDYPGEPFILHHSTSYNSK 2 CD45 extracellular domain O isotype QSPTPSPTDAYLNASETTTLSPSGSAVISTTTIATTPSKPTCDEKYANITVDYLYNKETKLFTAKLNVNENVECGNNTCTNNEVHNLTECKNASVSISHNSCTAPDKTLILDVPPGVEKFQLHDCTQVEKADTTICLKWKNIETFTCDTQNITYRFQCGNMIFDNKEIKLENLEPEHEYKCDSEILYNNHKFTNASKIIKTDFGSPGEPQIIFCRSEAAHQGVITWNPPQRSFHNFTLCYIKETEKDCLNLDKNLIKYDLQNLKPYTKYVLSLHAYIIAKVQRNGSAAMCHFTTKSAPPSQVWNMTVSMTSDNSMHVKCRPPRDRNGPHERYHLEVEAGNTLVRNESHKNCDFRVKDLQYSTDYTFKAYFHNGDYPGEPFILHHSTSYNSK 3 CD45 extracellular domain AB isotype QSPTPSPTGLTTAKMPSVPLSSDPLPTHTTAFSPASTFERENDFSETTTSLSPDNTSTQVSPDSLDNASAFNTTGVSSVQTPHLPTHADSQTPSAGTDTQTFSGSAANAKLNPTPGSNAISDAYLNASETTTLSPSGSAVISTTTIATTPSKPTCDEKYANITVDYLYNKETKLFTAKLNVNENVECGNNTCTNNEVHNLTECKNASVSISHNSCTAPDKTLILDVPPGVEKFQLHDCTQVEKADTTICLKWKNIETFTCDTQNITYRFQCGNMIFDNKEIKLENLEPEHEYKCDSEILYNNHKFTNASKIIKTDFGSPGEPQIIFCRSEAAHQGVITWNPPQRSFHNFTLCYIKETEKDCLNLDKNLIKYDLQNLKPYTKYVLSLHAYIIAKVQRNGSAAMCHFTTKSAPPSQVWNMTVSMTSDNSMHVKCRPPRDRNGPHERYHLEVEAGNTLVRNESHKNCDFRVKDLQYSTDYTFKAYFHNGDYPGEPFILHHSTSYNSK 4 CD45 extracellular domain AC isoform QSPTPSPTGLTTAKMPSVPLSSDPLPTHTTAFSPASTFERENDFSETTTSLSPDNTSTQVSPDSLDNASAFNTTDVPGERSTASTFPTDPVSPLTTTLSLAHHSSAALPARTSNTTITANTSDAYLNASETTTLSPSGSAVISTTTIATTPSKPTCDEKYANITVDYLYNKETKLFTAKLNVNENVECGNNTCTNNEVHNLTECKNASVSISHNSCTAPDKTLILDVPPGVEKFQLHDCTQVEKADTTICLKWKNIETFTCDTQNITYRFQCGNMIFDNKEIKLENLEPEHEYKCDSEILYNNHKFTNASKIIKTDFGSPGEPQIIFCRSEAAHQGVITWNPPQRSFHNFTLCYIKETEKDCLNLDKNLIKYDLQNLKPYTKYVLSLHAYIIAKVQRNGSAAMCHFTTKSAPPSQVWNMTVSMTSDNSMHVKCRPPRDRNGPHERYHLEVEAGNTLVRNESHKNCDFRVKDLQYSTDYTFKAYFHNGDYPGEPFILHHSTSYNSK 5 CD45 extracellular domain BC isoform QSPTPSPTGVSSVQTPHLPTHADSQTPSAGTDTQTFSGSAANAKLNPTPGSNAISDVPGERSTASTFPTDPVSPLTTTLSLAHHSSAALPARTSNTTITANTSDAYLNASETTTLSPSGSAVISTTTIATTPSKPTCDEKYANITVDYLYNKETKLFTAKLNVNENVECGNNTCTNNEVHNLTECKNASVSISHNSCTAPDKTLILDVPPGVEKFQLHDCTQVEKADTTICLKWKNIETFTCDTQNITYRFQCGNMIFDNKEIKLENLEPEHEYKCDSEILYNNHKFTNASKIIKTDFGSPGEPQIIFCRSEAAHQGVITWNPPQRSFHNFTLCYIKETEKDCLNLDKNLIKYDLQNLKPYTKYVLSLHAYIIAKVQRNGSAAMCHFTTKSAPPSQVWNMTVSMTSDNSMHVKCRPPRDRNGPHERYHLEVEAGNTLVRNESHKNCDFRVKDLQYSTDYTFKAYFHNGDYPGEPFILHHSTSYNSK 6 CD45 extracellular domain A isoform QSPTPSPTGLTTAKMPSVPLSSDPLPTHTTAFSPASTFERENDFSETTTSLSPDNTSTQVSPDSLDNASAFNTTDAYLNASETTTLSPSGSAVISTTTIATTPSKPTCDEKYANITVDYLYNKETKLFTAKLNVNENVECGNNTCTNNEVHNLTECKNASVSISHNSCTAPDKTLILDVPPGVEKFQLHDCTQVEKADTTICLKWKNIETFTCDTQNITYRFQCGNMIFDNKEIKLENLEPEHEYKCDSEILYNNHKFTNASKIIKTDFGSPGEPQIIFCRSEAAHQGVITWNPPQRSFHNFTLCYIKETEKDCLNLDKNLIKYDLQNLKPYTKYVLSLHAYIIAKVQRNGSAAMCHFTTKSAPPSQVWNMTVSMTSDNSMHVKCRPPRDRNGPHERYHLEVEAGNTLVRNESHKNCDFRVKDLQYSTDYTFKAYFHNGDYPGEPFILHHSTSYNSK 7 CD45 extracellular domain B isoform QSPTPSPTGVSSVQTPHLPTHADSQTPSAGTDTQTFSGSAANAKLNPTPGSNAISDAYLNASETTTLSPSGSAVISTTTIATTPSKPTCDEKYANITVDYLYNKETKLFTAKLNVNENVECGNNTCTNNEVHNLTECKNASVSISHNSCTAPDKTLILDVPPGVEKFQLHDCTQVEKADTTICLKWKNIETFTCDTQNITYRFQCGNMIFDNKEIKLENLEPEHEYKCDSEILYNNHKFTNASKIIKTDFGSPGEPQIIFCRSEAAHQGVITWNPPQRSFHNFTLCYIKETEKDCLNLDKNLIKYDLQNLKPYTKYVLSLHAYIIAKVQRNGSAAMCHFTTKSAPPSQVWNMTVSMTSDNSMHVKCRPPRDRNGPHERYHLEVEAGNTLVRNESHKNCDFRVKDLQYSTDYTFKAYFHNGDYPGEPFILHHSTSYNSK 8 CD45 extracellular domain C isoform QSPTPSPTDVPGERSTASTFPTDPVSPLTTTLSLAHHSSAALPARTSNTTITANTSDAYLNASETTTLSPSGSAVISTTTIATTPSKPTCDEKYANITVDYLYNKETKLFTAKLNVNENVECGNNTCTNNEVHNLTECKNASVSISHNSCTAPDKTLILDVPPGVEKFQLHDCTQVEKADTTICLKWKNIETFTCDTQNITYRFQCGNMIFDNKEIKLENLEPEHEYKCDSEILYNNHKFTNASKIIKTDFGSPGEPQIIFCRSEAAHQGVITWNPPQRSFHNFTLCYIKETEKDCLNLDKNLIKYDLQNLKPYTKYVLSLHAYIIAKVQRNGSAAMCHFTTKSAPPSQVWNMTVSMTSDNSMHVKCRPPRDRNGPHERYHLEVEAGNTLVRNESHKNCDFRVKDLQYSTDYTFKAYFHNGDYPGEPFILHHSTSYNSK 9 CD45 extracellular domain consensus sequence DAYLNASETTTLSPSGSAVISTTTIATTPSKPTCDEKYANITVDYLYNKETKLFTAKLNVNENVECGNNTCTNNEVHNLTECKNASVSISHNSCTAPDKTLILDVPPGVEKFQLHDCTQVEKADTTICLKWKNIETFTCDTQNITYRFQCGNMIFDNKEIKLENLEPEHEYKCDSEILYNNHKFTNASKIIKTDFGSPGEPQIIFCRSEAAHQGVITWNPPQRSFHNFTLCYIKETEKDCLNLDKNLIKYDLQNLKPYTKYVLSLHAYIIAKVQRNGSAAMCHFTTKSAPPSQVWNMTVSMTSDNSMHVKCRPPRDRNGPHERYHLEVEAGNTLVRNESHKNCDFRVKDLQYSTDYTFKAYFHNGDYPGEPFILHHSTSYNSK 10 Full-length CD34 MLVRRGARAGPRMPRGWTALCLLSLLPSGFMSLDNNGTATPELPTQGTFSNVSTNVSYQETTTPSTLGSTSLHPVSQHGNEATTNITETTVKFTSTSVITSVYGNTNSSVQSQTSVISTVFTTPANVSTPETTLKPSLSPGNVSDLSTTSTSLATSPTKPYTSSSPILSDIKAEIKCSGIREVKLTQGICLEQNKTSSCAEFKKDRGEGLARVLCGEEQADADAGAQVCSLLLAQSEVRPQCLLLVLANRTEISSKLQLMKKHQSDLKKLGILDFTEQDVASHQSYSQKTLIALVTSGALLAVLGITGYFLMNRRSWSPTGERLGEDPYYTENGGGQGYSSGPGTSPEAQGKASVNRGAQENGTGQATSRNGHSARQHVVADTEL 11 Full-length CD45 MTMYLWLKLLAFGFAFLDTEVFVTGQSPTPSPTGLTTAKMPSVPLSSDPLPTHTTAFSPASTFERENDFSETTTSLSPDNTSTQVSPDSLDNASAFNTTGVSSVQTPHLPTHADSQTPSAGTDTQTFSGSAANAKLNPTPGSNAISDVPGERSTASTFPTDPVSPLTTTLSLAHHSSAALPARTSNTTITANTSDAYLNASETTTLSPSGSAVISTTTIATTPSKPTCDEKYANITVDYLYNKETKLFTAKLNVNENVECGNNTCTNNEVHNLTECKNASVSISHNSCTAPDKTLILDVPPGVEKFQLHDCTQVEKADTTICLKWKNIETFTCDTQNITYRFQCGNMIFDNKEIKLENLEPEHEYKCDSEILYNNHKFTNASKIIKTDFGSPGEPQIIFCRSEAAHQGVITWNPPQRSFHNFTLCYIKETEKDCLNLDKNLIKYDLQNLKPYTKYVLSLHAYIIAKVQRNGSAAMCHFTTKSAPPSQVWNMTVSMTSDNSMHVKCRPPRDRNGPHERYHLEVEAGNTLVRNESHKNCDFRVKDLQYSTDYTFKAYFHNGDYPGEPFILHHSTSYNSKALIAFLAFLIIVTSIALLVVLYKIYDLHKKRSCNLDEQQELVERDDEKQLMNVEPIHADILLETYKRKIADEGRLFLAEFQSIPRVFSKFPIKEARKPFNQNKNRYVDILPYDYNRVELSEINGDAGSNYINASYIDGFKEPRKYIAAQGPRDETVDDFWRMIWEQKATVIVMVTRCEEGNRNKCAEYWPSMEEGTRAFGDVVVKINQHKRCPDYIIQKLNIVNKKEKATGREVTHIQFTSWPDHGVPEDPHLLLKLRRRVNAFSNFFSGPIVVHCSAGVGRTGTYIGIDAMLEGLEAENKVDVYGYVVKLRRQRCLMVQVEAQYILIHQALVEYNQFGETEVNLSELHPYLHNMKKRDPPSEPSPLEAEFQRLPSYRSWRTQHIGNQEENKSKNRNSNVIPYDYNRVPLKHELEMSKESEHDSDESSDDDSDSEEPSKYINASFIMSYWKPEVMIAAQGPLKETIGDFWQMIFQRKVKVIVMLTELKHGDQEICAQYWGEGKQTYGDIEVDLKDTDKSSTYTLRVFELRHSKRKDSRTVYQYQYTNWSVEQLPAEPKELISMIQVVKQKLPQKNSSEGNKHHKSTPLLIHCRDGSQQTGIFCALLNLLESAETEEVVDIFQVVKALRKARPGMVSTFEQYQFLYDVIASTYPAQNGQVKKNNHQEDKIEFDNEVDKVKQDANCVNPLGAPEKLPEAKEQAEGSEPTSGTEGPEHSVNGPASPALNQGS 12

[0188] Tables 2 and 3 provide additional biomolecules that can be used with the disclosed hydrogel beads and the methods disclosed herein.

[0189] Table 2: Exemplary target cells and representative cell surface markers

[0190]

[0191] Table 3. Cell surface markers used in conjunction with the hydrogel beads described herein.

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228] In another embodiment, this disclosure relates to a population of hydrogel beads, each hydrogel bead comprising: a) a polymeric monomer and a bifunctional monomer; and b) a predetermined amount of protein antigen bound to the hydrogel bead. In some embodiments, the amount of protein antigen in each hydrogel bead varies by less than 30%, 20%, 10%, 5%, or 2%. In some embodiments, the protein antigen is bound to a commercial cell staining agent.

[0229] In another embodiment, the protein antigen exhibits at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity with CD34 (e.g., human CD34).

[0230] In some implementations, the protein antigen exhibits at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity with CD45 (e.g., human CD45).

[0231] In some embodiments, the protein antigen exhibits at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity with UniProtKB Registry No. P08575 Gln26 to Lys577 or SEQ ID NO: 2. In some embodiments, the protein antigen exhibits at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity with any of SEQ ID NO: 2-9. In some embodiments, the protein antigen exhibits at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity with SEQ ID NO: 10.

[0232] In some implementations, the protein antigen exhibits at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity with UniProtKB accession number P28906 Ser32 to Thr290 or SEQ ID NO: 1.

[0233] In some embodiments, the biomarker is attached to the matrix of the hydrogel beads via a linker. The biomarker may be covalently linked to the matrix (e.g., via a covalent linker, or directly covalently conjugated) or non-covalently linked to the matrix (e.g., via a tag non-covalently bound to a binder on the matrix, such as biotin-streptavidin). In some embodiments, the matrix of the hydrogel beads is formed of polymeric monomers and / or bifunctional monomers.

[0234] As mentioned elsewhere in this document, in some embodiments, this disclosure provides hydrogel beads having various predetermined levels of CD34 and CD45 biomarkers, which can be used as controls for various assays (e.g., flow cytometry). In such embodiments, populations of hydrogels are designed to mimic corresponding animal cell populations. Thus, in some embodiments, these populations of hydrogel beads are configured with appropriate CD34 and / or CD45 biomarkers (or their extracellular domains) that approximate the concentrations of those biomarkers in various animal cell populations. In some embodiments, the approximate concentration of the biomarkers is assessed by the ability of the hydrogel beads to generate similar levels of signal from various labeled biomarkers (e.g., by having substantially similar MFIs).

[0235] In some implementations, the hydrogel beads are capable of generating a signal of any of the stated biomarkers that is substantially similar to a comparable signal from target cells. As used herein, the term “signal” refers to any measurable signal arising from the presence of a biomarker, whether inherent to the biomarker itself or achievable through one or more labeling methods (e.g., fluorescence from an antibody-fluorophore conjugate targeting a biomarker).

[0236] Therefore, in some embodiments, the biomarkers in the hydrogel beads of this disclosure exhibit values ​​of 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 25000, 30000, 35000, 40000, 45000, 50000, 55000, 60000, 65000, 70000, 75000, 80000, 85000, 90000, 95000, 100000, 150000, 175000, 200000, 225000, 250000, 275000, 300000, 325000. MFIs of 350000, 375000, 400000, 425000, 450000, 475000, 500000, 525000, 550000, 575000, 600000, 625000, 650000, 675000, 700000, 725000, 750000, 775000, 800000, 825000, 850000, 875000, 900000, 925000, 950000, 975000, 1000000, 1500000, 2000000, 2500000, 3000000, or 3500000, including all ranges and subranges in between.

