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CN-121986254-A - CD34 stem cell mimics

CN121986254ACN 121986254 ACN121986254 ACN 121986254ACN-121986254-A

Abstract

Hydrogel beads with quantifiable attached biomolecules and their use as cell mimics in cell counting applications are described. The cell mimics described herein are selectively tunable to have at least one optical property that is substantially similar to at least one optical property of a target cell (e.g., a cd34+ stem cell). The present disclosure further relates to methods of using the disclosed cell mimics as enrichment controls in cell counting applications.

Inventors

  • J.Jin
  • A.T.Ruan
  • Subanipu Bisgas
  • Kanwar Para

Assignees

  • 弹弓生物科学公司

Dates

Publication Date
20260505
Application Date
20240828
Priority Date
20230829

Claims (20)

  1. 1. A composition comprising a first population of hydrogel beads, the hydrogel beads comprising: a) A polymerizable monomer and a difunctional monomer, and B) A cell surface biomarker profile comprising: i) CD34 and CD45 extracellular domains.
  2. 2. The composition of claim 1, comprising a second population of hydrogel beads comprising: c) A polymerizable monomer and a difunctional monomer, and D) A cell surface biomarker profile comprising: i) CD45 extracellular domain, but lacks CD34 extracellular domain.
  3. 3. The composition of claim 1, comprising a second population of hydrogel beads comprising: c) A polymerizable monomer and a difunctional monomer, and D) A cell surface biomarker profile comprising: i) The extracellular domain of CD45, Wherein each hydrogel bead in the hydrogel beads in the second population comprises no more than 10% of the median number of CD34 extracellular domains comprised in the hydrogel beads of the first population.
  4. 4. The composition of claim 3, wherein each hydrogel bead in the hydrogel beads in the second population 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 population.
  5. 5. The composition of any one of claims 1-4, wherein each hydrogel bead in the hydrogel beads in the first population comprises about 10% to about 400% of the amount of CD45 extracellular domain present on the cell surface of a target cell.
  6. 6. The composition of claim 5, wherein each hydrogel bead in the first population 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 CD45 extracellular domain present on the cell surface of a target cell.
  7. 7. The composition of any one of claims 1-6, wherein each hydrogel bead in the hydrogel beads in the first population comprises about 10% to about 400% of the amount of CD34 extracellular domain present on the cell surface of a target cell.
  8. 8. The composition of claim 7, wherein each hydrogel bead in the first population 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 CD34 extracellular domain present on the cell surface of a target cell.
  9. 9. The composition of any one of claims 2-8, wherein each hydrogel bead in the second population comprises about 10% to about 400% of the amount of CD45 extracellular domain present on the cell surface of a target cell.
  10. 10. The composition of claim 9, wherein each hydrogel bead in the second population 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 CD45 extracellular domain present on the cell surface of a target cell.
  11. 11. The composition of any one of claims 2 to 10, wherein each hydrogel bead in the second population comprises no more than 10% of the amount of CD34 extracellular domain present on the cell surface of a target cell.
  12. 12. The composition of claim 11, wherein each hydrogel bead in the hydrogel beads in the second population 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 amount of CD34 extracellular domain present on the cell surface of a target cell.
  13. 13. The composition of any one of claims 5 to 12, wherein the amount of CD34 and/or CD45 extracellular domain present on the cell surface of the target cell is the median amount of CD34 and/or CD45 extracellular domain present on the surface of cells in a cd34+ cell enriched leukocyte pack treated with protocol H.
  14. 14. The composition of any one of claims 5 to 13, wherein the target cell is a hematopoietic stem cell.
  15. 15. The composition of any one of claims 5 to 14, wherein the target cells are CD45dim positive (CD 45 dim+) and CD34 positive (cd34+) stem cells.
  16. 16. The composition of any one of claims 5 to 15, wherein the target cell is a lymphocyte.
  17. 17. The composition of any one of claims 5 to 16, wherein the amount of the CD45 and/or CD34 extracellular domain present in the hydrogel and/or on the cell surface is measured based on fluorescence intensity using flow cytometry.
  18. 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. 19. The composition of claim 18, wherein the binding molecule comprises a monoclonal antibody or antigen-binding fragment thereof.
  20. 20. The composition of any one of claims 18 to 19, wherein the CD 34-specific binding molecule is selected from Phycoerythrin (PE) -labeled anti-CD 34 antibody clone 8G12, phycoerythrin (PE) -labeled anti-CD 34 antibody clone AC136, allophycocyanin (APC) -labeled anti-CD 34 antibody clone 4H11, and BrilliantTM Violet (BV 421) -labeled anti-CD 34 antibody clone 581.

