3D cell viability assay
Cells are grown in 3D culture and topological features obtained by photomicrography are correlated to cell viability and cell cell interactions.
1. A wound closure assay, comprising:
a) perforating a plurality of samples of magnetized cell clusters levitating in a magnetic field or magnetically patterning a plurality of samples of magnetized cells levitating in a magnetic field to form levitating 3D cell clusters having a hole therein, one or more of said plurality of samples being control samples without a test agent, and one or more of said plurality of samples being test samples with one or more concentrations of said test agent;
b) taking photomicrographs of said plurality of samples of said levitating 3D cell clusters with said hole at one or more times; and
c) analyzing said photomicrographs to measure wound closure, wherein:
wound
closure
(
%
)
=
100
%
-
(
area
of
wound
t
area
of
wound
t
0
×
100
%
)
,
wherein said area of wound t0 is an area of said hole before said test agent is added and said area of wound t is an area of said hole at a given time t after addition of said test agent.
2. The assay of claim 1 , further including adding varying amounts of said test agent to said test samples.
3. The assay of claim 2 , comprising taking a plurality of photomicrographs of said plurality of samples at a plurality of times.
4. The assay of claim 2 , further comprising washing out said test agent and taking a further plurality of photomicrographs of said test samples at a further plurality of times.
5. The assay of claim 1 , wherein said magnetized cells or said magnetized cell clusters are magnetized with a composition comprising:
i) negatively-charged nanoparticles;
ii) positively-charged nanoparticles; and
iii)) support molecules,
wherein one of i) or ii) contains a magnetically responsive element, and wherein said support molecules hold said negatively-charged nanoparticles and said positively-charged nanoparticles in an intimate admixture.
6. The assay of claim 5 , wherein said support molecules comprise peptides, polysaccharides, nucleic acids, and mixtures thereof, said negatively-charged nanoparticles comprise gold nanoparticles, and said positively-charged nanoparticles comprise iron oxide nanoparticles.
7. The assay of claim 6 , wherein said support molecules comprise poly-lysine.
8. A cell assay comprising:
a) culturing a plurality of samples of magnetized cells levitating in a magnetic field to form levitating 3D cell clusters, one or more of said plurality of samples being control samples without a test agent, and one or more of said plurality of samples being test samples with one or more concentrations of said test agent;
b) taking photomicrographs of said plurality of samples of said levitating 3D cell clusters at one or more times; and
c) analyzing said photomicrographs to measure fractal dimension (D f ) and one or more of i) number of cell clusters, ii) cell cluster size, iii) total area of cell clusters, and iv) rate of cell cluster contraction;
wherein said fractal dimension and said cell cluster size are directly proportional to cell viability and cell-cell interactions, and said number of cell clusters and said total area of cell clusters are inversely proportional to said cell viability and said cell-cell interactions.
9. The assay of claim 8 , wherein said fractal dimension =D f =[log(N)−log (kg)]/log (L/a), wherein 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 that covers a pixel of said photomicrographs.
10. The assay of claim 8 , wherein
cell
viability
=
D
fsample
-
D
f
100
%
cell
death
D
fcontrol
-
D
f
100
%
cell
death
,
and D fsample is said fractal dimension of said test samples, and D fcontrol is said fractal dimension of said control samples.
11. A cell assay comprising:
a) culturing a plurality of samples of levitating 3D cell cultures, one or more of said plurality of samples being control samples without a test agent, and one or more of said plurality of samples being test samples with one or more concentrations of said test agent;
b) taking photomicrographs of said plurality of samples of said levitating 3D cell cultures at one or more times;
c) analyzing said photomicrographs to measure fractal dimension (D f ) and one or more of:
c-i) number of cell clusters,
c-ii) cell cluster size,
c-iii) total area of cell clusters, and
c-iv) rate of cell cluster contraction;
d) wherein said fractal dimension and said cell cluster size are directly proportional to cell viability and cell-cell interactions, and said number of cell clusters and said total area of cell clusters are inversely proportional to said cell viability and said cell-cell interactions;
e) wherein said levitating 3D cell cultures are prepared by levitating cells with a composition comprising:
e-i) negatively-charged nanoparticles;
e-ii) positively-charged nanoparticles; and
e-iii) support molecules,
wherein one of e-i) and e-ii) includes magnetically responsive iron or iron oxide, and wherein said support molecules hold said negatively-charged nanoparticles and said positively-charged nanoparticles in an intimate admixture.
12. The assay of claim 11 , wherein said plurality of samples of 3D cultures each have a hole and wound closure in response to said test agent is measured, wherein
wound
closure
(
%
)
=
100
%
-
(
area
of
wound
t
area
of
wound
t
0
×
100
%
)
,
wherein said area of wound t0 is an area of said hole before said test agent is added and said area of wound t is an area of said hole at a given time t after addition of said test agent.
13. The assay of claim 11 , wherein said support molecules comprise peptides, polysaccharides, nucleic acids, and mixtures thereof, wherein said negatively-charged nanoparticles comprise gold nanoparticles, and wherein said positively-charged nanoparticles comprise iron oxide nanoparticles.
14. The assay of claim 13 , wherein said support molecules comprise poly-lysine, fibronectin, collagen, laminin, BSA, hyaluronan, glycosaminoglycan, anionic, non-sulfated glycosaminoglycan, gelatin, extracellular matrix protein mixtures, antibody, and mixtures thereof.
15. The assay of claim 11 , wherein said support molecules comprise poly-lysine, wherein said negatively-charged nanoparticles comprise gold nanoparticles, and wherein said positively-charged nanoparticles comprise iron oxide nanoparticles.
16. The assay of claim 11 , wherein said test agent is a drug, a cell type different from that of said 3D cell cultures, a cell component, a toxin, or an environmental agent.