Cell capture and expansion
Inventors
Jones, Mark E. • Sethi, Dalip • Hlavinka, Dennis J. • Felt, Thomas J.
Assignees
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Publication Number
US-12043823-B2
Publication Date
2024-07-23
Expiration Date
Abstract
Implementations are described that relate to methods and systems for growing cells in a hollow fiber bioreactor. In implementations, the cells may be exposed to a number of growth factors including a combination of recombinant growth factors. In other implementations, the cells may be grown in co-culture with other cells, e.g., hMSC's. In implementations, the cells may include CD34+ cells. A coated membrane includes a membrane having a first coating configured to promote cellular adhesion to the membrane and a second coating that includes a soluble protein moiety.
Core Innovation
The invention provides a method of expanding CD34+ hematopoietic stem cells using a hollow fiber bioreactor having semi-permeable hollow fibers. A coating is applied to an interior lumen of a plurality of hollow fibers, where the coating includes fibronectin configured to adhere to the interior lumen and stromal cell-derived factor 1 (SDF-1) configured to both adhere to the fibronectin and retain CD34+ hematopoietic stem cells thereto. This lumen-coating arrangement supports retention of CD34+ cells in the bioreactor.
Cells are introduced as a first plurality of cells into the plurality of hollow fibers, with the first plurality including CD34+ hematopoietic stem cells derived from at least one of cord blood, bone marrow, and peripheral blood. The method maintains and concentrates the first plurality within the hollow fibers by directing fluid into opposite ends of the hollow fiber bioreactor simultaneously. The first plurality of cells is exposed to growth conditions within the hollow fibers.
Expansion is performed to generate a second plurality of expanded target cells including CD34+ hematopoietic stem cells that are expanded at least 50-fold. The expanded target cells are generated by contacting the first plurality within the hollow fibers with soluble growth supporting cytokines including at least one of SCF, TPO, Flt-3L, IL-3, and IL-6, an aryl receptor antagonist, and glial cell-derived neurotrophic factor (GDNF). The approach combines the fibronectin/SDF-1 retention coating with specified soluble growth conditions and cellular expansion within the hollow fiber bioreactor.
Claims Coverage
Independent claim coverage centers on 5 inventive features: lumen-applied fibronectin/SDF-1 retention, introduction of CD34+ hematopoietic stem cells from specified sources, simultaneous fluid directing at opposite ends to maintain and concentrate cells, soluble growth conditions with specified cytokines plus an aryl receptor antagonist and GDNF, and at least 50-fold expansion.
Fibronectin and SDF-1 coated interior lumen for retaining CD34+ hematopoietic stem cells
Applying a coating to an interior lumen of a plurality of semi-permeable hollow fibers of a hollow fiber bioreactor, where the coating includes fibronectin configured to adhere to the interior lumen and stromal cell-derived factor 1 (SDF-1) configured to both adhere to the fibronectin and retain CD34+ hematopoietic stem cells thereto.
Introducing CD34+ hematopoietic stem cells derived from cord blood, bone marrow, and/or peripheral blood
Introducing a first plurality of cells into the plurality of hollow fibers of the hollow fiber bioreactor, the first plurality of cells including CD34+ hematopoietic stem cells derived from at least one of cord blood, bone marrow, and peripheral blood.
Simultaneous fluid directing at opposite ends to maintain and concentrate cells within the hollow fibers
Maintaining and concentrating the first plurality of cells within the plurality of hollow fibers by directing fluid into opposite ends of the hollow fiber bioreactor simultaneously.
Soluble growth conditions with specified cytokines, aryl receptor antagonist, and GDNF to achieve at least 50-fold expansion
Expanding at least a portion of the first plurality of cells in the hollow fibers of the bioreactor to generate a second plurality of expanded target cells including CD34+ hematopoietic stem cells expanded at least 50-fold, by introducing into the plurality of hollow fibers, and into contact with the first plurality of cells, soluble growth supporting cytokines including at least one of SCF, TPO, Flt-3L, IL-3, and IL-6, an aryl receptor antagonist, and glial cell-derived neurotrophic factor (GDNF).
The claims are directed to retaining CD34+ hematopoietic stem cells in a fibronectin/SDF-1 coated lumen, maintaining and concentrating the cells by simultaneous fluid delivery at opposite ends, and expanding the cells with specified soluble growth supporting cytokines, an aryl receptor antagonist, and GDNF to at least 50-fold expansion.
Stated Advantages
Expanded target cells including CD34+ hematopoietic stem cells expanded at least 50-fold.
Retention of CD34+ hematopoietic stem cells by SDF-1 configured to adhere to fibronectin in the interior lumen.
Documented Applications
Expanding cord blood-derived CD34+ HSCs using a perfusion-based hollow fiber bioreactor with coated hollow fiber membranes, with reported outcomes including fold expansion and viability.
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