Mixed cell populations for tissue repair and separation technique for cell processing
Inventors
Hampson, Brian • Goltry, Kristin • Smith, Doug • Rowley, Jonathan A. • Venturi, Naia
Assignees
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Publication Number
US-7871605-B2
Publication Date
2011-01-18
Expiration Date
Abstract
The present invention provides a fluid exchange cell culture technique and tissue repair cells (TRCs) made by these methods, as well as methods using these cells. The method includes a new wash step which increases the tissue repair properties of the TRCs of the invention. This wash step allows for the production of TRC populations with greater tissue repair and anti -inflammatory capabilities. Embodiments of the present invention include a post-culture process for cultured cells that preferably includes the steps of: a wash process for removing unwanted residual culture components, a volume reduction process, and a harvesting process to remove cultured cells. Preferably, all these steps are performed within a aseptically closed cell culture chamber by implementing a separation method that minimizes mechanical disruption of the cells and is simple to automate. The harvested cells may then be concentrated to a final volume for the intended use. In such embodiments, the final composition is a substantially purified and concentrated cell mixture suspended in a physiologic solution suitable for immediate use in humans without further washing, volume reduction, or processing. Embodiments are also applicable to harvesting (and/or washing) particles within a liquid or solution within a chamber.
Core Innovation
The invention provides an isolated cell composition for tissue repair comprising a mixed population of cells of hematopoietic, mesenchymal and endothelial lineage. The composition is defined by viability and by proportions of viable CD90+ and CD45+ cells, while also imposing limits on residual culture components and enzymatically active harvest reagent carryover.
A central aspect of the invention is the specification of quantitative quality attributes for tissue-repair cells. The composition is characterized as at least 80% viable, contains about 5-75% viable CD90+ cells with the remaining cells being CD45+, contains less than 2 μg/mL of bovine serum albumin, and contains less than 1 μg/mL of an enzymatically active harvest reagent.
The disclosed tissue-repair cell composition also includes optional further characterizations tied to cell phenotype and functional immunoregulatory markers. The composition may produce reduced levels of one or more pro-inflammatory cytokines, may be defined by expression of indoleamine 2,3-dioxygenase (IDO) or PD-L1, and may be characterized by CD45+ cells that co-express CD14, CD34, or VEGFR1.
The composition is further characterized as substantially free of mycoplasm, endotoxin, and microbial contamination, and may be substantially free of horse serum and/or fetal bovine serum.
Claims Coverage
The document provides one independent claim directed to an isolated tissue-repair cell composition defined by mixed hematopoietic, mesenchymal, and endothelial lineage, quantitative viability and marker proportions (CD90+ and CD45+), strict residual component limits (bovine serum albumin and enzymatically active harvest reagent), and substantial freedom from mycoplasm, endotoxin, and microbial contamination. Dependent claims refine cytokine production, immunoregulatory marker expression (IDO/PD-L1), CD45+ co-expression markers, total viable cell number range, and additional serum carryover exclusions.
Isolated mixed-lineage tissue-repair cell composition
An isolated cell composition for tissue repair comprising a mixed population of cells of hematopoietic, mesenchymal and endothelial lineage.
Viability and lineage marker proportions
The cell composition is characterized as being at least 80% viable, containing about 5-75% viable CD90+ cells with the remaining cells being CD45+.
Low residual bovine serum albumin
The cell composition contains less than 2 μg/mL of bovine serum albumin.
Low enzymatically active harvest reagent
The cell composition contains less than 1 μg/mL of an enzymatically active harvest reagent.
Substantially free of contamination
The cell composition is substantially free of mycoplasm, endotoxin, and microbial contamination.
Reduced pro-inflammatory cytokine production
The cell composition may produce reduced levels of one or more pro-inflammatory cytokines.
Expression of indoleamine 2,3-dioxygenase or PD-L1
The cell composition may be defined by expression of indoleamine 2,3-dioxygenase or PD-L1.
CD45+ co-expression markers
CD45+ cells may co-express CD14, CD34, or VEGFR1.
Total viable cell number range
The claimed cell composition may include a total number of viable cells ranging from 35 million to 300 million.
Substantially free of horse serum and/or fetal bovine serum
The cell composition may be substantially free of horse serum and/or fetal bovine serum.
Across the provided claims, the coverage centers on a mixed hematopoietic, mesenchymal, and endothelial tissue-repair cell composition defined by high viability, specific CD90+/CD45+ proportions, low residual bovine serum albumin and low enzymatically active harvest reagent, and substantial freedom from mycoplasm, endotoxin, and microbial contamination. Dependent characterizations further define cytokine-production reduction, immunoregulatory phenotype via IDO or PD-L1, CD45+ co-expression markers, a total viable cell count range, and additional serum exclusions.
Stated Advantages
Provides a tissue-repair cell composition that is at least 80% viable.
Contains less than 2 μg/mL of bovine serum albumin.
Contains less than 1 μg/mL of an enzymatically active harvest reagent.
Is substantially free of mycoplasm, endotoxin, and microbial contamination.
May produce reduced levels of one or more pro-inflammatory cytokines.
May express indoleamine-2,3-dioxygenase or PD-L1.
May have CD45+ cells co-express CD14, CD34, or VEGFR1.
May have a total number of viable cells ranging from 35 million to 300 million.
May be substantially free of horse serum and/or fetal bovine serum.
Documented Applications
Bone regeneration and vascular regeneration are described as therapeutic administration and clinical use cases.
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