Description

CD34 stem cell mimics Cross Reference to Related Applications The application claims the benefit of U.S. provisional application No. 63/535,233, filed on 8/29 2023, the contents of which are incorporated herein by reference in their entirety. Reference to an electronic sequence Listing The contents of the electronic sequence listing (SLIN _024_01wo_seqlist_st26.Xml; size: 17,366 bytes; and date of creation: 2024, 8, 19 days) are incorporated herein by reference in their entirety. Technical Field The present disclosure relates to compositions of matter and methods that allow for calibration of stem cells and experimental controls. Background Stem cells expressing CD34 are increasingly used in cell therapies and other applications. Stem cells expressing CD34 are typically enriched from donor-derived blood samples and phenotypically characterized using flow cytometry, for example, to characterize the starting material or for quality control before and/or after enrichment. Flow cytometry allows measurement of forward scatter ("FSC") and side scatter ("SSC"), which are parameters related to cell volume and internal complexity of particles (e.g., shape of nucleus, amount and type of cytoplasmic particles, or membrane roughness), respectively, and analysis of cell surface markers indicative of cell status. Using flow cytometry, CD34 expressing cells in a heterogeneous cell population can be sorted, counted, and/or characterized, allowing them to be used for a desired application. The control was used to calibrate the flow cytometer parameters in order to distinguish the optical properties of cells expressing CD34 from other cells in the 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 (resulting in long sampling times or large control populations required to collect enough events), suffer from supply inconsistencies (introduction costs and/or batch-to-batch variability), and/or are characterized by poor shelf life of the closed vials (resulting in more frequent bridging studies required 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. Thus, there is a need in the art for cell-free compositions that mimic CD 34-expressing stem cells in order to calibrate devices (such as flow cytometers) for analyzing populations containing such cells. Disclosure of Invention In some aspects, the present disclosure provides compositions comprising a first population of hydrogel beads comprising a) polymerized monomers and difunctional monomers, and b) a cell surface biomarker profile comprising i) CD34 and CD45 extracellular domains. In some embodiments, the composition further comprises a second population of hydrogel beads comprising c) a polymeric monomer and a difunctional monomer, and d) a cell surface biomarker profile comprising i) a CD45 extracellular domain, but lacking a CD34 extracellular domain. In some embodiments, the composition comprises a second population of hydrogel beads comprising c) polymerized monomers and difunctional monomers, and d) a cell surface biomarker profile comprising i) CD45 extracellular domains, wherein each hydrogel bead in the second population comprises 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 composition comprises a second population of hydrogel beads comprising c) polymerized monomers and difunctional monomers, and d) a cell surface biomarker profile comprising i) CD45 extracellular domains, wherein the hydrogel beads in the second population comprise no more than 10% of the median number of CD34 extracellular domains comprised in the hydrogel beads of the first population. In some embodiments, the hydrogel beads in the second population comprise 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 comprised in the hydrogel beads of the first population. In some embodiments, each hydrogel bead in the second population 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 comprised in the hydrogel beads of the first population. In some embodiments, the hydrogel beads in the first population comprise from about 10% to about 400% of the amount of CD45 extracellular domain present on the cell surface of the target cells. In some embodiments, each hydrogel bead in the first population comprises about 1