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US 12,725,435 B2Granted
FIM-CNN for detection of viable cells and/or particulate impurities
AssigneeCoriolis Pharma Research GmbH
InventorsAndrea Hawe, Tim Menzen, Adam Grabarek, Alexandra Roesch
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Abstract
The disclosure presented herein provides methods for quantifying viable cells and particulate cell impurities in a cell-based product sample. The method is implemented on a convolutional neural network (CNN) that learns to classify flow-imaging microscopy (FIM) images. The CNN learning is accomplished by using a training set of classified images of viable cells and different types of impurities.
Claims8
A method for quantifying single viable cells and/or particulate impurities in a cell-based product sample, said method comprising:
- a) providing a training set of classified flow-imaging microscopy (FIM) images comprising single viable cells, debris, and at least one of:
- necrotic cells, apoptotic cells, doublet cells, magnetic beads, or adducts;
- b) training a convolutional neural network (CNN) with the classified images of step (a);
- c) capturing FIM images from said product sample;
- d) identifying single viable cells, debris, and at least one of:
- necrotic cells, apoptotic cells, doublet cells, magnetic beads, or adducts, by applying the trained CNN obtained in step (b) to the FIM images of step (c);
- and
- e) quantifying the single viable cells and/or the particulate impurities identified in step (d):
- wherein said classified images of step (a) are obtained by a method comprising:
- i) providing a cell suspension;
- ii) applying to said cell suspension conditions that induce necrosis, apoptosis, cell debris, adducts, or any combination thereof;
- iii) optionally labeling the cells;
- iv) obtaining FIM images of said cell suspension;
- and
- v) classifying said FIM images;
- and wherein:
- i) said conditions that induce necrosis comprise incubating cells at 55° C. for 90 min, or at 37° C. for 90 min in a cell culture comprising 10% ethanol;
- ii) said conditions that induce apoptosis comprise incubating the cells with 1 μM staurosporin for 3 hours at 37° C.;
- iii) said conditions that induce debris comprise vortexing cells in a 2-mL microcentrifuge tube for 2 min, or submitting the cells to two freeze-thaw cycles from −140° C. to 37° C.;
- and
- iv) said conditions that induce adducts comprise incubating magnetic beads with cells in a 1:1 ratio for 1.5 hours at 37° C.
18 more elements
A method for quantifying single viable cells and/or particulate impurities in a cell-based product sample, said method comprising:
- a) providing a cell suspension comprising single viable cells, necrotic cells, and/or apoptotic cells, wherein conditions that induce necrosis, apoptosis, cell debris, adducts, or any combination thereof were applied to said cell suspension;
- b) simultaneously obtaining a brightfield image and a fluorescent signal from said cell suspension using an imaging flow cytometer;
- wherein said fluorescent signal is used to attribute a classification label to said brightfield image, according to whether said brightfield image comprises a viable cell, a necrotic cell, or an apoptotic cell;
- c) training a convolutional neural network (CNN) with the brightfield images of step (b);
- d) capturing flow-image microscopy (FIM) images from said product sample;
- e) identifying single viable cells, debris, and at least one of:
- necrotic cells, apoptotic cells, doublet cells, magnetic beads, or adducts, by applying the trained CNN obtained in step (c) to the FIM images of step (d);
- f) quantifying the single viable cells and/or the particulate impurities identified in step (e);
- wherein:
- i) said conditions that induced necrosis in said cell suspension comprise incubating cells at 55° C. for 90 min, or at 37° C. for 90 min in a cell culture comprising 10% ethanol;
- ii) said conditions that induced apoptosis in said cell suspension comprise incubating the cells with 1 μM staurosporin for 3 hours at 37° C.;
- iii) said conditions that induced debris in said cell suspension comprise vortexing cells in a 2-mL microcentrifuge tube for 2 min, or submitting the cells to two freeze-thaw cycles from −140° C. to 37° C.;
- and
- iv said conditions that induced adducts in said cell suspension comprise incubating magnetic beads with cells in a 1:1 ratio for 1.5 hours at 37° C.
11 more elements
Dependencies are inferred from the claim text.