[0237] In some embodiments, the hydrogel beads of this disclosure exhibit values ​​of 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 25000, 30000, 35000, 40000, 45000, 50000, 55000, 60000, 65000, 70000, 75000, 80000, 85000, 90000, 95000, 100000, 150000, 175000, 200000, 225000, 250000, 275000, 300000, and 325. 000, 350000, 375000, 400000, 425000, 450000, 475000, 500000, 525000, 550000, 575000, 600000, 625000, 650000, 675000, 700000, 725000, 750000, 775000, 800000, 825000, 850000, 875000, 900000, 925000, 950000, 975000, 1000000, 1500000, 2000000, 2500000, 3000000 or 3500000 The CD34 MFI of the MFI, including all ranges and subranges therein, as measured by any of the antibodies in Table 4.

[0238] In some embodiments, the hydrogel beads of this disclosure exhibit CD34 MFI of 30,000 to 100,000, 50,000 to 80,000, or 60,000 to 70,000, as measured by any of the antibodies in Table 4.

[0239] In some embodiments, for hydrogel beads, the amount of the CD34 extracellular domain in the first group of hydrogel beads is about 100 to about 300, about 300 to about 1,000, about 1,000 to about 3,000, about 3,000 to about 10,000, about 10,000 to about 30,000, about 30,000 to about 100,000, about 100,000 to about 300,000, about 300,000 to about 1,000,000, about 1,000,000 to about 3,000,000, about 3,000,000 to about 10,000,000, about 100 to about 1,000, about 300 to about 3,000, about 1,000 to about 1,000, about 300 to about 3,000, about 1,000 to about 1,000, about 1 ... 10,000, about 3,000 to about 30,000, about 10,000 to about 100,000, about 30,000 to about 300,000, about 100,000 to about 1,000,000, about 300,000 to about 3,000,000, about 1,000,000 to about 10,000,000, about 100 to about 3,000, about 300 to about 10,000, about 1,000 to about 30,000, about 3,000 to about 100,000, about 10,000 to about 300,000, about 30,000 to about 1,000,000, about 100,000 to about 3,000,000, about 300,000 to about 10,000,000, about 100 to about 10,000, about 300 to about 30,000, about 1,000 to about 100,000, about 3,000 to about 300,000, about 10,000 to about 1,000,000, about 30,000 to about 3,000,000, about 100,000 to about 10,000,000, about 100 to about 30,000, about 300 to about 100,000, about 1,000 to about 300,000, about 3,000 to about 1,000,000, about 10,000 to about 3,000,000, about 30,000 to about 10,000,000, about 100 to about 100,000 Approximately 300 to approximately 300,000, approximately 1,000 to approximately 1,000,000, approximately 3,000 to approximately 3,000,000, approximately 10,000 to approximately 10,000,000, approximately 100 to approximately 300,000, approximately 300 to approximately 1,000,000, approximately 1,000 to approximately 3,000,000, approximately 3,000 to approximately 10,000,000, approximately 100 to approximately 1,000,000, approximately 300 to approximately 3,000,000, approximately 1,000 to approximately 10,000,000, approximately 100 to approximately 3,000,000, approximately 300 to approximately 10,000,000, or approximately 100 to approximately 10,000,000 copies.In some implementations, the amount of CD34 extracellular domain in each hydrogel bead of the first population is within this range.

[0240] In some embodiments, the amount of the CD34 extracellular domain in the first group of hydrogel beads is about 100 to about 1,000 copies. In some embodiments, the amount of the CD34 extracellular domain in the first group of hydrogel beads is about 250 to about 2,500 copies. In some embodiments, the amount of the CD34 extracellular domain in the first group of hydrogel beads is about 500 to about 5,000 copies. In some embodiments, the amount of the CD34 extracellular domain in the first group of hydrogel beads is about 1,000 to about 10,000 copies. In some embodiments, the amount of the CD34 extracellular domain in the first group of hydrogel beads is about 2,500 to about 25,000 copies. In some embodiments, the amount of the CD34 extracellular domain in the first group of hydrogel beads is about 5,000 to about 50,000 copies. In some embodiments, the amount of the CD34 extracellular domain in the first group of hydrogel beads is about 10,000 to about 100,000 copies. In some embodiments, the amount of the CD34 extracellular domain in the first group of hydrogel beads is about 25,000 to about 250,000 copies. In some embodiments, the amount of the CD34 extracellular domain in the first group of hydrogel beads is about 50,000 to about 500,000 copies. In some embodiments, the amount of the CD34 extracellular domain in the first group of hydrogel beads is about 100,000 to about 1,000,000 copies. In some embodiments, the amount of the CD34 extracellular domain in the first group of hydrogel beads is about 250,000 to about 2,500,000 copies. In some embodiments, the amount of the CD34 extracellular domain in the first group of hydrogel beads is about 500,000 to about 5,000,000 copies. In some implementations, the amount of CD34 extracellular domain in the first group of hydrogel beads is about 1,000,000 to about 10,000,000 copies.

[0241] In some embodiments, the amount of the CD34 extracellular domain in the second population of hydrogel beads does not exceed 3 copies, 10 copies, 30 copies, 100 copies, 300 copies, 1,000 copies, 3,000 copies, 10,000 copies, 30,000 copies, 100,000 copies, 300,000 copies, 1,000,000 copies, or 3,000,000 copies. In some embodiments, the amount of the CD34 extracellular domain in the second population of hydrogel beads does not exceed 3 copies. In some embodiments, the amount of the CD34 extracellular domain in the second population of hydrogel beads does not exceed 10 copies. In some embodiments, the amount of the CD34 extracellular domain in the second population of hydrogel beads does not exceed 30 copies. In some embodiments, the amount of the CD34 extracellular domain in the second population of hydrogel beads does not exceed 100 copies. In some embodiments, the amount of the CD34 extracellular domain in the second group of hydrogel beads does not exceed 300 copies. In some embodiments, the amount of the CD34 extracellular domain in the second group of hydrogel beads does not exceed 1,000 copies. In some embodiments, the amount of the CD34 extracellular domain in the second group of hydrogel beads does not exceed 3,000 copies. In some embodiments, the amount of the CD34 extracellular domain in the second group of hydrogel beads does not exceed 10,000 copies. In some embodiments, the amount of the CD34 extracellular domain in the second group of hydrogel beads does not exceed 30,000 copies. In some embodiments, the amount of the CD34 extracellular domain in the second group of hydrogel beads does not exceed 1,000,000 copies. In some implementations, the amount of CD34 extracellular domain in the second group of hydrogel beads does not exceed 3,000,000 copies.

[0242] In some embodiments, for each hydrogel bead in the first group of hydrogel beads, the amount of the CD34 extracellular domain in the first group of hydrogel beads is about 100 to about 1,000 copies. In some embodiments, for each hydrogel bead in the first group of hydrogel beads, the amount of the CD34 extracellular domain in the first group of hydrogel beads is about 250 to about 2,500 copies. In some embodiments, for each hydrogel bead in the first group of hydrogel beads, the amount of the CD34 extracellular domain in the first group of hydrogel beads is about 500 to about 5,000 copies. In some embodiments, for each hydrogel bead in the first group of hydrogel beads, the amount of the CD34 extracellular domain in the first group of hydrogel beads is about 1,000 to about 10,000 copies. In some embodiments, for each hydrogel bead in the first group of hydrogel beads, the amount of the CD34 extracellular domain in the first group of hydrogel beads is about 2,500 to about 25,000 copies. In some embodiments, for each hydrogel bead in the first group of hydrogel beads, the amount of the CD34 extracellular domain in the first group of hydrogel beads is about 5,000 to about 50,000 copies. In some embodiments, for each hydrogel bead in the first group of hydrogel beads, the amount of the CD34 extracellular domain in the first group of hydrogel beads is about 10,000 to about 100,000 copies. In some embodiments, for each hydrogel bead in the first group of hydrogel beads, the amount of the CD34 extracellular domain in the first group of hydrogel beads is about 25,000 to about 250,000 copies. In some embodiments, for each hydrogel bead in the first group of hydrogel beads, the amount of the CD34 extracellular domain in the first group of hydrogel beads is about 50,000 to about 500,000 copies. In some embodiments, for each hydrogel bead in the first group of hydrogel beads, the amount of the CD34 extracellular domain in the first group of hydrogel beads is about 100,000 to about 1,000,000 copies. In some embodiments, for each hydrogel bead in the first group of hydrogel beads, the amount of the CD34 extracellular domain in the first group of hydrogel beads is about 250,000 to about 2,500,000 copies. In some embodiments, for each hydrogel bead in the first group of hydrogel beads, the amount of the CD34 extracellular domain in the first group of hydrogel beads is about 500,000 to about 5,000,000 copies. In some embodiments, for each hydrogel bead in the first group of hydrogel beads, the amount of the CD34 extracellular domain in the first group of hydrogel beads is about 1,000,000 to about 10,000,000 copies.

[0243] In some embodiments, for each hydrogel bead in the second group of hydrogel beads, the amount of the CD34 extracellular domain in the second group of hydrogel beads does not exceed 3 copies, 10 copies, 30 copies, 100 copies, 300 copies, 1,000 copies, 3,000 copies, 10,000 copies, 30,000 copies, 100,000 copies, 300,000 copies, 1,000,000 copies, or 3,000,000 copies. In some embodiments, for each hydrogel bead in the second group of hydrogel beads, the amount of the CD34 extracellular domain in the second group of hydrogel beads does not exceed 3 copies. In some embodiments, for each hydrogel bead in the second group of hydrogel beads, the amount of the CD34 extracellular domain in the second group of hydrogel beads does not exceed 10 copies. In some embodiments, for each hydrogel bead in the second group of hydrogel beads, the amount of the CD34 extracellular domain in the second group of hydrogel beads does not exceed 30 copies. In some embodiments, for each hydrogel bead in the second group of hydrogel beads, the amount of the CD34 extracellular domain in the second group of hydrogel beads does not exceed 100 copies. In some embodiments, for each hydrogel bead in the second group of hydrogel beads, the amount of the CD34 extracellular domain in the second group of hydrogel beads does not exceed 300 copies. In some embodiments, for each hydrogel bead in the second group of hydrogel beads, the amount of the CD34 extracellular domain in the second group of hydrogel beads does not exceed 1,000 copies. In some embodiments, for each hydrogel bead in the second group of hydrogel beads, the amount of the CD34 extracellular domain in the second group of hydrogel beads does not exceed 3,000 copies. In some embodiments, for each hydrogel bead in the second group of hydrogel beads, the amount of the CD34 extracellular domain in the second group of hydrogel beads does not exceed 10,000 copies. In some embodiments, for each hydrogel bead in the second group of hydrogel beads, the amount of the CD34 extracellular domain in the second group of hydrogel beads does not exceed 30,000 copies. In some embodiments, for each hydrogel bead in the second group of hydrogel beads, the amount of the CD34 extracellular domain in the second group of hydrogel beads does not exceed 100,000 copies. In some embodiments, for each hydrogel bead in the second group of hydrogel beads, the amount of the CD34 extracellular domain in the second group of hydrogel beads does not exceed 300,000 copies. In some embodiments, for each hydrogel bead in the second group of hydrogel beads, the amount of the CD34 extracellular domain in the second group of hydrogel beads does not exceed 1,000,000 copies.In some implementations, for each hydrogel bead in the second population, the amount of the CD34 extracellular domain in the hydrogel beads does not exceed 3,000,000 copies.

[0244] In some embodiments, the hydrogel beads of this disclosure exhibit values ​​of 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 25000, 30000, 35000, 40000, 45000, 50000, 55000, 60000, 65000, 70000, 75000, 80000, 85000, 90000, 95000, 100000, 150000, 175000, 200000, 225000, 250000, 275000, 300000, and 325. 000, 350000, 375000, 400000, 425000, 450000, 475000, 500000, 525000, 550000, 575000, 600000, 625000, 650000, 675000, 700000, 725000, 750000, 775000, 800000, 825000, 850000, 875000, 900000, 925000, 950000, 975000, 1000000, 1500000, 2000000, 2500000, 3000000 or 3500000 The CD45 MFI of the MFI, including all ranges and subranges therein, as measured by any of the antibodies in Table 4.

[0245] In some embodiments, the hydrogel beads of this disclosure exhibit CD45 MFI of 300,000 to 1,000,000, 600,000 to 900,000, or 70,000 to 85,000, as measured by any of the antibodies in Table 4.

[0246] In some embodiments, for hydrogel beads, the amount of the CD45 extracellular domain in the first group of hydrogel beads and / or the second group of hydrogel beads is about 100 to about 300, about 300 to about 1,000, about 1,000 to about 3,000, about 3,000 to about 10,000, about 10,000 to about 30,000, about 30,000 to about 100,000, about 100,000 to about 300,000, about 300,000 to about 1,000,000, about 1,000,000 to about 3,000,000, about 3,000,000 to about 10,000,000, about 100 to about 1,000, about 300 to about 3,000. 0, about 1,000 to about 10,000, about 3,000 to about 30,000, about 10,000 to about 100,000, about 30,000 to about 300,000, about 100,000 to about 1,000,000, about 300,000 to about 3,000,000, about 1,000,000 to about 10,000,000, about 100 to about 3,000, about 300 to about 10,000, about 1,000 to about 30,000, about 3,000 to about 10,000, about 3,000 to about 100,000, about 10,000 to about 300,000, about 30,000 to about 1,000,000, about 100,000 to about 3,000,000, about 300 From approximately 10,000 to 10,000,000; from approximately 100 to 10,000; from approximately 300 to 30,000; from approximately 1,000 to 100,000; from approximately 3,000 to 300,000; from approximately 10,000 to 1,000,000; from approximately 30,000 to 3,000,000; from approximately 100,000 to 10,000,000; from approximately 100 to 30,000; from approximately 3,000 to 1,000,000; from approximately 10,000 to 3,000,000; from approximately 30,000 to 10,000,000; from approximately 100 to 100,000. 000, about 300 to about 300,000, about 1,000 to about 1,000,000, about 3,000 to about 3,000,000, about 10,000 to about 10,000,000, about 100 to about 300,000, about 300 to about 1,000,000, about 1,000 to about 3,000,000, about 3,000 to about 10,000,000, about 100 to about 1,000,000, about 300 to about 3,000,000, about 1,000 to about 10,000,000, about 100 to about 3,000,000, about 300 to about 10,000,000 or about 100 to about 10,000,000 copies.In some implementations, the amount of CD45 extracellular domain in each hydrogel bead of the first population is within this range.

[0247] In some embodiments, the amount of the CD45 extracellular domain in the first group of hydrogel beads and / or the second group of hydrogel beads is about 100 to about 1,000 copies. In some embodiments, the amount of the CD45 extracellular domain in the first group of hydrogel beads and / or the second group of hydrogel beads is about 250 to about 2,500 copies. In some embodiments, the amount of the CD45 extracellular domain in the first group of hydrogel beads and / or the second group of hydrogel beads is about 500 to about 5,000 copies. In some embodiments, the amount of the CD45 extracellular domain in the first group of hydrogel beads and / or the second group of hydrogel beads is about 1,000 to about 10,000 copies. In some embodiments, the amount of the CD45 extracellular domain in the first group of hydrogel beads and / or the second group of hydrogel beads is about 2,500 to about 25,000 copies. In some embodiments, the amount of the CD45 extracellular domain in the first group of hydrogel beads and / or the second group of hydrogel beads is about 5,000 to about 50,000 copies. In some embodiments, the amount of the CD45 extracellular domain in the first group of hydrogel beads and / or the second group of hydrogel beads is about 10,000 to about 100,000 copies. In some embodiments, the amount of the CD45 extracellular domain in the first group of hydrogel beads and / or the second group of hydrogel beads is about 25,000 to about 250,000 copies. In some embodiments, the amount of the CD45 extracellular domain in the first group of hydrogel beads and / or the second group of hydrogel beads is about 50,000 to about 500,000 copies. In some embodiments, the amount of the CD45 extracellular domain in the first group of hydrogel beads and / or the second group of hydrogel beads is about 100,000 to about 1,000,000 copies. In some embodiments, the amount of the CD45 extracellular domain in the first group of hydrogel beads and / or the second group of hydrogel beads is from about 250,000 to about 2,500,000 copies. In some embodiments, the amount of the CD45 extracellular domain in the first group of hydrogel beads and / or the second group of hydrogel beads is from about 500,000 to about 5,000,000 copies. In some embodiments, the amount of the CD45 extracellular domain in the first group of hydrogel beads and / or the second group of hydrogel beads is from about 1,000,000 to about 10,000,000 copies.

