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Publication Number
US-11124771-B2
Publication Date
2021-09-21
Expiration Date
Abstract
Methods for selecting lots of bone marrow stromal cells (MSCs) having a high proliferative capacity are provided. Such methods are useful in the manufacture of therapeutic derivatives of MSCs.
Core Innovation
The described approach solves selection of bone-marrow mesenchymal stromal cell (MSC) lots with high proliferative capacity from among a plurality of lots. Single colonies are formed by separately plating samples at low density, and after colony formation the method measures α-smooth muscle actin (αSMA) levels and lactate dehydrogenase (LDH) levels in each colony to assess colony characteristics relevant to subsequent growth.
Large colonies are defined using an LDH-based criterion, where a large colony is a colony in which the sum of intracellular LDH values of all cells in the colony is greater than 0.4 mU/ml. For each large colony, a normalized metric is determined by forming an αSMA/LDH ratio (normalized αSMA value), and the method evaluates an average value across large colonies, Av(αSMA/LDH)LC, together with the percentage of large colonies in the culture.
A lot is selected by applying criteria to Av(αSMA/LDH)LC and to the percentage of large colonies, and the selected MSCs are then grown in mass culture. The selection is represented as predicting subsequent mass-culture growth based on the rapid colony-stage αSMA/LDH predictor, and the concept is described as an ALC assay involving same-colony marker and cell-number surrogate measurements to distinguish slow versus fast growers.
Claims Coverage
The partial document includes two independent claims (clm-00001 and clm-00005). Each independent claim covers a lot-selection method using low-density single-colony culture, colony-level αSMA and LDH measurements, an LDH threshold to define large colonies, normalization of αSMA to LDH to compute a normalized αSMA/LDH metric, and selection based on cutoff criteria for both the normalized metric and the percentage of large colonies, followed by mass culture of the selected MSCs.
Low-density single-colony plating and colony marker/number measurement
Separately plating a sample of cells or MSCs from each lot of bone marrow suspension at low density to form single colonies, then measuring α-smooth muscle actin (αSMA) levels in each colony and measuring lactate dehydrogenase (LDH) levels in each colony.
LDH threshold definition of large colonies
Determining the percentage of large colonies in the culture, wherein a large colony is a colony in which the sum of the intracellular LDH values of all cells in the colony is greater than 0.4 mU/ml or greater than 0.4 milliUnits/ml.
Normalized αSMA/LDH ratio in large colonies and average metric function of large-colony percentage
Determining the αSMA/LDH ratio (normalized αSMA value) in each large colony and determining an average of the normalized αSMA values for all large colonies in the culture (Av(αSMA/LDH)LC) expressed as a function of the percentage of large colonies in the culture.
Selection criteria for high-proliferative-capacity MSC lots and mass culture growth
Selecting a culture with an Av(αSMA/LDH)LC value of 100 or less and a percentage of large colonies that is 40% or greater, representing a lot of MSCs with high proliferative capacity, and further comprising growing the selected MSCs in mass culture.
Across clm-00001 and clm-00005, the inventive concept is concentrated in forming low-density single-colony cultures; measuring αSMA and LDH at the colony level; defining large colonies by an LDH threshold; normalizing αSMA to LDH to compute an αSMA/LDH metric and expressing an average normalized value as a function of the percentage of large colonies; then selecting lots meeting specified cutoff values and growing the selected MSCs in mass culture.
Stated Advantages
Predicting subsequent mass-culture growth based on rapid colony-stage αSMA/LDH measurements.
Selecting bone-marrow MSC lots with high proliferative capacity.
Documented Applications
Selecting and growing mesenchymal stromal cell (MSC) lots with high proliferative capacity in subsequent mass culture.
Using selected MSC lots to manufacture a therapeutic derivative of MSCs (dependent claims clm-00003/clm-00007).
Performing, during mass culture of selected MSCs, transfection with a polynucleotide encoding a Notch intracellular domain rather than full-length Notch (dependent claims clm-00004/clm-00008).
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