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US 12,044,676 B2Granted
3D cell viability assay
AssigneeGreiner Bio-One North America, Inc.
InventorGlauco R. Souza
Life of this patent
Estimating the term…
Abstract
Cells are grown in 3D culture and topological features obtained by photomicrography are correlated to cell viability and cell interactions.
Claims18
A label-free method of determining cell viability or cell-cell interaction, comprising:
- a) culturing a sample 3D cell culture comprising a 3D cell culture and a test agent and a control 3D cell culture comprising the 3D cell culture without the test agent, wherein cells of the sample and control 3D cell cultures are levitated in a magnetic field with a composition comprising:
- i) a negatively charged nanoparticle;
- ii) a positively charged nanoparticle;
- and
- iii) a support molecule,
- wherein one of said negatively charged nanoparticle or positively charged nanoparticle contains a magnetically responsive element, and wherein said support molecule holds said negatively charged nanoparticle and said positively charged nanoparticle in an intimate admixture;
- b) taking photomicrographs of said sample and control 3D cell cultures respectively at one or more times while being levitated in a magnetic field;
- c) analyzing said photomicrographs to measure fractal dimension of the sample and control 3D cell cultures;
- wherein Df=[log(N)−log(kg)]/log(L/a) and Df is a fractal dimension which is a statistical indication of how completely a fractal fills space, wherein said statistical indication is used to approximate a number of cells in a 3D cell culture, kg is a structure prefactor and is a constant, N is a number of cells, a is a diameter of a cell, and L is a length of a box used for box-counting,
- d) determining cell viability based on the measured fractal dimension, wherein
- Cell
-
- viability
- =
- D
- f
-
- s
-
- a
-
- m
-
- p
-
- l
-
- e
- -
- D
- f
-
- 1
-
- 00
-
- %
-
- cell
-
- death
- D
- f
-
- c
-
- o
-
- n
-
- t
-
- r
-
- o
-
- l
- -
- D
- f
-
- 1
-
- 00
-
- %
-
- cell
-
- death
- ,
- and Dfsample is a fractal dimension of the sample 3D cell culture, Dfcontrol is a fractal dimension of the control 3D cell culture, and Df100% cell death is a fractal dimension of the sample 3D cell culture at 100% cell death and is not zero,
- wherein fractal dimension is directly proportional to cell viability or cell-cell interactions.
70 more elements
A label-free method of determining cell viability or cell-cell interaction in the presence of a test agent, comprising:
- a) culturing a plurality of magnetic cells levitated in a magnetic field to form a plurality of 3D cell cultures, one or more of said plurality of the 3D cell cultures being without a test agent representing one or more control 3D cell cultures, and one or more of said plurality of the 3D cell cultures being with one or more concentrations of said test agent representing one or more sample 3D cell cultures;
- b) taking photomicrographs of said one or more control 3D cell cultures or sample 3D cell cultures levitated in a magnetic field at one or more times;
- c) analyzing said photomicrographs to measure fractal dimension of said one or more control 3D cell cultures or sample 3D cell cultures;
- wherein Df=[log(N)−log(kg)]/log(L/a) and Df is a fractal dimension, which is a statistical indication of how completely a fractal fills space, wherein said statistical indication is used to approximate a number of cells in a 3D cell culture, kg is a structure prefactor and is a constant, N is a number of cells, a is a diameter of a cell, and L is a length of a box used for box-counting,
- d) determining cell viability based on the measured fractal dimension, wherein
- Cell
-
- viability
- =
- D
- f
-
- s
-
- a
-
- m
-
- p
-
- l
-
- e
- -
- D
- f
-
- 1
-
- 00
-
- %
-
- cell
-
- death
- D
- f
-
- c
-
- o
-
- n
-
- t
-
- r
-
- o
-
- l
- -
- D
- f
-
- 1
-
- 00
-
- %
-
- cell
-
- death
- ,
- and Dfsample is a fractal dimension of the sample 3D cell culture, Dfcontrol is a fractal dimension of the control 3D cell culture, and Df100% cell death is a fractal dimension of the sample 3D cell culture at 100% cell death and is not zero,
- wherein fractal dimension is directly proportional to cell viability or cell-cell interactions.
65 more elements
A label-free method for determining cell-cell interaction or viability in the presence of a test agent, comprising:
- a) culturing a plurality of 3D cell cultures, one or more of said plurality of 3D cell cultures being without a test agent representing one or more control 3D cell cultures, and one or more of said plurality of 3D cell cultures being with one or more concentrations of said test agent representing one or more sample 3D cell cultures;
- b) taking photomicrographs of said plurality of said one or more control 3D cell cultures or sample 3D cell cultures at one or more times;
- c) analyzing said photomicrographs to measure fractal dimension of said one or more control 3D cells cultures or sample 3D cell cultures,
- wherein Df=[log(N)−log(kg)]/log(L/a) and Df is a fractal dimension, which is a statistical indication of how completely a fractal fills space, wherein said statistical indication is used to approximate a number of cells in a 3D cell culture, kg is a structure prefactor and is a constant, N is a number of cells, a is a diameter of a cell, and L is a length of a box used for box-counting,
- d) determining cell viability based on the measured fractal dimension, wherein
- Cell
-
- viability
- =
- D
- f
-
- s
-
- a
-
- m
-
- p
-
- l
-
- e
- -
- D
- f
-
- 1
-
- 00
-
- %
-
- cell
-
- death
- D
- f
-
- c
-
- o
-
- n
-
- t
-
- r
-
- o
-
- l
- -
- D
- f
-
- 1
-
- 00
-
- %
-
- cell
-
- death
- ,
- and Dfsample is a fractal dimension of the sample 3D cell culture, Dfcontrol is a fractal dimension of the control 3D cell culture, and Df100% cell death is a fractal dimension of the sample 3D cell culture at 100% cell death and is not zero,
- wherein fractal dimension is directly proportional to cell-cell interaction and viability,
- and wherein a decrease in cell-cell interaction indicates that said test agent is inhibitory, and wherein an increase in cell-cell interactions indicates that said test agent is stimulatory;
- e) wherein said plurality of 3D cell cultures are prepared by levitating cells in a magnetic field with a composition comprising:
- a) a negatively charged nanoparticle;
- b) a positively charged nanoparticle;
- and c) a support molecule, wherein said negatively charged nanoparticle or positively charged nanoparticle contains a magnetically responsive iron or iron oxide, and wherein said support molecule holds said negatively charged nanoparticle and said positively charged nanoparticle in an intimate admixture.
70 more elements
Dependencies are inferred from the claim text.