[0248] In some embodiments, for each hydrogel bead in the first group of hydrogel beads and / or the second group of hydrogel beads, the amount of the CD45 extracellular domain is about 100 to about 1,000 copies. In some embodiments, for each hydrogel bead in the first group of hydrogel beads and / or the second group of hydrogel beads, the amount of the CD45 extracellular domain is about 250 to about 2,500 copies. In some embodiments, for each hydrogel bead in the first group of hydrogel beads and / or the second group of hydrogel beads, the amount of the CD45 extracellular domain is about 500 to about 5,000 copies. In some embodiments, for each hydrogel bead in the first group of hydrogel beads and / or the second group of hydrogel beads, the amount of the CD45 extracellular domain is about 1,000 to about 10,000 copies. In some embodiments, for each hydrogel bead in the first group of hydrogel beads and / or the second group of hydrogel beads, the amount of the CD45 extracellular domain is about 2,500 to about 25,000 copies. In some embodiments, for each hydrogel bead in the first group of hydrogel beads and / or the second group of hydrogel beads, the amount of the CD45 extracellular domain is about 5,000 to about 50,000 copies. In some embodiments, for each hydrogel bead in the first group of hydrogel beads and / or the second group of hydrogel beads, the amount of the CD45 extracellular domain is about 10,000 to about 100,000 copies. In some embodiments, for each hydrogel bead in the first group of hydrogel beads and / or the second group of hydrogel beads, the amount of the CD45 extracellular domain is about 25,000 to about 250,000 copies. In some embodiments, for each hydrogel bead in the first group of hydrogel beads and / or the second group of hydrogel beads, the amount of the CD45 extracellular domain is from about 50,000 to about 500,000 copies. In some embodiments, for each hydrogel bead in the first group of hydrogel beads and / or the second group of hydrogel beads, the amount of the CD45 extracellular domain is from about 100,000 to about 1,000,000 copies. In some embodiments, for each hydrogel bead in the first group of hydrogel beads and / or the second group of hydrogel beads, the amount of the CD45 extracellular domain is from about 250,000 to about 2,500,000 copies. In some implementations, for each hydrogel bead in the first group of hydrogel beads and / or the second group of hydrogel beads, the amount of the CD45 extracellular domain is about 500,000 to about 5,000,000 copies.In some implementations, for each hydrogel bead in the first group of hydrogel beads and / or the second group of hydrogel beads, the amount of the CD45 extracellular domain is about 1,000,000 to about 10,000,000 copies.

[0249] In some embodiments, when comparatively labeled, the hydrogel beads in the first population exhibit about 10% to about 400% of the CD34 signal exhibited on the target cells. In some embodiments, when comparatively labeled, the hydrogel beads in the first population exhibit about 10% to about 400% of the CD45 signal exhibited by the target cells. In some embodiments, when comparatively labeled, the hydrogel beads in the first population exhibit approximately 10% to 50%, approximately 10% to 70%, approximately 10% to 100%, approximately 10% to 120%, approximately 10% to 150%, approximately 10% to 200%, approximately 10% to 250%, approximately 10% to 300%, approximately 10% to 400%, approximately 20% to 50%, approximately 20% to 70%, approximately 20% to 100%, approximately 20% to 120%, approximately 20% to 150%, approximately 20% to 200%, and approximately 20% to 200% of the CD34 and / or CD45 signaling exhibited by the target cells. 0% to approximately 250%, approximately 20% to approximately 300%, approximately 20% to approximately 400%, approximately 30% to approximately 50%, approximately 30% to approximately 70%, approximately 30% to approximately 100%, approximately 30% to approximately 120%, approximately 30% to approximately 150%, approximately 30% to approximately 200%, approximately 30% to approximately 250%, approximately 30% to approximately 300%, approximately 30% to approximately 400%, approximately 40% to approximately 50%, approximately 40% to approximately 70%, approximately 40% to approximately 100%, approximately 40% to approximately 120%, approximately 40% to approximately 150%, approximately 40% to approximately 200%, approximately 40% to approximately 250%, approximately 40% to approximately 300%, approximately 40% Approximately 400%, approximately 50% to approximately 70%, approximately 50% to approximately 100%, approximately 50% to approximately 120%, approximately 50% to approximately 150%, approximately 50% to approximately 200%, approximately 50% to approximately 250%, approximately 50% to approximately 300%, approximately 50% to approximately 400%, approximately 60% to approximately 70%, approximately 60% to approximately 100%, approximately 60% to approximately 120%, approximately 60% to approximately 150%, approximately 60% to approximately 200%, approximately 60% to approximately 250%, approximately 60% to approximately 300%, approximately 60% to approximately 400%, approximately 70% to approximately 100%, approximately 70% to approximately 120%, approximately 70% to approximately 150%, approximately 70% About 200%, about 70% to about 250%, about 70% to about 300%, about 70% to about 400%, about 80% to about 100%, about 80% to about 120%, about 80% to about 150%, about 80% to about 200%, about 80% to about 250%, about 80% to about 300%, about 80% to about 400%, about 90% to about 100%, about 90% to about 120%, about 90% to about 150%, about 90% to about 200%, about 90% to about 250%, about 90% to about 300%, about 90% to about 400%, about 100% to about 120%, about 100% to about 150%.Approximately 100% to approximately 200%, approximately 100% to approximately 250%, approximately 100% to approximately 300%, approximately 100% to approximately 400%, approximately 120% to approximately 150%, approximately 120% to approximately 200%, approximately 120% to approximately 250%, approximately 120% to approximately 300%, approximately 120% to approximately 400%, approximately 150% to approximately 200%, approximately 150% to approximately 250%, approximately 150% to approximately 300%, or approximately 150% to approximately 400%, including all ranges and subranges therein. In some embodiments, each hydrogel bead in the first group exhibits such CD34 and / or CD45 signals when comparablely labeled.

[0250] In some embodiments, this disclosure provides a second population of hydrogel beads, each comprising a cell surface biomarker profile containing a CD45 extracellular domain but at a lower level than that of the first population of hydrogel beads. In some embodiments, when comparablely labeled, the hydrogel beads in the second population contain no more than 10% of the CD34 signal exhibited by the hydrogel beads in the first population. In some embodiments, when comparablely labeled, the hydrogel beads in the second population exhibit no more than 0.1%, no more than 0.2%, no more than 0.3%, no more than 0.5%, no more than 0.7%, no more than 1%, no more than 2%, no more than 3%, no more than 5%, or no more than 7% of the CD34 signal exhibited by the hydrogel beads in the first population. In some embodiments, the cell surface biomarker profile of each hydrogel bead in the second population does not exhibit any CD34 signal above the background. In some embodiments, each hydrogel bead in the second population exhibits such a CD34 signal when comparablely labeled.

[0251] In some embodiments, when comparatively labeled, the hydrogel beads in the second population exhibit approximately 10% to approximately 400% of the CD45 signal expressed by the target cells. In some embodiments, when comparatively labeled, the hydrogel beads in the second population exhibit approximately 10% to approximately 50%, approximately 10% to approximately 70%, approximately 10% to approximately 100%, approximately 10% to approximately 120%, approximately 10% to approximately 150%, approximately 10% to approximately 200%, approximately 10% to approximately 250%, approximately 10% to approximately 300%, approximately 10% to approximately 400%, approximately 20% to approximately 50%, approximately 20% to approximately 70%, approximately 20% to approximately 100%, approximately 20% to approximately 120%, approximately 20% to approximately 150%, approximately 20% to approximately 200%, approximately 20% to approximately 250%, approximately 20% to approximately 300%, and approximately 20% of the CD45 signal expressed by the target cells. About 400%, about 30% to about 50%, about 30% to about 70%, about 30% to about 100%, about 30% to about 120%, about 30% to about 150%, about 30% to about 200%, about 30% to about 250%, about 30% to about 300%, about 30% to about 400%, about 40% to about 50%, about 40% to about 70%, about 40% to about 100%, about 40% to about 120%, about 40% to about 150%, about 40% to about 200%, about 40% to about 250%, about 40% to about 300%, about 40% to about 400%, about 50% to about 70%, about 50% to about 100%, about 50% to about 120%, about 50% to about 150%, about 50% to about 200%, about 50% to about 250%, about 50% to about 300%, about 50% to about 400%, about 60% to about 70%, about 60% to about 100%, about 60% to about 120%, about 60% to about 150%, about 60% to about 200%, about 60% to about 250%, about 60% to about 300%, about 60% to about 400%, about 70% to about 100%, about 70% to about 120%, about 70% to about 150%, about 70% to about 200%, about 70% to about 250%, about 70% to about 300%, about 70% to about 400%, about 80% to about 100%, about 80% to About 120%, about 80% to about 150%, about 80% to about 200%, about 80% to about 250%, about 80% to about 300%, about 80% to about 400%, about 90% to about 100%, about 90% to about 120%, about 90% to about 150%, about 90% to about 200%, about 90% to about 250%, about 90% to about 300%, about 90% to about 400%, about 100% to about 120%, about 100% to about 150%, about 100% to about 200%, about 100% to about 250%, about 100% to about 300%, about 100% to about 400%, about 120% to about 150%, about 120% to about 200%.Approximately 120% to approximately 250%, approximately 120% to approximately 300%, approximately 120% to approximately 400%, approximately 150% to approximately 200%, approximately 150% to approximately 250%, approximately 150% to approximately 300%, or approximately 150% to approximately 400%, including all ranges and subranges therein. In some embodiments, each hydrogel bead in the second population exhibits such a CD45 signal when comparablely labeled.

[0252] In some embodiments, when comparatively labeled, the hydrogel beads in the second population exhibit a lower CD34 signal than those exhibited by the hydrogel beads in the first population. In some embodiments, when comparatively labeled, the hydrogel beads in the second population exhibit no more than 10% of the CD34 signal exhibited by the target cells. In some embodiments, when comparatively labeled, the hydrogel beads in the second population exhibit no more than 50%, no more than 40%, no more than 30%, no more than 20%, or no more than 10% of the CD34 signal exhibited by the target cells. In some embodiments, when comparatively labeled, the hydrogel beads in the second population exhibit no more than 0.1%, no more than 0.2%, no more than 0.3%, no more than 0.5%, no more than 0.7%, no more than 1%, no more than 2%, no more than 3%, no more than 5%, or no more than 7% of the CD34 signal exhibited by the target cells.

[0253] In some embodiments, the extracellular CD34 and / or CD45 domains present on the cell surface of the target cells are the median amount of CD34 and / or CD45 domains present on the cell surface of cells in a leukocyte package containing CD34+ cells treated with Protocol H. In some embodiments, when comparatively labeled, the CD34 and / or CD45 signal exhibited by the target cells is the median amount of CD34 and / or CD45 signal exhibited by cells in a leukocyte package containing CD34+ cells treated with Protocol H. In another embodiment, this disclosure provides a means for determining the resolution and / or sensitivity of an intracellular protein quantification assay. In one embodiment, hydrogel beads are encapsulated with known amounts of protein at various concentrations and subsequently stained with a suitable protein antibody. The fluorescence of the various beads is measured to determine the sensitivity and / or dynamic range of the assay. The fluorescence values ​​can then be compared with values ​​obtained from cells in a sample to determine the presence of target cells and whether they contain intracellular proteins and the amount of protein.

[0254] In one embodiment, individual hydrogel beads are tuned to possess at least one optical property substantially similar to circulating tumor cells or fetal cells present in maternal blood. Each bead encapsulates a known amount of the biomolecule of interest. The beads are used to generate a standard curve for the assay of a specific cell type's biomolecule.

[0255] In some embodiments, the hydrogel particles are formed by polymerizing at least one bifunctional monomer, and after formation, the hydrogel particles include one or more functional groups that can be used for further attachment of cell surface markers, epitope-binding regions of cell surface markers, fluorescent dyes, or combinations thereof. In one embodiment, the free functional group is an amine group, a carboxyl group, a hydroxyl group, or a combination thereof. Depending on the desired functionalization, it should be understood that multiple bifunctional monomers can be used, for example, to functionalize the particles using different chemicals and different molecules.

[0256] Hydrogel particles can be functionalized with any fluorescent dye known in the art, including those listed in The MolecularProbes® Handbook - A Guide to Fluorescent Probes and Labeling Technologies, which is incorporated herein by reference in its entirety for all purposes. Functionalization can be mediated by compounds containing free amine groups, such as allylamine, which can be incorporated into bifunctional monomers used to form the hydrogel, as discussed above.

[0257] Non-limiting examples of known fluorescent dyes that can be used to functionalize the surface of the particles described herein include: 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein succinimide; 5-(6)-carboxyeosin; 5-carboxyfluorescein; 6-carboxyfluorescein; 5-(6)-carboxyfluorescein; S-carboxyfluorescein-bis-(5-carboxymethoxy-2-nitrobenzyl) ester, β-alanine-formamide or succinimide ester; 5-carboxyfluorescein succinimide; 6-carboxyfluorescein Succinimide ester; 5-(6)-carboxyfluorescein succinimide ester; 5-(4,6-dichlorotriazinyl)aminofluorescein; 2',7'-difluorofluorescein; Eosin-5-isothiocyanate; Erythrosin-5-isothiocyanate; 6-(fluorescein-5-formamide)hexanoic acid or succinimide ester; 6-(fluorescein-5-(6)-formamide)hexanoic acid or succinimide ester; fluorescein-S-EX succinimide ester; fluorescein-5-isothiocyanate; fluorescein-6-isothiocyanate; Oregon Oregon Green® 488 carboxylic acid or succinimide ester; Oregon Green® 488 isothiocyanate; Oregon Green® 488-X succinimide ester; Oregon Green® 500 carboxylic acid; Oregon Green® 500 carboxylic acid, succinimide ester, or triethylammonium salt; Oregon Green® 514 carboxylic acid; Oregon Green® 514 carboxylic acid or succinimide ester; Rhodamine Green™ carboxylic acid, succinimide ester, or hydrochloride; Rhodamine Green™ carboxylic acid, trifluoroacetamide, or succinimide ester; Rhodamine Green™-X succinimide ester or hydrochloride; RhodolGreen™ carboxylic acid, N,O-bis-(trifluoroacetyl) or succinimide ester; bis-(4-carboxypiperidinyl)sulfone rhodamine or bis(succinimide ester); 5-(6)-carboxynaphthalene fluorescein, 5-(6)-carboxynaphthalene fluorescein succinimide ester; 5-carboxyrhodamine 6G hydrochloride; 6-carboxyrhodamine 6G hydrochloride, 5-carboxyrhodamine 6G succinimide ester; 6-carboxyrhodamine 6G succinimide ester; 5-(6)-carboxyrhodamine 6G succinimide ester;5-Carboxy-2',4',5',7'-Tetrabromosulfone fluorescein succinimide ester or bis-(diisopropylethylammonium) salt; 5-Carboxytetramethylrhodamine; 6-Carboxytetramethylrhodamine; 5-(6)-Carboxytetramethylrhodamine; 5-Carboxytetramethylrhodamine succinimide ester; 6-Carboxytetramethylrhodamine succinimide ester; 5-(6)-Carboxytetramethylrhodamine succinimide ester ; 6-Carboxy-X-Rhodamine; 5-Carboxy-X-Rhodamine succinimide ester; 6-Carboxy-X-Rhodamine succinimide ester; 5-(6)-Carboxy-X-Rhodamine succinimide ester; 5-Carboxy-X-Rhodamine triethylammonium salt; Lissamine™ Rhodamine B sulfonyl chloride; Malachite green; Isothiocyanate; NANOGOLD® mono(sulfosuccinimide ester); QSY® 21. Carboxylic acid or succinimide ester; QSY® 7. Carboxylic acid or succinimide ester; Rhodamine Red™-X succinimide ester; 6-(tetramethylrhodamine-5-(6)-formamide)hexanoic acid; succinimide ester; tetramethylrhodamine-5-isothiocyanate; tetramethylrhodamine-6-isothiocyanate; tetramethylrhodamine-5-(and-6)-isothiocyanate; Texas Red® sulfonyl; Texas Red® sulfonyl chloride; Texas Red®-X STP ester or sodium salt; Texas Red®-X succinimide ester; Texas Red®-X succinimide ester; and X-rhodamine-5-(6)isothiocyanate.

