Selection and use of umbilical cord cell fractions suitable for transplantation
Inventors
Peled, Tony • Harati, Dorit • LANDAU, Efrat
Assignees
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Publication Number
US-11730771-B2
Publication Date
2023-08-22
Expiration Date
Abstract
Methods of selecting umbilical cord blood units for ex-vivo expansion, separation of CD133+/CD34+ positive and uncultured CD133+/CD34+ negative fractions, methods for expanding the selected CD133+/CD34+ fraction, selection of expanded populations of CD133+/CD34+ cord blood cells for transplantation to subjects in need thereof and the therapeutic use of suitable selected, ex-vivo expanded CD 133+/CD34+ and unselected CD133/CD34 negative cord blood fractions for transplantation in the clinical setting, for treatment of hematological malignancies are provided. The present invention also envisions kits comprising the expanded and unselected cord blood fractions.
Core Innovation
The invention provides a method for preparing a single thawed umbilical cord blood unit for transplantation into a subject by separating the unit into a first selected blood cell fraction comprising CD133+/CD34+ selected cells and a second unselected blood cell fraction comprising CD133 negative and CD34 negative CD133/CD34 negative cells. The selected CD133+/CD34+ fraction is defined for ex-vivo expansion and transplantation into the subject by meeting post-separation parameters including total cells, viability, CD133+ and CD34+ percentages, selection yield, CFU per 1000 cells, and endotoxin less than 3 Eu/ml.
After separation, the invention includes ex vivo culturing the first blood cell fraction comprising CD133+/CD34+ selected cells under conditions allowing cell proliferation, including providing nutrients, serum, and cytokines comprising stem cell factor, thrombopoietin, FLt3 ligand and IL-6, together with nicotinamide in an amount between 1.0 mM to 10 mM. The culture conditions support ex-vivo expansion of the CD133+/CD34+ cord blood fraction for transplantation.
The post-separation definition of the selected CD133+/CD34+ fraction requires quantitative and quality constraints comprising about 2.0×10^5 to about 7.5×10^6 total cells, about 70% to about 85% viability, about 70% to about 85% CD133+ cells, about 70% to about 85% CD34+ cells, about 0.02% to about 1% cell yield post CD133+/CD34+ selection, about 15 to about 75 CFU per 1000 cells, and less than 3 Eu/ml endotoxin. The method also separates out an unselected CD133/CD34 negative fraction for the subject.
Claims Coverage
The patent includes one independent claim directed to a complete method for preparing a cord blood unit by separating selected and unselected fractions and ex vivo culturing the selected CD133+/CD34+ fraction with specified culture components and acceptance parameters. Dependent claims further define additional selection criteria, culture compositions, post-expansion release determinations, and separate cryopreservation.
Separating a thawed umbilical cord blood unit into CD133+/CD34+ selected and CD133/CD34 negative unselected fractions
Separating a single, thawed umbilical cord blood unit suitable for transplantation into a first, selected blood cell fraction comprising CD133+/CD34+ selected cells and a second, unselected blood cell fraction comprising CD133 negative and CD34 negative CD133/CD34 negative cells.
Ex vivo culturing CD133+/CD34+ selected cells with nutrients, serum, cytokines and nicotinamide
Ex vivo culturing the first blood cell fraction comprising CD133+/CD34+ selected cells under conditions allowing for cell proliferation, the conditions comprising providing nutrients, serum and cytokines including stem cell factor, thrombopoietin, FLt3 ligand and IL-6 and nicotinamide in an amount between 1.0 mM to 10 mM.
Defining the post-separation acceptance parameters for the CD133+/CD34+ fraction
Selecting the first, selected CD133+/CD34+ blood cell fraction according to post-separation parameters including total cells, viability, CD133+ cells, CD34+ cells, cell yield post selection, CFU per 1000 cells, and endotoxin less than 3 Eu/ml.
Determining additional selection and acceptance criteria and excluding contaminated fractions
Determining specific cell-number and marker-viability parameters in an ex-vivo expanded ex-vivo cultured umbilical cord blood fraction, excluding fractions that do not meet all criteria including no mycoplasma or bacterial/yeast/mold growth, and selecting the resulting ex-vivo cultured CD133+/CD34+ umbilical cord blood fraction for transplantation into the subject.
Separately cryopreserving the ex-vivo cultured selected fraction and the unselected fraction
Separately cryopreserving an ex-vivo cultured first selected blood cell fraction of CD133+/CD34+ cells and a second unselected blood cell fraction of CD133/CD34 negative cells.
Overall claim coverage centers on preparing a transplantation-suitable cord blood unit by separating a thawed unit into CD133+/CD34+ selected and CD133/CD34 negative unselected fractions, culturing the selected fraction with a nutrient/serum/cytokine regimen including stem cell factor, thrombopoietin, FLt3 ligand, IL-6, and nicotinamide, and requiring defined post-separation quality and acceptance parameters for the selected fraction, with further dependent-claim refinement for unit selection, post-expansion release determination including contamination exclusions, and separate cryopreservation.
Stated Advantages
Faster platelet engraftment (median ~34 days), with associated statistical correlations with total viable cells.
Documented Applications
Transplantation into a subject using prepared umbilical cord blood units for hematological malignancies, including ALL, AML, CML, MDS, Hodgkin lymphoma, and non-Hodgkin lymphoma.
Use of kits/compositions comprising expanded and unselected cord blood fractions.
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