[0258] Other examples of fluorescent dyes that can be used with the particles described herein include, but are not limited to, BODIPY® dyes commercially available from Invitrogen, including, but not limited to, BODIPY® FL; BODIPY® TMR STP ester; BODIPY® TR-X STP ester; BODIPY® 630 / 650-X STP ester; and BODIPY® 650 / 665-X. STP ester; 6-dibromo-4,4-difluoro-5,7-dimethyl-4-boron-3a,4a-diaza-s-indaminon-3-propionic acid or succinimide ester; 4,4-difluoro-4-boron-3a,4a-diaza-s-indaminon-3,5-dipropionic acid; 4,4-difluoro-5,7-dimethyl-4-boron-3a,4a-diaza-s-indaminon-3-pentanoic acid; 4,4-difluoro-5,7-dimethyl-4-boron-3a,4a-diaza-s-indaminon-3-pentanoic acid or succinimide ester; 4,4-difluoro-5,7-dimethyl-4-boron-3a,4a- Diaza-S-indaminozide-3-propionic acid; 4,4-difluoro-5,7-dimethyl-4-boron-3a,4a-diaza-S-indaminozide-3-propionic acid succinimide ester; 4,4-difluoro-5,7-dimethyl-4-boron-3a,4a-diaza-S-indaminozide-3-propionic acid; sulfosuccinimide ester or sodium salt; 6-((4,4-difluoro-5,7-dimethyl-4-boron-3a,4a-diaza-S-indaminozide-3-propionic acid)amino)hexanoic acid; 6-((4,4-difluoro-5,7-dimethyl-4-boron-3a,4a-diaza-S-indaminozide-3-propionic acid) (4,4-difluoro-5,7-dimethyl-4-boron-3a,4a-diaza-s-indaminone-3-propionyl)cysteine, succinimide, or triethylammonium salt; 6-4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-boron-3a,4a-4,4-difluoro-5,7-diphenyl-4-boron-3a,4a-diaza-s-indaminone-3-propionic acid; 4,4-difluoro-5,7-diphenyl-4-boron-3a,4a-diaza-s-indaminone-3-propionic acid or succinimide ester; 4 4-Difluoro-5-phenyl-4-boron-3a,4a-diaza-s-indaminone-3-propionic acid or succinimide ester; 6-((4,4-difluoro-5-phenyl-4-boron-3a,4a-diaza-s-indaminone-3-propionyl)amino)hexanoic acid or succinimide ester; 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-boron-3a,4a-diaza-s-indaminone-3-propionic acid or succinimide ester; 4,4-difluoro-5-(2-pyrrole)-4-boron-3a,4a-diaza-s-indaminone-3-propionic acid or succinimide ester;6-(((4,4-difluoro-5-(2-pyrrolidinyl)-4-boron-3a,4a-diaza-s-indane-3-yl)styryloxy)acetyl)aminohexanoic acid or succinimide ester; 4,4-difluoro-5-styryl-4-boron-3a,4a-diaza-s-indane-3-propionic acid; 4,4-difluoro-5-styryl-4-boron-3a,4a-diaza-s-indane-3-propionic acid or succinimide ester; 4,4-difluoro-1,3,5,7-tetramethyl-4-boron-3a,4a-diaza-s-indane-8-propionic acid; 4,4-difluoro-1,3,5,7-tetramethyl-4-boron-3a,4a-diaza-s-indane-8-propionic acid; 4,4-difluoro-1,3,5,7-tetramethyl-4-boron -3a,4a-diaza-s-indaminozide-8-propionic acid or succinimide ester; 4,4-difluoro-5-(2-thienyl)-4-boron-3a,4a-diaza-s-indaminozide-3-propionic acid or succinimide ester; 6-(((4-(4,4-difluoro-5-(2-thienyl)-4-boron-3a,4a-diaza-s-indaminozide-3-yl)phenoxy)acetyl)amino)hexanoic acid or succinimide ester; and 6-(((4,4-difluoro-5-(2-thienyl)-4-boron-3a,4a-diaza-s-indaminozide-3-yl)styryloxy)acetyl)aminohexanoic acid or succinimide ester. ;

[0259] In one embodiment, the fluorescent dye used for deriving the surface of one or more hydrogel particles includes, but is not limited to, Alexa fluorescent dyes commercially available from Invitrogen, including, but not limited to, Alexa Fluor® 350 carboxylic acid; Alexa Fluor® 430 carboxylic acid; Alexa Fluor® 488 carboxylic acid; Alexa Fluor® 532 carboxylic acid; Alexa Fluor® 546 carboxylic acid; Alexa Fluor® 555 carboxylic acid; Alexa Fluor® 568 carboxylic acid; Alexa Fluor® 594 carboxylic acid; Alexa Fluor® 633 carboxylic acid; Alexa Fluor® 647 carboxylic acid; Alexa Fluor® 660 carboxylic acid; and Alexa Fluor® 680 carboxylic acid. In another embodiment, the fluorescent dye used with the hydrogel particles and methods described herein includes cyanine dyes commercially available from Amersham-Pharmacia Biotech, including, but not limited to, Cy3 NHS esters; Cy5 NHS esters; Cy5.5 NHS esters; and Cy7 NHS esters.

[0260] Selecting one or more suitable dyes based on the desired spectral excitation and emission properties of hydrogel particles is within the scope of common techniques in the art.

[0261] Composition comprising hydrogel beads of first group and hydrogel beads of second group

[0262] In some embodiments, this disclosure provides a composition comprising both the first group of hydrogel beads and the second group of hydrogel beads of this disclosure.

[0263] In some embodiments, the active portion of the composition further comprises a third group of hydrogel beads (e.g., those containing a third cell surface biomarker but lacking any CD45 or CD34 extracellular domains). In some other embodiments, the active portion of the composition consists of a first group of hydrogel beads and a second group of hydrogel beads as disclosed herein.

[0264] In some embodiments, the first group of hydrogel beads and the second group of hydrogel beads together constitute at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% of the number of hydrogel beads in the active portion of the composition. In some embodiments, a small fraction of the hydrogel beads may not be labeled with the target biomarker profile during the manufacturing process and / or long-term storage (e.g., the amount of biomarker falls outside the specified range). This is one of the reasons why, in some embodiments, the first group of hydrogel beads and the second group of hydrogel beads together may comprise a fraction smaller than the entire active portion of the composition. In some embodiments, the first group of hydrogel beads and the second group of hydrogel beads together may constitute about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 95% to about 97%, about 97% to about 98%, about 98% to about 99%, or about 99% to 100% of the active portion of the composition.

[0265] Hydrogel beads as cell mimics of cell populations without CD34 enrichment

[0266] In some embodiments, the ratio of the number of hydrogel beads in the first group to the number of hydrogel beads in the second group does not exceed 1, 0.5, 0.3, 0.2, 0.1, 0.07, 0.05, 0.03, 0.02, 0.018, 0.017, 0.016, 0.015, 0.01, 0.005, or 0.001. In some embodiments, the ratio of the number of hydrogel beads in the first group to the number of hydrogel beads in the second group is not greater than 0.0183.

[0267] In some embodiments, the ratio of the number of hydrogel beads in the first group to the number of hydrogel beads in the second group is about 1 to about 0.5, about 0.5 to about 0.2, about 0.2 to about 0.1, about 0.1 to about 0.05, about 0.05 to about 0.02, about 0.02 to about 0.015, about 0.015 to about 0.01, about 0.01 to about 0.005, about 0.005 to about 0.002, or about 0.002 to about 0.001, including all ranges and subranges therein. In some embodiments, the ratio of the number of hydrogel beads in the first group to the number of hydrogel beads in the second group is about 0.0183 to about 0.0142.

[0268] In some embodiments, the first group of hydrogel beads constitutes 0.1%-30%, 0.2%-15%, 0.3%-10%, 0.5%-6%, 1%-3%, or 1.2%-2% of the total number of hydrogel beads in the active portion of the composition, including all ranges and subranges therein. In some embodiments, the first group of hydrogel beads constitutes 1%-3% of the total number of hydrogel beads in the active portion of the composition. In some embodiments, the second group of hydrogel beads constitutes 40%-99.9%, 50%-99%, 60%-99%, 70%-99%, 80%-99%, 90%-99%, or 95%-99% of the total number of hydrogel beads in the active portion of the composition, including all ranges and subranges therein. In some embodiments, the second group of hydrogel beads constitutes 97%-99% of the total number of hydrogel beads in the active portion of the composition. In some embodiments, the first group of hydrogel beads constitutes 1%-3% of the total number of hydrogel beads in the active portion of the composition, and the second group of hydrogel beads constitutes 97%-99% of the total number of hydrogel beads in the active portion of the composition. In some embodiments, the first group of hydrogel beads constitutes 1.4%-1.8% of the total number of hydrogel beads in the active portion of the composition, and the second group of hydrogel beads constitutes 98.2%-98.6% of the total number of hydrogel beads in the active portion of the composition. In some embodiments, the combination of the first group of hydrogel beads and the second group of hydrogel beads together constitutes at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% of the total number of hydrogel beads in the active portion of the composition.

[0269] Hydrogel beads as cell mimics of CD34-enriched cell populations

[0270] In some embodiments, the ratio of the number of hydrogel beads in the first group to the number of hydrogel beads in the second group is at least 1, at least 2, at least 5, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 50, at least 70, or at least 100. In some embodiments, the ratio of the number of hydrogel beads in the first group to the number of hydrogel beads in the second group is at least 9.

[0271] In some embodiments, the ratio of the number of hydrogel beads in the first group to the number of hydrogel beads in the second group is about 1 to about 2, about 2 to about 5, about 5 to about 8, about 8 to about 10, about 10 to about 12, about 12 to about 15, about 15 to about 20, about 20 to about 50, about 50 to about 100, including all ranges and subranges therein. In some embodiments, the ratio of the number of hydrogel beads in the first group to the number of hydrogel beads in the second group is about 9 to about 19.

[0272] In some embodiments, the first group comprises 10%-99%, 20%-99%, 30%-99%, 40%-99%, 50%-99%, 60%-99%, 70%-99%, 80%-99%, or 90%-99% of the number of hydrogel beads in the active portion of the composition, including all ranges and subranges therein. In some embodiments, the first group comprises 70%-95%, 80%-95%, or 90%-95% of the number of hydrogel beads in the active portion of the composition, including all ranges and subranges therein. In some embodiments, the first group comprises 80%-95% of the number of hydrogel beads in the active portion of the composition. In some embodiments, the second group comprises 1%-90%, 1%-80%, 1%-70%, 1%-60%, 1%-50%, 1%-40%, 1%-30%, 1%-20%, 1%-10%, or 1%-5% of the number of hydrogel beads in the active portion of the composition, including all ranges and subranges therein. In some embodiments, the second group comprises 5%-90%, 5%-70%, 5%-50%, 5%-30%, or 5%-20% of the number of hydrogel beads in the active portion of the composition, including all ranges and subranges therein. In some embodiments, the second group comprises 5%-20% of the number of hydrogel beads in the active portion of the composition. In some embodiments, the second group comprises 5%-10% of the number of hydrogel beads in the active portion of the composition. In some embodiments, the first group comprises 80%-95% of the number of hydrogel beads in the active portion of the composition, and the second group comprises 5%-20% of the number of hydrogel beads in the active portion of the composition. In some embodiments, the first group comprises 90%-95% of the number of hydrogel beads in the active portion of the composition, and the second group comprises 5%-10% of the number of hydrogel beads in the active portion of the composition. In some embodiments, the combination of the first group of hydrogel beads and the second group of hydrogel beads together comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% of the number of hydrogel beads in the active portion of the composition.

[0273] Kits and Compositions

[0274] In some aspects, this disclosure provides kits comprising one or more quantitative hydrogel particles as disclosed herein. In some embodiments, the kit includes instructions / reagents for linking such molecules as needed.

[0275] In some aspects, the kit comprises at least two compositions: a first composition containing hydrogel beads as a cell mimic for a cell population without CD34 enrichment, and a second composition containing hydrogel beads as a cell mimic for a CD34-enriched cell population, as described above. In some embodiments, the two compositions are stored separately in separate containers of the kit, namely a first container and a second container. In some embodiments, the first container (containing cell mimics for a cell population without CD34 enrichment) contains approximately 5 × 10⁻⁶ cells / mL. 5 Approximately 2×10 6 Each bead contains approximately 2.5 × 10⁻⁶ beads, and the second container (containing cell mimics for CD34-enriched cell populations) contains approximately 2.5 × 10⁻⁶ beads. 5 Each bead. In some embodiments, the ratio of the total number of hydrogel beads of the first group and the second group in (b) the first container to the total number of hydrogel beads of the first group and the second group in (b) the second container is about 1 to about 10, about 2 to about 8, about 3 to about 6, or about 4, including all ranges and subranges therein.

[0276] method

[0277] In some aspects, this disclosure provides a method for calibrating an apparatus for detecting target cells in a cell population, comprising sampling a composition or kit of this disclosure and calibrating the apparatus based on at least one property of the hydrogel beads in the composition or kit. In some embodiments, the apparatus is a cytometer. In some embodiments, the target cells are cells expressing CD34. In some embodiments, the composition comprises both a first population and a second population of hydrogel beads of this disclosure. In some embodiments, sampling comprises sampling at least two different compositions of this disclosure, wherein these different compositions contain different ratios of first population and second population hydrogel beads. In some embodiments, calibration is based on at least one optical property of the hydrogel beads.

[0278] In some embodiments, this disclosure provides a method for enriching target cells, comprising sampling a composition or kit of this disclosure, forming a selection scheme based on at least one property of the hydrogel beads of the composition or kit, and selecting target cells from a cell population based on the selection scheme. In some embodiments, the method uses a cytometer to enrich the target cells. In some embodiments, the selection scheme includes a gating scheme. In some embodiments, the composition comprises both a first population and a second population of hydrogel beads of this disclosure. In some embodiments, sampling comprises sampling at least two different compositions of this disclosure, wherein these different compositions contain different ratios of the first population and the second population of hydrogel beads. In some embodiments, the target cells comprise cells expressing CD34. In some embodiments, at least one property comprises at least one optical property of the hydrogel beads.

[0279] In some embodiments, the method includes sampling a cell population and obtaining cells that possess at least one property of the cells (e.g., optical properties). In some embodiments, the method includes forming a gating scheme based on at least one property of the cells (e.g., optical properties).

[0280] In some embodiments, at least one optical property includes the fluorescence intensity of FSC, SSC, CD45, CD34, or any combination thereof. In some embodiments, at least one optical property includes at least two, at least three, or all of the optical properties of fluorescence intensity of FSC, SSC, CD45, and CD34. In some embodiments, at least one optical property comprises the fluorescence intensity of CD45 and the fluorescence intensity of CD34.

[0281] In some embodiments, cells expressing CD34 express reduced amounts of CD45. In some embodiments, the cells expressing CD34 are CD34-expressing stem cells.

[0282] Further numbered implementation plan

[0283] Further numbered embodiments of the present invention are provided below:

[0284] Implementation Scheme 1. A composition comprising a first group of hydrogel beads, the hydrogel beads comprising:

[0285] a) Polymer monomers and bifunctional monomers; and

[0286] b) A spectrum of cell surface biomarkers, which includes:

[0287] i) CD34 and CD45 extracellular domains.

[0288] Implementation Scheme 2. The composition of Implementation Scheme 1, comprising a second group of hydrogel beads, the hydrogel beads comprising:

[0289] c) Polymer monomers and bifunctional monomers; and

[0290] d) Cell surface biomarker profile, which includes:

[0291] i) CD45 extracellular domain, but lacks CD34 extracellular domain.

[0292] Implementation Scheme 3. The composition of Implementation Scheme 1, comprising a second group of hydrogel beads, the hydrogel beads comprising:

[0293] c) Polymer monomers and bifunctional monomers; and

[0294] d) Cell surface biomarker profile, which includes:

[0295] i) CD45 extracellular domain

[0296] The hydrogel beads in the second group comprise no more than 10% of the median number of CD34 extracellular domains contained in the hydrogel beads of the first group.

[0297] Implementation Scheme 3.1. The composition of Implementation Scheme 1, comprising a second group of hydrogel beads, the hydrogel beads comprising: c) a polymeric monomer and a bifunctional monomer; and d) a cell surface biomarker profile comprising: i) a CD45 extracellular domain, wherein each hydrogel bead in the second group comprises no more than 10% of the median number of CD34 extracellular domains contained in the hydrogel beads of the first group.

[0298] Implementation Scheme 4. The composition as described in Implementation Scheme 3 or Implementation Scheme 3.1, wherein the hydrogel beads in the second group comprise a median number of CD34 extracellular domains contained in the hydrogel beads of the first group of no more than 0.1%, no more than 0.2%, no more than 0.3%, no more than 0.5%, no more than 0.7%, no more than 1%, no more than 2%, no more than 3%, no more than 5%, or no more than 7%.

[0299] Implementation Scheme 4.1. The composition as described in Implementation Scheme 3 or Implementation Scheme 3.1, wherein each hydrogel bead in the second group comprises no more than 0.1%, no more than 0.2%, no more than 0.3%, no more than 0.5%, no more than 0.7%, no more than 1%, no more than 2%, no more than 3%, no more than 5%, or no more than 7% of the median number of CD34 extracellular domains contained in the hydrogel beads of the first group.

[0300] Implementation Scheme 5. The composition of any one of Implementation Schemes 1 to 4.1, wherein the hydrogel beads in the first group comprise about 10% to about 400% of the amount of the CD45 extracellular domain present on the cell surface of the target cells.

[0301] Implementation Scheme 5.1. The composition of any one of Implementation Schemes 1 to 4.1, wherein each of the hydrogel beads in the first group comprises about 10% to about 400% of the amount of the CD45 extracellular domain present on the cell surface of the target cell.

[0302] Implementation Scheme 6. The composition of Implementation Scheme 5, wherein the hydrogel beads in the first group comprise about 10% to about 300%, about 20% to about 400%, about 20% to about 300%, about 20% to about 200%, or about 50% to about 200% of the amount of the CD45 extracellular domain present on the cell surface of the target cells.

[0303] Implementation Scheme 6.1. The composition of Implementation Scheme 5.1, wherein each of the hydrogel beads in the first group contains about 10% to about 300%, about 20% to about 400%, about 20% to about 300%, about 20% to about 200%, or about 50% to about 200% of the amount of the extracellular domain of CD45 present on the cell surface of the target cell.

[0304] Implementation Scheme 7. The composition of any one of Implementation Schemes 1 to 6.1, wherein the hydrogel beads in the first group comprise about 10% to about 400% of the amount of the CD34 extracellular domain present on the cell surface of the target cells.

[0305] Implementation Scheme 7.1. The composition of any one of Implementation Schemes 1 to 6.1, wherein each of the hydrogel beads in the first group comprises about 10% to about 400% of the amount of the extracellular domain of CD34 present on the cell surface of the target cell.

[0306] Implementation Scheme 8. The composition of Implementation Scheme 7, wherein the hydrogel beads in the first group comprise about 10% to about 300%, about 20% to about 400%, about 20% to about 300%, about 20% to about 200%, or about 50% to about 200% of the amount of the extracellular domain of CD34 present on the cell surface of the target cells.

[0307] Implementation Scheme 8.1. The composition of Implementation Scheme 7.1, wherein each of the hydrogel beads in the first group contains about 10% to about 300%, about 20% to about 400%, about 20% to about 300%, about 20% to about 200%, or about 50% to about 200% of the amount of the extracellular domain of CD34 present on the cell surface of the target cell.

[0308] Implementation Scheme 9. The composition of any one of Implementation Schemes 2 to 8.1, wherein the hydrogel beads in the second population comprise about 10% to about 400% of the amount of the CD45 extracellular domain present on the cell surface of the target cells.

[0309] Implementation Scheme 9.1. The composition of any one of Implementation Schemes 2 to 8.1, wherein each hydrogel bead in the second group comprises about 10% to about 400% of the amount of the CD45 extracellular domain present on the cell surface of the target cell.

[0310] Implementation Scheme 10. The composition of Implementation Scheme 9, wherein the hydrogel beads in the second group comprise about 10% to about 300%, about 20% to about 400%, about 20% to about 300%, about 20% to about 200%, or about 50% to about 200% of the amount of the extracellular domain of CD45 present on the cell surface of the target cells.

[0311] Implementation Scheme 10.1. The composition of Implementation Scheme 9.1, wherein each of the hydrogel beads in the second group contains about 10% to about 300%, about 20% to about 400%, about 20% to about 300%, about 20% to about 200%, or about 50% to about 200% of the amount of the extracellular domain of CD45 present on the cell surface of the target cell.

[0312] Implementation Scheme 11. The composition of any one of Implementation Schemes 2 to 10.1, wherein the hydrogel beads in the second group contain no more than 10% of the amount of the CD34 extracellular domain present on the cell surface of the target cells.

[0313] Implementation Scheme 11.1. The composition of any one of Implementation Schemes 2 to 10.1, wherein each of the hydrogel beads in the second group contains no more than 10% of the amount of the CD34 extracellular domain present on the cell surface of the target cell.

[0314] Implementation Scheme 12. The composition of Implementation Scheme 11, wherein the hydrogel beads in the second population contain an amount of CD34 extracellular domain present on the cell surface of the target cells not exceeding 0.1%, not exceeding 0.2%, not exceeding 0.3%, not exceeding 0.5%, not exceeding 0.7%, not exceeding 1%, not exceeding 2%, not exceeding 3%, not exceeding 5%, or not exceeding 7%.

[0315] Implementation Scheme 12.1. The composition as described in Implementation Scheme 11.1, wherein each of the hydrogel beads in the second group contains an amount of CD34 extracellular domain present on the cell surface of the target cell not exceeding 0.1%, not exceeding 0.2%, not exceeding 0.3%, not exceeding 0.5%, not exceeding 0.7%, not exceeding 1%, not exceeding 2%, not exceeding 3%, not exceeding 5%, or not exceeding 7%.

[0316] Implementation Scheme 13. The composition of any one of Implementation Schemes 5 to 12.1, wherein the amount of CD34 and / or CD45 extracellular domains present on the cell surface of the target cells is the median amount of CD34 and / or CD45 extracellular domains present on the cell surface of cells in a leukocyte package of CD34+ enriched cells treated with Scheme H.

[0317] Implementation Scheme 14. The composition of any one of Implementation Schemes 5 to 13, wherein the target cell is a hematopoietic stem cell.

[0318] Implementation Scheme 15. The composition of any one of Implementation Schemes 5 to 14, wherein the target cells are CD45dim-positive (CD45dim+) and CD34-positive (CD34+) stem cells.

[0319] Implementation Scheme 16. The composition of any one of Implementation Schemes 5 to 15, wherein the target cell is a lymphocyte.

[0320] Implementation Scheme 17. The composition of any one of Implementation Schemes 5 to 16, wherein flow cytometry is used to measure the amount of the extracellular domains of CD45 and / or CD34 present in the hydrogel and / or on the cell surface based on fluorescence intensity.

[0321] Implementation Scheme 18. The composition of Implementation Scheme 17, wherein the fluorescence intensity of the CD45 extracellular domain is measured using a fluorophore-labeled CD45-specific binding molecule, and / or wherein the fluorescence intensity of the CD34 extracellular domain is measured using a fluorophore-labeled CD34-specific binding molecule.

[0322] Implementation Scheme 19. The composition of Implementation Scheme 18, wherein the binding molecule comprises a monoclonal antibody or an antigen-binding fragment thereof.

[0323] Implementation Scheme 20. The composition of any one of Implementation Schemes 18 to 19, wherein the CD34-specific binding molecule is selected from: phycoerythrin (PE)-labeled anti-CD34 antibody clone 8G12, phycoerythrin (PE)-labeled anti-CD34 antibody clone AC136, allophycocyanin (APC)-labeled anti-CD34 antibody clone 4H11, and Brilliant™ Violet 421 (BV421)-labeled anti-CD34 antibody clone 581.

[0324] Implementation Scheme 21. The composition of any one of Implementation Schemes 18 to 20, wherein the CD45-specific binding molecule is selected from: FITC-labeled anti-CD45 antibody clone 2D1, PerCP-Cyanine® 5.5 (PerCP-Cy5.5)-labeled anti-CD45 antibody clone 2D1, PE-labeled anti-CD45 antibody clone MEM-28, and BD Horizon™ V500-labeled anti-CD45 antibody clone HI30.

[0325] Implementation Scheme 22. The composition of any one of Implementation Schemes 18 to 21, wherein the combination of the CD34-specific binding molecule and the CD45-specific binding molecule is selected from one group of Table 4; optionally, the combination is group 1 of Table 4.

[0326] Implementation Scheme 23. The composition of any one of Implementation Schemes 1 to 22, wherein the biomarker is connected to the matrix of the hydrogel beads via a connector.

[0327] Implementation Scheme 24. The composition of any one of Implementation Schemes 1 to 23, wherein the biomarker is covalently linked to the matrix of the hydrogel beads.

[0328] Implementation Scheme 25. The composition of any one of Implementation Schemes 1 to 23, wherein the biomarker is non-covalently linked to the matrix of the hydrogel beads.

[0329] Implementation Scheme 26. The composition of any one of Implementation Schemes 1 to 25, wherein the hydrogel beads of the first group and / or the second group have an average diameter of about 1 μm to about 40 μm, about 20 μm to about 30 μm, about 3 μm to about 20 μm, or about 4-10 μm.

[0330] Implementation Scheme 27. The composition of any one of Implementation Schemes 2 to 26, wherein the active portion of the composition comprises or consists of the first group of hydrogel beads and the second group of hydrogel beads.

[0331] Implementation Scheme 28. The composition of Implementation Scheme 27, wherein the first group comprises 1%-3% of the number of hydrogel beads in the active portion of the composition.

[0332] Implementation Scheme 29. The composition of any one of Implementation Schemes 27 to 28, wherein the second group comprises 50% to 99% of the number of hydrogel beads in the active portion of the composition; wherein any remaining portion of the active portion of the composition comprises hydrogel beads lacking CD34 and CD45.

[0333] Implementation Scheme 30. The composition as described in Implementation Scheme 27 or 28, wherein the second group comprises 97%-99% of the number of hydrogel beads in the active portion of the composition; wherein any remaining portion of the active portion of the composition comprises hydrogel beads lacking CD34 and CD45.

[0334] Implementation Scheme 31. The composition as described in Implementation Scheme 27 or 28, wherein the first group comprises 1%-3% of the number of hydrogel beads in the active portion of the composition; and wherein the second group comprises 97%-99% of the number of hydrogel beads in the active portion of the composition.

[0335] Implementation Scheme 32. The composition of Implementation Scheme 27 or 28, wherein the first group comprises 1.4%-1.8% of the number of hydrogel beads in the active portion of the composition; and wherein the second group comprises 98.2%-98.6% of the number of hydrogel beads in the active portion of the composition.

[0336] Implementation Scheme 33. The composition of any one of Implementation Schemes 27 to 32, wherein the first group of hydrogel beads and the second group of hydrogel beads together account for at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% of the number of hydrogel beads in the active portion of the composition.

[0337] Implementation Scheme 34. The composition of any one of Implementation Schemes 2 to 33, wherein the ratio of the number of hydrogel beads of the first group to the number of hydrogel beads of the second group is not more than 1, not more than 0.5, not more than 0.3, not more than 0.2, not more than 0.1, not more than 0.07, not more than 0.05, not more than 0.03, not more than 0.02, not more than 0.018, not more than 0.017, not more than 0.016, not more than 0.015, not more than 0.01, not more than 0.005, or not more than 0.001.

[0338] Implementation Scheme 35. The composition of Implementation Scheme 34, wherein the ratio of the number of hydrogel beads in the first group to the number of hydrogel beads in the second group is not greater than 0.0183.

[0339] Implementation Scheme 36. The composition of any one of Implementation Schemes 2 to 35, wherein the ratio of the number of hydrogel beads of the first group to the number of hydrogel beads of the second group is about 1 to about 0.5, about 0.5 to about 0.2, about 0.2 to about 0.1, about 0.1 to about 0.05, about 0.05 to about 0.02, about 0.02 to about 0.015, about 0.015 to about 0.01, about 0.01 to about 0.005, about 0.005 to about 0.002, or about 0.002 to about 0.001, including all ranges and subranges therein.

[0340] Embodiment 37. The composition of Embodiment 36, wherein the ratio of the number of hydrogel beads in the first group to the number of hydrogel beads in the second group is from about 0.0183 to about 0.0142.

[0341] Implementation Scheme 38. The composition of Implementation Scheme 27, wherein the first group comprises 10%-95% of the number of hydrogel beads in the active portion of the composition.

[0342] Implementation Scheme 39. The composition of Implementation Scheme 27, wherein the first group comprises 80%-95% of the number of hydrogel beads in the active portion of the composition.

[0343] Implementation Scheme 40. The composition of any one of Implementation Schemes 27 and 38 to 39, wherein the second group comprises 5% to 90% of the number of hydrogel beads in the active portion of the composition; wherein any remaining portion of the active portion of the composition comprises hydrogel beads lacking CD34 or CD45.

[0344] Implementation Scheme 41. The composition of any one of Implementation Schemes 27 and 38 to 39, wherein the second group comprises 5% to 20% of the number of hydrogel beads in the active portion of the composition.

[0345] Implementation Scheme 42. The composition of Implementation Scheme 27, wherein the first group comprises 80%-95% of the number of hydrogel beads in the active portion of the composition; and wherein the second group comprises 5%-20% of the number of hydrogel beads in the active portion of the composition.

[0346] Implementation Scheme 43. The composition of Implementation Scheme 27, wherein the first group comprises 90%-95% of the number of hydrogel beads in the active portion of the composition; and wherein the second group comprises 5%-10% of the number of hydrogel beads in the active portion of the composition.

[0347] Implementation Scheme 44. The composition of any one of Implementation Schemes 27 and 38 to 43, wherein the first group of hydrogel beads and the second group of hydrogel beads together constitute at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% of the number of hydrogel beads in the active portion of the composition.

[0348] Implementation Scheme 45. The composition of any one of Implementation Schemes 2 to 27 and 38 to 44, wherein the ratio of the number of hydrogel beads of the first group to the number of hydrogel beads of the second group is at least 1, at least 2, at least 5, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 50, at least 70, or at least 100.

[0349] Implementation Scheme 46. The composition of Implementation Scheme 45, wherein the ratio of the number of hydrogel beads in the first group to the number of hydrogel beads in the second group is at least 9.

[0350] Implementation Scheme 47. The composition of any one of Implementation Schemes 2 to 27 and 38 to 46, wherein the ratio of the number of hydrogel beads of the first group to the number of hydrogel beads of the second group is about 1 to about 2, about 2 to about 5, about 5 to about 8, about 8 to about 10, about 10 to about 12, about 12 to about 15, about 15 to about 20, about 20 to about 50, about 50 to about 100, including all ranges and subranges therein.

[0351] Embodiment 48. The composition of Embodiment 47, wherein the ratio of the number of hydrogel beads of the first group to the number of hydrogel beads of the second group is about 9 to about 19.

[0352] Implementation Scheme 49. A reagent kit comprising:

[0353] A first container comprising the composition as described in any one of embodiments 28 to 37; and

[0354] The second container contains the composition as described in any one of embodiments 38 to 48.

[0355] Implementation Scheme 50. The kit as described in Implementation Scheme 49, wherein the first container contains at least 2, 3, 4, 5, 6, 7, 8 or more times the number of the hydrogel beads as the second container.

[0356] Implementation Scheme 51. The kit as described in Implementation Scheme 49 or 50, wherein the first container contains about 5 × 10⁵ to about 2 × 10⁶ beads, and the second container contains about 1 × 10⁵ to about 4 × 10⁵ beads.

[0357] Implementation Scheme 52. The kit as described in any one of Implementation Schemes 49 to 51, wherein the first container contains about 1 × 10⁶ beads and the second container contains about 2.5 × 10⁵ beads.

[0358] Implementation Scheme 53. The kit of any one of Implementation Schemes 49 to 52, wherein (a) the ratio of the total number of the first group of hydrogel beads and the second group of hydrogel beads in the first container to (b) the total number of the first group of hydrogel beads and the second group of hydrogel beads in the second container is about 1 to about 10, about 2 to about 8, about 3 to about 6, or about 4, including all ranges and subranges therein.

[0359] Implementation Scheme 54. The composition or kit of any one of Implementation Schemes 1 to 53, wherein the CD34 and CD45 cell surface markers each contain a fluorophore.

[0360] Implementation Scheme 55. The composition or kit of any one of Implementation Schemes 1 to 54, wherein the CD34 and CD45 cell surface markers each contain different fluorophores.

[0361] Implementation Scheme 56. The composition or kit as described in Implementation Scheme 54 or 55, wherein each fluorophore is independently selected from any of the following: polydinophytin-chlorophyll-cyanin 5.5 dye (PerCP-Cy5.5); phycoerythrin-cyanin 7 (PE Cy7); allophycocyanin-cyanin 7 (APC-Cy7); fluorescein isothiocyanate (FITC); phycoerythrin (PE); allophycocyanin (APC); 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein succinimide ester; 5-(and-6)-carboxyeosin; 5-carboxyfluorescein; 6-carboxyfluorescein; 5-(and-6)-carboxyfluorescein; S-carboxyfluorescein-bis-(5-carboxymethoxy-2-nitrobenzyl) ether, -alanine-formamide or succinimide ester; 5-carboxyfluorescein succinimide ester; 6 -Carboxyfluorescein succinimide; 5-(and-6)-carboxyfluorescein succinimide; 5-(4,6-dichlorotriazinyl)aminofluorescein; 2',7'-difluorofluorescein; Eosin-5-isothiocyanate; Erythrosin-5-isothiocyanate; 6-(fluorescein-5-formylamino)hexanoic acid or succinimide; 6-(fluorescein-5-(and-6)-formylamino)hexanoic acid or succinimide; fluorescein-S-EX succinimide; fluorescein-5-isothiocyanate; fluorescein-6-isothiocyanate; OregonGreen® 488 carboxylic acid or succinimide ester; Oregon Green® 488 isothiocyanate; Oregon Green® 488-X succinimide ester; Oregon Green® 500 carboxylic acid; Oregon Green® 500 carboxylic acid, succinimide ester, or triethylammonium salt; Oregon Green® 514 carboxylic acid; Oregon Green® 514 carboxylic acid or succinimide ester; RhodamineGreen™ carboxylic acid, succinimide ester, or hydrochloride; Rhodamine Green™ carboxylic acid, trifluoroacetamide, or succinimide ester; Rhodamine Green™-X succinimide ester or hydrochloride; RhodolGreen™ carboxylic acid, N,O-bis-(trifluoroacetyl) or succinimide ester; bis-(4-carboxypiperidinyl)sulfonylrhodamine or di(succinimide ester); 5-(and-6)carboxynaphthalene fluorescein, 5-(and-6)carboxynaphthalene fluorescein succinimide ester; 5-carboxyrhodamine 6G hydrochloride; 6 -Carboxyrhodamine 6G hydrochloride, 5-carboxyrhodamine 6G succinimide ester; 6-carboxyrhodamine 6G succinimide ester; 5-(and -6)-carboxyrhodamine 6G succinimide ester; 5-carboxy-2',4',5',7'-tetrabromosulfone fluorescein succinimide ester or bis-(diisopropylethylammonium) salt; 5-carboxytetramethylrhodamine; 6-carboxytetramethylrhodamine;5-(and-6)-Carboxytetramethylrhodamine; 5-Carboxytetramethylrhodamine succinimide ester; 6-Carboxytetramethylrhodamine succinimide ester; 5-(and-6)-Carboxytetramethylrhodamine succinimide ester; 6-Carboxy-X-rhodamine; 5-Carboxy-X-rhodamine succinimide ester; 6-Carboxy-X-rhodamine succinimide ester; 5-(and-6)-Carboxy-X-rhodamine succinimide ester; 5-Carboxy-X-rhodamine triethylammonium salt; Lissamine; TMRhodamine B sulfonyl chloride; Malachite green; Isothiocyanate; NANOGOLD® mono(sulfosuccinimide); QSY® 21 carboxylic acid or succinimide; QSY® 7 carboxylic acid or succinimide; Rhodamine Red™-X succinimide; 6-(tetramethylrhodamine-5-(and-6)-formamide)hexanoic acid; succinimide; tetramethylrhodamine-5-isothiocyanate; tetramethylrhodamine-6-isothiocyanate; tetramethylrhodamine-5-(and-6)-isothiocyanate; Texas Red® sulfonyl; Texas Red® sulfonyl chloride; Texas Red®-X STP ester or sodium salt; Texas Red®-X succinimide; Texas Red®-X succinimide; X-rhodamine-5-(and-6)isothiocyanate, BODIPY® FL; BODIPY® TMR STP ester; BODIPY® TR-X STP ester; BODIPY® 630 / 650-X STP ester; BODIPY® 650 / 665-X STP ester; 6-Dibromo-4,4-difluoro-5,7-dimethyl-4-boron-3a,4a-diaza-s-indaminon-3-propionic acid succinimide ester; 4,4-difluoro-4-boron-3a,4a-diaza-s-indaminon-3,5-dipropionic acid; 4,4-difluoro-5,7-dimethyl-4-boron-3a,4a-diaza-s-indaminon-3-pentanoic acid; 4,4-difluoro-5,7-dimethyl-4-boron-3a,4a-diazon-s-indaminon-3-pentanoic acid; 4,4-difluoro-5,7-dimethyl-4-boron-3a 4,4-Diaza-S-indaminozide-3-pentanoic acid succinimide; 4,4-Difluoro-5,7-dimethyl-4-boron-3a,4a-diaza-S-indaminozide-3-propionic acid; 4,4-Difluoro-5,7-dimethyl-4-boron-3a,4a-diaza-S-indaminozide-3-propionic acid succinimide; 4,4-Difluoro-5,7-dimethyl-4-boron-3a,4a-diaza-S-indaminozide-3-propionic acid; sulfosuccinate Imide esters or sodium salts; 6-((4,4-difluoro-5,7-dimethyl-4-boron-3a,4a-diaza-s-indane-3-propionyl)amino)hexanoic acid; 6-((4,4-difluoro-5,7-dimethyl-4-boron-3a,4a-diaza-s-indane-3-propionyl)amino)hexanoic acid or succinimide esters; N-(4,4-difluoro-5,7-dimethyl-4-boron-3a,4a-diaza-s-indane) (3-propionyl)cysteine, succinimide ester or triethylammonium salt; 6-4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-boron-3a,4a-4,4-difluoro-5,7-diphenyl-4-boron-3a,4a-diaza-s-indaminone-3-propionic acid; 4,4-difluoro-5,7-diphenyl-4-boron-3a,4a-diaza-s-indaminone-3-propionic acid succinimide ester;4,4-Difluoro-5-phenyl-4-boron-3a,4a-diaza-s-indaminone-3-propionic acid; succinimide ester; 6-((4,4-difluoro-5-phenyl-4-boron-3a,4a-diaza-s-indaminone-3-propionyl)amino)hexanoic acid or succinimide ester; 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-boron-3a,4a-diaza-s-indaminone-3-propionic acid succinimide ester; 4,4- Difluoro-5-(2-pyrrolidinyl)-4-boron-3a,4a-diaza-s-indane-3-propionic acid succinimide ester; 6-(((4,4-difluoro-5-(2-pyrrolidinyl)-4-boron-3a,4a-diaza-s-indane-3-yl)styryloxy)acetyl)aminohexanoic acid or succinimide ester; 4,4-difluoro-5-styryl-4-boron-3a,4a-diaza-s-indane-3-propionic acid; 4,4-difluoro-5- Styrenico-4-boron-3a,4a-diaza-sinden-3-propionic acid; succinimide ester; 4,4-difluoro-1,3,5,7-tetramethyl-4-boron-3a,4a-diaza-sinden-8-propionic acid; 4,4-difluoro-1,3,5,7-tetramethyl-4-boron-3a,4a-diaza-sinden-8-propionic acid succinimide ester; 4,4-difluoro-5-(2-thienyl)-4-boron-3a,4a-diaza- s-indaminozide-3-propanoic acid succinimide; 6-(((4-(4,4-difluoro-5-(2-thienyl)-4-boron-3a,4a-diaza-s-indaminozide-3-yl)phenoxy)acetyl)amino)hexanoic acid or succinimide; and 6-(((4,4-difluoro-5-(2-thienyl)-4-boron-3a,4a-diaza-s-indaminozide-3-yl)styryloxy)acetyl)aminohexanoic acid or succinimide, Alexa Alexa Fluor® 350 carboxylic acid; Alexa Fluor® 430 carboxylic acid; Alexa Fluor® 488 carboxylic acid; Alexa Fluor® 532 carboxylic acid; Alexa Fluor® 546 carboxylic acid; Alexa Fluor® 555 carboxylic acid; Alexa Fluor® 568 carboxylic acid; Alexa Fluor® 594 carboxylic acid; Alexa Fluor® 633 carboxylic acid; Alexa Fluor® 647 carboxylic acid; Alexa Fluor® 660 carboxylic acid; Alexa Fluor® 680 carboxylic acid; Cy3 NHS ester; Cy5 NHS ester; Cy5.5 NHS ester; and Cy7 NHS ester.

[0362] Implementation Scheme 57. The composition or kit of any one of Implementation Schemes 54 to 56, wherein the fluorophore is conjugated to an antibody or a fragment thereof, and the antibody or a fragment thereof binds to an epitope within the polymer beads.

[0363] Implementation Scheme 58. The composition or kit of any one of Implementation Schemes 1 to 57, wherein the CD34 is derived from Homo sapiens.

[0364] Implementation Scheme 59. The composition or kit of any one of Implementation Schemes 1 to 58, wherein the CD45 is derived from Homo sapiens.

[0365] Implementation Scheme 60. The composition or kit of any one of Implementation Schemes 1 to 59, wherein the CD34 extracellular domain comprises Ser32-Thr290 of uniprot P28906, or a sequence containing at least 70%, 75%, 80%, 85%, 90%, 95%, or 97.5% identity with SEQ ID NO: 1.

[0366] Implementation Scheme 61. The composition or kit of any one of Implementation Schemes 1 to 60, wherein the CD45 extracellular domain comprises Gln26-Lys577 of uniprot P08575, or a sequence comprising at least 70%, 75%, 80%, 85%, 90%, 95%, or 97.5% identity with any of SEQ ID NO: 2-9.

[0367] Implementation Scheme 62. The composition or kit of any one of Implementation Schemes 1 to 61, wherein the CD45 extracellular domain comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 97.5% identical to SEQ ID NO:10.

[0368] Implementation Scheme 63. The composition or kit of any one of Implementation Schemes 1 to 62, wherein the hydrogel beads exhibit at least one optical property substantially similar to the corresponding optical properties of the target cells.

[0369] Implementation Scheme 64. The composition or kit as described in Implementation Scheme 63, wherein the at least one optical property comprises lateral scattering.

[0370] Implementation Scheme 65. The composition or kit as described in Implementation Scheme 63, wherein the at least one optical property comprises forward scattering.

[0371] Implementation Scheme 66. The composition or kit as described in Implementation Scheme 63, wherein the at least one optical property comprises lateral scattering and forward scattering.

[0372] Implementation Scheme 67. The composition or kit of any one of Implementation Schemes 63 to 66, wherein the target cells are hematopoietic stem cells.

[0373] Implementation Scheme 68. The composition or kit of any one of Implementation Schemes 63 to 66, wherein the target cells are lymphocytes.

[0374] Implementation Scheme 69. A method for calibrating a cytometer for detecting cells expressing CD34 and / or CD45 in a cell population, comprising sampling a composition or kit as described in any one of Implementation Schemes 1 to 68, and calibrating the cytometer based on at least one optical property of the hydrogel beads of the composition.

[0375] Implementation Scheme 70. The method of Implementation Scheme 69, further comprising sampling the cell population and obtaining cells containing the at least one optical property.

[0376] Implementation Scheme 71. The method of any one of Implementation Schemes 69 to 70, wherein the method includes forming a gating scheme based on the at least one optical property.

[0377] Implementation Scheme 72. A method for enriching cells expressing CD34, comprising sampling a composition or kit as described in any one of Implementation Schemes 1 to 68, forming a gating scheme based on at least one optical property of the hydrogel beads of the composition, and selecting cells expressing CD34 from a cell population based on the gating scheme.

[0378] Implementation Scheme 73. The method of any one of Implementation Schemes 69 to 72, wherein the cells expressing CD34 express a reduced amount of CD45.

[0379] Implementation Scheme 74. The method of any one of Implementation Schemes 69 to 73, wherein the CD34-expressing cell is a CD34-expressing stem cell.

[0380] Implementation Scheme 75. The method of any one of Implementation Schemes 69 to 74, wherein the at least one optical property includes FSC, SSC, median fluorescence intensity (MFI) of CD45, MFI of CD34, or any combination thereof.

[0381] Example

[0382] Example 1: Generation of hydrogel beads

[0383] Hydrogel beads were prepared to substantially match the SSCs and FSCs of lymphocytes. The method used to prepare the hydrogel beads is as follows. The photomask used for UV lithography was sourced from CADart Services Inc. and designed using AutoCad (AutoDesk, Inc.). SU-8 photoresist (Microchem, Inc.) was photocrosslinked on a silicon wafer using a collimated UV light source (OAI, Inc.) to produce a master for fabrication of microfluidic devices. PDMS (polydimethylsiloxane, Sigma Aldrich, Inc.) was prepared and formed using standard published methods for soft lithography and microfluidic device fabrication (see McDonald JC et al., 2000, Electrophoresis 21:27-40).

[0384] Flow-focusing geometry was used to form droplets, in which two oil channels focused the central flow of an aqueous monomer solution to disrupt droplets in a water-in-oil emulsion. A fluorocarbon-oil (Novec 7500 3M, Inc.) was used as the outer continuous phase liquid for droplet formation. To stabilize the droplets prior to polymerization, a surfactant was added at 0.5% w / w to the oil phase (ammonium carboxylate of Krytox 157 FSH, DuPont). To prepare basic polyacrylamide gel beads, a central phase of an aqueous monomer solution containing N-acrylamide (1%–20% w / v), a crosslinking agent (N,N'-bisacrylamide, 0.05%–1% w / v), an accelerator, and ammonium persulfate (1% w / v) was used. An accelerator (N,N,N',N'-tetramethylethylenediamine (2% vol%)) was added to the oil phase to initiate the polymerization of the hydrogel beads after droplet formation.

[0385] Several comonomers are added to the alkaline gel formulation to increase functionality. Allylamine provides primary amine groups for secondary labeling after gel formation. Forward scattering is modulated by adjusting the refractive index of the gel through the addition of allyl acrylate and allyl methacrylate comonomers. Lateral scattering of the droplets is modulated by adding a colloidal suspension of silica nanobeads and / or PMMA (poly(methyl methacrylate)) beads (approximately 100 nm) to the central aqueous phase prior to polymerization.

[0386] Stoichiometric reuse of hydrogel beads is achieved by using comonomers containing chemically orthogonal side groups (amines, carboxyl groups, maleimides, epoxides, alkynes, etc.) for secondary labeling.

[0387] Droplets formed at an average rate of 5 kHz and collected in a fluorocarbon oil phase. Polymerization was completed at 50°C for 30 minutes, and the resulting hydrogel beads were washed from the oil into an aqueous solution.

[0388] Example 2: Generation and visualization of hydrogel beads

[0389] Water containing 5% acrylamide, 0.25% bisacrylamide, 0.05% allylamine, and 0.1% ammonium persulfate was flowed through a central channel and focused through a 10-micron nozzle onto oil containing 0.1% TEMED to produce 10 µm hydrogel beads, such as... Figure 1A As shown in the image. After polymerization, the beads are washed in water, as... Figure 1B As shown in the figure, and conjugated with the dye of interest. Fluorescent hydrogel beads were observed using a fluorescence microscope, as shown in... Figure 1C As shown in the image.

[0390] Example 3: Multidimensional modulation of the optical properties of hydrogel beads

[0391] like Figures 2A to 2C As described, unlike polystyrene beads, hydrogel beads are tuned in multiple dimensions to match specific cell types. This is achieved using optical parameters such as FSC and SSC. Figure 2A Cells are deconvoluted using a combination of secondary markers or secondary markers. Unlike size- and side-scatter-restricted polystyrene beads (brown), hydrogel beads are tuned to match the SSC and FSC of specific cell types. Figure 2B The hydrogel beads are further functionalized with specific chemical side groups and secondary markers with stoichiometric ratios, allowing for precise cell type matching without the biological noise associated with fixed cell lines. Figure 2C ).

[0392] Example 4: CD45 and CD34 cell mimics as enrichment controls

[0393] Cell mimics of CD45dim-positive (CD45dim+) and CD34-positive (CD34+) stem cells were prepared to generate gating schemes for pre- and post-enrichment stem cell phenotypic characterization. The cell mimic used as a pre-enrichment control contained a first mixture of 98.5% CD45dim+ hydrogel beads (i.e., hydrogel beads of this disclosure containing only the CD45 cell surface marker) and 1.5% CD45dim+ / CD34+ hydrogel beads (i.e., hydrogel beads of this disclosure containing both CD45 and CD34 cell surface markers). This first mixture is referred to herein as the "CD34 Lo" cell mimic. The cell mimic used as a post-enrichment control contained a second mixture of 10% CD45dim+ hydrogel beads (i.e., hydrogel beads of this disclosure containing only the CD45 cell surface marker) and 90% CD45dim+ / CD34+ hydrogel beads (i.e., hydrogel beads of this disclosure containing both CD45 and CD34 cell surface markers). This second mixture is referred to herein as the "CD34 Hi" cell mimic. The preparation of the CD34 Lo and CD34 Hi cell mimics is as follows.

[0394] To generate CD45dim+ hydrogel beads, a population of hydrogel beads prepared as described in Examples 1 to 3 was functionalized with the human CD45 extracellular domain (huCD45 ECD). huCD45 ECD corresponds to amino acid residues 26 to 577 of huCD45 according to UniProt accession number P08575.

[0395] To generate CD45dim+ / CD34+ hydrogel beads, a population of hydrogel beads prepared as described in Examples 1 to 3 was functionalized with the huCD45 ECD and human CD34 extracellular domain (huCD34 ECD) as described above. huCD34ECD corresponds to amino acid residues 32 to 290 of huCD34 according to UniProt accession number P28906.

[0396] Hydrogel beads were functionalized with huCD45 ECD and huCD34 ECD at a level that produces MFI equivalent to that of biorelevant samples. CD34 Lo and CD34 Hi cell mimics were prepared by mixing CD45dim+ hydrogel beads and CD45dim+ / CD34+ hydrogel beads at the ratios indicated above.

[0397] Three batches of lyophilized hydrogel bead mixtures representing the CD34 Lo and CD34 Hi cell mimics prepared as described above were provided in storage vials. Tests were performed from three vials of each batch. Preparations contained approximately 1 × 10⁻⁶ beads. 6A vial containing approximately 0.25 × 10⁶ hydrogel beads of CD34 Lo cell mimicry was prepared. 6 A vial containing one hydrogel bead of CD34 Hi cell mimicry. Gently tap the vial to ensure all lyophilized material is collected at the bottom. Add 500 µL of 1X PBS buffer, being careful not to touch / interfere with the lyophilized material, until the precipitate is submerged in the buffer. Gently pipette the mixture up and down to mix and ensure all contents are completely dissolved, then proceed with transfer to Eppendorf or FACS tubes.

[0398] Add another 500 µL of 1X PBS buffer to rinse the vial with any remaining material and transfer it to the same tube. Centrifuge the tube at 500 xg for 5 minutes and remove the supernatant without disturbing the bead precipitation.

[0399] The four antibody groups were evaluated as shown in Table 4. 100 µL of staining buffer containing an appropriate amount of staining antibody was added, and the mixture was vortexed at high speed for 3 seconds to ensure thorough mixing, following the staining reagent manufacturer's instructions.

[0400] Incubate the mixture in the dark at room temperature for 15 to 30 minutes. To wash the beads, add 1 mL of 1x PBS to the tube, vortex, and then centrifuge at 500 x g for 5 minutes. Remove the supernatant without disturbing the bead precipitation and repeat the washing to prevent nonspecific binding.

[0401] Table 4: Groups of anti-CD45 antibody and anti-CD34 antibody

[0402]

[0403] Labeled cell mimics were analyzed on two different flow cytometers (Cytek Aurora and BD Lyric). Cell mimic populations were selected using a sequential gating strategy according to the ISHAGE guidelines (see Sutherland DR et al., J Hematother. (1996) 5:213-26; Keeney M et al., International Society of Hematotherapy and Graft Engineering. Cytometry. (1998) 34:61-70; Whitby A et al., Cytometry B Clin Cytom. (2012) 82B:9-17). Representative gating schemes generated using CD34 Lo or CD34 Hi populations labeled with Group 1 and measured on the Cytek Aurora are shown in the figure. Figures 3A to 3BLateral and forward scattering data were gated on functionalized hydrogel beads existing as singlet states. Anti-CD45 labeling was analyzed from data collected from singlet populations to gate CD45-positive bead populations. Anti-CD34 labeling was analyzed from data collected from CD45-positive bead populations, and a threshold gate was set to distinguish between CD34+ and CD34-negative bead populations. The proportion of beads in the average CD34 Hi and CD34 Lo cell mimic populations across the three batches showed low batch-to-batch variability.

[0404] Example 5: Flow cytometry analysis of biological samples using CD34 Lo and CD34 Hi cell mimics

[0405] Gating protocols using CD34 Lo and CD34 Hi populations were applied to biological samples. The biological samples evaluated included (i) immobilized leukocyte packages containing a pre-enriched CD34+ cell population with a relatively low percentage of CD34+ cells in the total cell count, and (ii) immobilized leukocyte packages rich in CD34+ cells treated with protocol H.

[0406] Biological samples were labeled using Group 1 while labeling CD34 Lo and CD34 Hi cell mimics. Data were collected on a Cytek Aurora and gating was performed as described in Example 4. Figure 4A The analysis of the pre-enrichment sample using a gating scheme generated with CD34 Lo cell mimics shows that CD45dim+ cells were identified as 2.5% of the total singlet population and CD34+ cells as 0.9% of the CD45dim+ population. Figure 4B The analysis of the enriched sample using a gating scheme generated with CD34 Hi cell mimics shows that CD45dim+ cells were identified as 92.4% of the total singlet cells, and CD34+ cells as 97.4% of the total CD45dim+ cells. These data demonstrate that CD34 Lo and CD34 Hi cell mimics are suitable pre- and post-enrichment controls for analyzing CD34+ cell populations without requiring the use of biological samples that introduce cost, handling, and safety concerns.

[0407] * * * * * * * *

[0408] All documents, patents, patent applications, publications, product descriptions, and solutions cited throughout this application are incorporated herein by reference in their entirety for all purposes. The following U.S. and PCT patent applications are expressly incorporated in their entirety for all purposes: US 2022 / 0178810; US 2020 / 0400546; US 2021 / 0341469; US 2021 / 0231552; US 2020 / 0400546; PCT / US2023 / 06668; and PCT / US2023 / 067893.

[0409] The embodiments shown and discussed in this specification are intended only to teach those skilled in the art to make and use the invention of this disclosure in the best manner known to the inventors. Modifications and variations of the above-described embodiments of this disclosure are possible without departing from the spirit of the invention, as understood by those skilled in the art based on the foregoing teachings. Therefore, it should be understood that the invention of this disclosure can be practiced in ways other than those specifically described within the scope of the claims and their equivalents.

Claims

1. A composition comprising a first group of hydrogel beads, the hydrogel beads comprising: a) Polymer monomers and bifunctional monomers; and b) A spectrum of cell surface biomarkers, which includes: i) CD34 and CD45 extracellular domains.

2. The composition of claim 1, comprising a second group of hydrogel beads, the hydrogel beads comprising: c) Polymer monomers and bifunctional monomers; and d) Cell surface biomarker profile, which includes: i) CD45 extracellular domain, but lacks CD34 extracellular domain.

3. The composition of claim 1, comprising a second group of hydrogel beads, the hydrogel beads comprising: c) Polymer monomers and bifunctional monomers; and d) Cell surface biomarker profile, which includes: i) CD45 extracellular domain Each hydrogel bead in the second group contains no more than 10% of the median number of CD34 extracellular domains contained in the hydrogel beads of the first group.

4. The composition of claim 3, wherein each hydrogel bead in the second group comprises no more than 0.1%, no more than 0.2%, no more than 0.3%, no more than 0.5%, no more than 0.7%, no more than 1%, no more than 2%, no more than 3%, no more than 5%, or no more than 7% of the median number of CD34 extracellular domains contained in the hydrogel beads of the first group.

5. The composition of any one of claims 1 to 4, wherein each of the hydrogel beads in the first group comprises about 10% to about 400% of the amount of the CD45 extracellular domain present on the cell surface of the target cell.

6. The composition of claim 5, wherein each of the hydrogel beads in the first group comprises about 10% to about 300%, about 20% to about 400%, about 20% to about 300%, about 20% to about 200%, or about 50% to about 200% of the amount of the CD45 extracellular domain present on the cell surface of the target cell.

7. The composition of any one of claims 1 to 6, wherein each of the hydrogel beads in the first group comprises about 10% to about 400% of the amount of the CD34 extracellular domain present on the cell surface of the target cell.

8. The composition of claim 7, wherein each of the hydrogel beads in the first group comprises about 10% to about 300%, about 20% to about 400%, about 20% to about 300%, about 20% to about 200%, or about 50% to about 200% of the amount of the extracellular domain of CD34 present on the cell surface of the target cell.

9. The composition of any one of claims 2 to 8, wherein each hydrogel bead in the second group comprises about 10% to about 400% of the amount of the CD45 extracellular domain present on the cell surface of the target cell.

10. The composition of claim 9, wherein each of the hydrogel beads in the second group comprises about 10% to about 300%, about 20% to about 400%, about 20% to about 300%, about 20% to about 200%, or about 50% to about 200% of the amount of the CD45 extracellular domain present on the cell surface of the target cell.

11. The composition of any one of claims 2 to 10, wherein each hydrogel bead in the second group contains no more than 10% of the amount of the CD34 extracellular domain present on the cell surface of the target cell.

12. The composition of claim 11, wherein each hydrogel bead in the second group contains an amount of CD34 extracellular domain present on the cell surface of the target cell not exceeding 0.1%, 0.2%, 0.3%, 0.5%, 0.7%, 1%, 2%, 3%, 5%, or 7%.

13. The composition of any one of claims 5 to 12, wherein the amount of CD34 and / or CD45 extracellular domains present on the cell surface of the target cells is the median amount of CD34 and / or CD45 extracellular domains present on the cell surface of cells in a leukocyte package of CD34+ cells enriched using scheme H.

14. The composition of any one of claims 5 to 13, wherein the target cell is a hematopoietic stem cell.

15. The composition of any one of claims 5 to 14, wherein the target cells are CD45dim-positive (CD45dim+) and CD34-positive (CD34+) stem cells.

16. The composition of any one of claims 5 to 15, wherein the target cell is a lymphocyte.

17. The composition of any one of claims 5 to 16, wherein the amount of the extracellular domains of CD45 and / or CD34 present in the hydrogel and / or on the cell surface is measured based on fluorescence intensity using flow cytometry.

18. The composition of claim 17, wherein the fluorescence intensity of the CD45 extracellular domain is measured using a fluorophore-labeled CD45-specific binding molecule, and / or wherein the fluorescence intensity of the CD34 extracellular domain is measured using a fluorophore-labeled CD34-specific binding molecule.

19. The composition of claim 18, wherein the binding molecule comprises a monoclonal antibody or an antigen-binding fragment thereof.

20. The composition of any one of claims 18 to 19, wherein the CD34-specific binding molecule is selected from: phycoerythrin (PE)-labeled anti-CD34 antibody clone 8G12, phycoerythrin (PE)-labeled anti-CD34 antibody clone AC136, allophycocyanin (APC)-labeled anti-CD34 antibody clone 4H11, and Brilliant™ Violet 421 (BV421)-labeled anti-CD34 antibody clone 581.

21. The composition of any one of claims 18 to 20, wherein the CD45-specific binding molecule is selected from: FITC-labeled anti-CD45 antibody clone 2D1, PerCP-Cyanine® 5.5 (PerCP-Cy5.5)-labeled anti-CD45 antibody clone 2D1, PE-labeled anti-CD45 antibody clone MEM-28, and BD Horizon™ V500-labeled anti-CD45 antibody clone HI30.

22. The composition of any one of claims 18 to 21, wherein the combination of the CD34-specific binding molecule and the CD45-specific binding molecule is selected from one group of Table 4; optionally, the combination is group 1 of Table 4.

23. The composition of any one of claims 1 to 22, wherein the biomarker is connected to the matrix of the hydrogel beads via a connector.

24. The composition of any one of claims 1 to 23, wherein the biomarker is covalently linked to the matrix of the hydrogel beads.

25. The composition of any one of claims 1 to 23, wherein the biomarker is non-covalently linked to the matrix of the hydrogel beads.

26. The composition of any one of claims 1 to 25, wherein the hydrogel beads of the first group and / or the second group have an average diameter of about 1 μm to about 40 μm, about 20 μm to about 30 μm, about 3 μm to about 20 μm, or about 4-10 μm.

27. The composition of any one of claims 2 to 26, wherein the active portion of the composition comprises or consists of the first group of hydrogel beads and the second group of hydrogel beads.

28. The composition of claim 27: The first group comprises 1%-3% of the number of hydrogel beads in the active portion of the composition.

29. The composition according to any one of claims 27 to 28: The second group comprises 50%-99% of the number of hydrogel beads in the active portion of the composition; wherein any remaining portion of the active portion of the composition contains hydrogel beads lacking CD34 and CD45.

30. The composition as claimed in claim 27 or 28: The second group comprises 97%-99% of the number of hydrogel beads in the active portion of the composition; wherein any remaining portion of the active portion of the composition contains hydrogel beads lacking CD34 and CD45.

31. The composition as described in claim 27 or 28: The first group comprises 1%-3% of the number of hydrogel beads in the active portion of the composition, and The second group comprises 97%-99% of the number of hydrogel beads in the active portion of the composition.

32. The composition as described in claim 27 or 28: The first group comprises 1.4%-1.8% of the number of hydrogel beads in the active portion of the composition, and The second group comprises 98.2%-98.6% of the number of hydrogel beads in the active portion of the composition.

33. The composition of any one of claims 27 to 32, wherein the first group of hydrogel beads and the second group of hydrogel beads together constitute at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% of the number of hydrogel beads in the active portion of the composition.

34. The composition of any one of claims 2 to 33, wherein the ratio of the number of hydrogel beads in the first group to the number of hydrogel beads in the second group is not more than 1, not more than 0.5, not more than 0.3, not more than 0.2, not more than 0.1, not more than 0.07, not more than 0.05, not more than 0.03, not more than 0.02, not more than 0.018, not more than 0.017, not more than 0.016, not more than 0.015, not more than 0.01, not more than 0.005, or not more than 0.

001.

35. The composition of claim 34, wherein the ratio of the number of hydrogel beads in the first group to the number of hydrogel beads in the second group is not greater than 0.0183.

36. The composition of any one of claims 2 to 35, wherein the ratio of the number of hydrogel beads of the first group to the number of hydrogel beads of the second group is about 1 to about 0.5, about 0.5 to about 0.2, about 0.2 to about 0.1, about 0.1 to about 0.05, about 0.05 to about 0.02, about 0.02 to about 0.015, about 0.015 to about 0.01, about 0.01 to about 0.005, about 0.005 to about 0.002, or about 0.002 to about 0.001, including all ranges and subranges therein.

37. The composition of claim 36, wherein the ratio of the number of hydrogel beads in the first group to the number of hydrogel beads in the second group is from about 0.0183 to about 0.0142.

38. The composition of claim 27: The first group comprises 10%-95% of the number of hydrogel beads in the active portion of the composition.

39. The composition of claim 27: The first group comprises 80%-95% of the number of hydrogel beads in the active portion of the composition.

40. The composition according to any one of claims 27 and 38 to 39: The second group comprises 5%-90% of the number of hydrogel beads in the active portion of the composition; wherein any remaining portion of the active portion of the composition contains hydrogel beads lacking CD34 or CD45.

41. The composition according to any one of claims 27 and 38 to 39: The second group comprises 5%-20% of the number of hydrogel beads in the active portion of the composition.

42. The composition of claim 27: The first group comprises 80%-95% of the number of hydrogel beads in the active portion of the composition, and The second group comprises 5%-20% of the number of hydrogel beads in the active portion of the composition.

43. The composition of claim 27: The first group comprises 90%-95% of the number of hydrogel beads in the active portion of the composition, and The second group comprises 5%-10% of the number of hydrogel beads in the active portion of the composition.

44. The composition of any one of claims 27 and 38 to 43, wherein the first group of hydrogel beads and the second group of hydrogel beads together constitute at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% of the number of hydrogel beads in the active portion of the composition.

45. The composition of any one of claims 2 to 27 and 38 to 44, wherein the ratio of the number of hydrogel beads in the first group to the number of hydrogel beads in the second group is at least 1, at least 2, at least 5, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 50, at least 70, or at least 100.

46. ​​The composition of claim 45, wherein the ratio of the number of hydrogel beads in the first group to the number of hydrogel beads in the second group is at least 9.

47. The composition of any one of claims 2 to 27 and 38 to 46, wherein the ratio of the number of hydrogel beads of the first group to the number of hydrogel beads of the second group is about 1 to about 2, about 2 to about 5, about 5 to about 8, about 8 to about 10, about 10 to about 12, about 12 to about 15, about 15 to about 20, about 20 to about 50, about 50 to about 100, including all ranges and subranges therein.

48. The composition of claim 47, wherein the ratio of the number of hydrogel beads in the first group to the number of hydrogel beads in the second group is about 9 to about 19.

49. A reagent kit comprising: A first container comprising the composition as described in any one of claims 28 to 37; and The second container contains the composition as described in any one of claims 38 to 48.

50. The kit of claim 49, wherein the first container contains at least 2, 3, 4, 5, 6, 7, 8 or more times the number of the hydrogel beads as the second container.

51. The kit of claim 49 or 50, wherein the first container contains approximately 5 × 10 5 Approximately 2×10 6 10 beads, and the second container contains approximately 1×10 5 Approximately 4×10 5 Each bead.

52. The kit according to any one of claims 49 to 51, wherein the first container contains about 1 × 10 6 The second container contains approximately 2.5 × 10⁶ beads. 5 Each bead.

53. The kit according to any one of claims 49 to 52, wherein (a) the ratio of the total number of the first group of hydrogel beads and the second group of hydrogel beads in the first container to (b) the total number of the first group of hydrogel beads and the second group of hydrogel beads in the second container is about 1 to about 10, about 2 to about 8, about 3 to about 6, or about 4, including all ranges and subranges therein.

54. The composition or kit according to any one of claims 1 to 53, wherein the CD34 and CD45 cell surface markers each comprise a fluorophore.

55. The composition or kit according to any one of claims 1 to 54, wherein the CD34 and CD45 cell surface markers each contain different fluorophores.

56. The composition or kit of claim 54 or 55, wherein each fluorophore is independently selected from any of the following: polydinophytin-chlorophyll-cyanin 5.5 dye (PerCP-Cy5.5); phycoerythrin-cyanin 7 (PE Cy7); allophycocyanin-cyanin 7 (APC-Cy7); fluorescein isothiocyanate (FITC); phycoerythrin (PE); allophycocyanin (APC); 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein succinimide ester; 5-(and-6)-carboxyeosin; 5-carboxyfluorescein; 6-carboxyfluorescein; 5-(and-6)-carboxyfluorescein; S-carboxyfluorescein-bis-(5-carboxymethoxy-2-nitrobenzyl) ether, -alanine-formamide or succinimide ester; 5-carboxyfluorescein succinimide ester; 6-carboxyfluorescein succinimide ester; Amino esters; 5-(and-6)-carboxyfluorescein succinimide ester; 5-(4,6-dichlorotriazinyl)aminofluorescein; 2',7'-difluorofluorescein; Eosin-5-isothiocyanate; Erythrosin-5-isothiocyanate; 6-(fluorescein-5-formamido)hexanoic acid or succinimide ester; 6-(fluorescein-5-(and-6)-formamido)hexanoic acid or succinimide ester; fluorescein-S-EX succinimide ester; fluorescein-5-isothiocyanate; fluorescein-6-isothiocyanate; OregonGreen®488 carboxylic acid or succinimide ester; Oregon Green® 488 isothiocyanate; Oregon Green® 488-X succinimide ester; Oregon Green® 500 carboxylic acid; Oregon Green® 500 carboxylic acid, succinimide ester, or triethylammonium salt; Oregon Green® 514 carboxylic acid; Oregon Green® 514 carboxylic acid or succinimide ester; RhodamineGreen™ carboxylic acid, succinimide ester, or hydrochloride; Rhodamine Green™ carboxylic acid, trifluoroacetamide, or succinimide ester; Rhodamine Green™-X succinimide ester or hydrochloride; RhodolGreen™ carboxylic acid, N,O-bis-(trifluoroacetyl) or succinimide ester; bis-(4-carboxypiperidinyl)sulfonylrhodamine or di(succinimide ester); 5-(and-6)carboxynaphthalene fluorescein, 5-(and-6)carboxynaphthalene fluorescein succinimide ester; 5-carboxyrhodamine 6G hydrochloride; 6 -Carboxyrhodamine 6G hydrochloride, 5-carboxyrhodamine 6G succinimide ester; 6-carboxyrhodamine 6G succinimide ester; 5-(and -6)-carboxyrhodamine 6G succinimide ester; 5-carboxy-2',4',5',7'-tetrabromosulfone fluorescein succinimide ester or bis-(diisopropylethylammonium) salt; 5-carboxytetramethylrhodamine; 6-carboxytetramethylrhodamine;5-(and-6)-Carboxytetramethylrhodamine; 5-Carboxytetramethylrhodamine succinimide ester; 6-Carboxytetramethylrhodamine succinimide ester; 5-(and-6)-Carboxytetramethylrhodamine succinimide ester; 6-Carboxy-X-rhodamine; 5-Carboxy-X-rhodamine succinimide ester; 6-Carboxy-X-rhodamine succinimide ester; 5-(and-6)-Carboxy-X-rhodamine succinimide ester; 5-Carboxy-X-rhodamine triethylammonium salt; Lissamine; TM Rhodamine B sulfonyl chloride; Malachite green; Isothiocyanate; NANOGOLD® mono(sulfosuccinimide); QSY® 21 carboxylic acid or succinimide; QSY® 7 carboxylic acid or succinimide; Rhodamine Red™-X succinimide; 6-(tetramethylrhodamine-5-(and-6)-formamide)hexanoic acid; succinimide; tetramethylrhodamine-5-isothiocyanate; tetramethylrhodamine-6-isothiocyanate; tetramethylrhodamine-5-(and-6)-isothiocyanate; Texas Red® sulfonyl; Texas Red® sulfonyl chloride; Texas Red®-X STP ester or sodium salt; Texas Red®-X succinimide; Texas Red®-X succinimide; X-rhodamine-5-(and-6)isothiocyanate, BODIPY® FL; BODIPY® TMR STP ester; BODIPY® TR-X STP ester; BODIPY® 630 / 650-X STP ester; BODIPY® 650 / 665-X STP ester; 6-Dibromo-4,4-difluoro-5,7-dimethyl-4-boron-3a,4a-diaza-s-indaminone-3-propionate succinimide ester; 4,4-difluoro-4-boron-3a,4a-diaza-s-indaminone-3,5-dipropionic acid; 4,4-difluoro-5,7-dimethyl-4-boron-3a,4a-diaza-s-indaminone-3-pentanoic acid; 4,4-difluoro-5,7-dimethyl-4-boron-3a,4a-diaza-s-indaminone-3-pentanoic acid; 4,4-Diaza-S-indaminozide-3-pentanoic acid succinimide; 4,4-Difluoro-5,7-dimethyl-4-boron-3a,4a-diaza-S-indaminozide-3-propionic acid; 4,4-Difluoro-5,7-dimethyl-4-boron-3a,4a-diaza-S-indaminozide-3-propionic acid succinimide; 4,4-Difluoro-5,7-dimethyl-4-boron-3a,4a-diaza-S-indaminozide-3-propionic acid; sulfosuccinate Imide esters or sodium salts; 6-((4,4-difluoro-5,7-dimethyl-4-boron-3a,4a-diaza-s-indane-3-propionyl)amino)hexanoic acid; 6-((4,4-difluoro-5,7-dimethyl-4-boron-3a,4a-diaza-s-indane-3-propionyl)amino)hexanoic acid or succinimide esters; N-(4,4-difluoro-5,7-dimethyl-4-boron-3a,4a-diaza-s-indane) (3-propionyl)cysteine, succinimide ester or triethylammonium salt; 6-4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-boron-3a,4a-4,4-difluoro-5,7-diphenyl-4-boron-3a,4a-diaza-s-indaminone-3-propionic acid; 4,4-difluoro-5,7-diphenyl-4-boron-3a,4a-diaza-s-indaminone-3-propionic acid succinimide ester;4,4-Difluoro-5-phenyl-4-boron-3a,4a-diaza-s-indaminone-3-propionic acid; succinimide ester; 6-((4,4-difluoro-5-phenyl-4-boron-3a,4a-diaza-s-indaminone-3-propionyl)amino)hexanoic acid or succinimide ester; 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-boron-3a,4a-diaza-s-indaminone-3-propionic acid succinimide ester; 4,4- Difluoro-5-(2-pyrrolidinyl)-4-boron-3a,4a-diaza-s-indane-3-propionic acid succinimide ester; 6-(((4,4-difluoro-5-(2-pyrrolidinyl)-4-boron-3a,4a-diaza-s-indane-3-yl)styryloxy)acetyl)aminohexanoic acid or succinimide ester; 4,4-difluoro-5-styryl-4-boron-3a,4a-diaza-s-indane-3-propionic acid; 4,4-difluoro-5- Styrenico-4-boron-3a,4a-diaza-sinden-3-propionic acid; succinimide ester; 4,4-difluoro-1,3,5,7-tetramethyl-4-boron-3a,4a-diaza-sinden-8-propionic acid; 4,4-difluoro-1,3,5,7-tetramethyl-4-boron-3a,4a-diaza-sinden-8-propionic acid succinimide ester; 4,4-difluoro-5-(2-thienyl)-4-boron-3a,4a-diaza- s-indaminozide-3-propanoic acid succinimide; 6-(((4-(4,4-difluoro-5-(2-thienyl)-4-boron-3a,4a-diaza-s-indaminozide-3-yl)phenoxy)acetyl)amino)hexanoic acid or succinimide; and 6-(((4,4-difluoro-5-(2-thienyl)-4-boron-3a,4a-diaza-s-indaminozide-3-yl)styryloxy)acetyl)aminohexanoic acid or succinimide, Alexa Alexa Fluor® 350 carboxylic acid; Alexa Fluor® 430 carboxylic acid; Alexa Fluor® 488 carboxylic acid; Alexa Fluor® 532 carboxylic acid; Alexa Fluor® 546 carboxylic acid; Alexa Fluor® 555 carboxylic acid; Alexa Fluor® 568 carboxylic acid; Alexa Fluor® 594 carboxylic acid; Alexa Fluor® 633 carboxylic acid; Alexa Fluor® 647 carboxylic acid; Alexa Fluor® 660 carboxylic acid; Alexa Fluor® 680 carboxylic acid; Cy3 NHS ester; Cy5 NHS ester; Cy5.5 NHS ester; and Cy7 NHS ester.

57. The composition or kit of any one of claims 54 to 56, wherein the fluorophore is conjugated to an antibody or a fragment thereof, and the antibody or a fragment thereof binds to an epitope within the polymer beads.

58. The composition or kit according to any one of claims 1 to 57, wherein the CD34 is derived from Homo sapiens.

59. The composition or kit according to any one of claims 1 to 58, wherein the CD45 is derived from Homo sapiens.

60. The composition or kit of any one of claims 1 to 59, wherein the CD34 extracellular domain comprises Ser32-Thr290 of uniprot P28906, or a sequence containing at least 70%, 75%, 80%, 85%, 90%, 95%, or 97.5% identity with SEQ ID NO:

1.

61. The composition or kit of any one of claims 1 to 60, wherein the CD45 extracellular domain comprises Gln26-Lys577 of uniprot P08575, or a sequence comprising at least 70%, 75%, 80%, 85%, 90%, 95%, or 97.5% identity with any of SEQ ID NO: 2-9.

62. The composition or kit of any one of claims 1 to 61, wherein the CD45 extracellular domain comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 97.5% identical to SEQ ID NO:

10.

63. The composition or kit of any one of claims 1 to 62, wherein the hydrogel beads exhibit at least one optical property substantially similar to the corresponding optical properties of the target cells.

64. The composition or kit of claim 63, wherein the at least one optical property comprises lateral scattering.

65. The composition or kit of claim 63, wherein the at least one optical property comprises forward scattering.

66. The composition or kit of claim 63, wherein the at least one optical property comprises lateral scattering and forward scattering.

67. The composition or kit of any one of claims 63 to 66, wherein the target cell is a hematopoietic stem cell.

68. The composition or kit of any one of claims 63 to 66, wherein the target cells are lymphocytes.

69. A method for calibrating a cytometer for detecting cells expressing CD34 and / or CD45 in a cell population, comprising sampling a composition or kit as described in any one of claims 1 to 68, and calibrating the cytometer based on at least one optical property of the hydrogel beads of the composition.

70. The method of claim 69, further comprising sampling the cell population and obtaining cells containing the at least one optical property.

71. The method of any one of claims 69 to 70, wherein the method includes forming a gating scheme based on the at least one optical property.

72. A method for enriching cells expressing CD34, comprising sampling a composition or kit as described in any one of claims 1 to 68, forming a gating scheme based on at least one optical property of the hydrogel beads of the composition, and selecting cells expressing CD34 from a cell population based on the gating scheme.

73. The method of any one of claims 69 to 72, wherein the cells expressing CD34 express a reduced amount of CD45.

74. The method of any one of claims 69 to 73, wherein the CD34-expressing cell is a CD34-expressing stem cell.

75. The method of any one of claims 69 to 74, wherein the at least one optical property comprises FSC, SSC, median fluorescence intensity (MFI) of CD45, MFI of CD34, or any combination thereof.

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