Cell growth rate determination by measurement of changes in cyanine dye levels in plasma membranes

Methods for determining growth rate of cells growing in vivo and in vitro. Cells are labelled with cyanine dyes and changes in plasma membrane cyanine dye levels are used to determine growth rate. Cell growth rate determinations are utilized to monitor transplanted bone marrow cell engraftment and post-surgical corneal epitheal cell growth. The invented methods also are used to determine tumor cell sensitivity to cancer therapeutic agents, yeast sensitivity to antifungal agents, and bacteria sensitivity to antibacterial agents.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to novel methods for measuring cell growth rate in 
vivo and in vitro. 
2. Background Information 
Currently, there are two popular methods for measuring cell growth. One 
method is to count the number of cells at the beginning of an analysis 
period, and then count the number of cells at the cell of that period to 
measure the increase in cell number. Cell counting can be achieved by 
using microscopic methods with a hemocytometer or by instrument aided 
methods using a Coulter Counter or other flow cytometer. another 
Methodology for measuring cell growth is to determine the uptake of 
tritiated thymidine using beta counting methods. In this methodology, the 
cell number is determined at the initiation of the experiment and then 
tritiated thymidine is placed in with the cells. At periodic intervals, 
aliquots of the culture are removed, counted, and washed free of unbound 
tritiated thymidine. These washed aliquots are then subjected to 
Trichloroacetic Acid (TCA) precipitation followed by scintillation 
counting of the radioactively labelled solid precipitate to measure 
tritiated thymidine incorporation into DNA. This methodology merely 
measures the rate of DNA synthesis and does not measure the cell growth 
per se. Because of the relative ease of this methodology, however, it 
generally is the methodology of choice when looking at cell stimulation. 
Another method used to determine cell proliferation activity is to look at 
the number of mitoses per hundred cells in any tissue under examination. 
This methodology is not extremely accurate because the preparation 
procedure causes loss of cells. In general, these assays work well in 
vitro but are difficult to apply to measurements of cell growth in vivo. 
Growth rate of tissues can be estimated by removing the tissue and 
monitoring in vitro pulse incorporation of tritiated thymidine. The tissue 
is sectioned into 30 micron sections and exposed to tritiated thymidine 
for thirty (30) minutes. The unincorporated tritiated thymidine is washed 
away and a nuclear emulsion is placed over the section where radioisotope 
disintegrations expose the film. The emulsion is developed and fixed; the 
tissue is stained with Hematoxylin and Eosin stain; then the section is 
examined microscopically to determine labelled fraction. This technique is 
labor intensive and time consuming. 
Cyanine dyes have been used in various biological applications. 
Dioxacarbocyanine dyes have been used in performing white blood cell 
differential counts. Gunter Valet, Max Planck Ges Wissensch; Patent 
Accession Number 84-102307/17, Simultaneous Quantitative Determination of 
Blood Cells by Selective Staining and Measuring Volume and Fluorescence. 
The dyes utilized in these studies, however, are short chain carbocyanine 
dyes (less than ten carbons) and respond to changes in membrane 
potentials. Furthermore, the short chain carbocyanine dyes enter the 
cell's mitochondria, are cytotoxic, and, when the cells are washed, these 
dyes easily leak out of the cell whether or not the membrane potential of 
the cell is changed. Other short aliphatic chain cyanine dyes are used in 
many other biological assays. The short chain molecules, however, respond 
to membrane potentials and cross the cell membrane, penetrating into the 
mitochondria. H. M. Shapiro, U.S. Pat. No. 4,343,782, Aug. 10, 1982. The 
short chain dyes also are toxic to cells and cannot be used to determine 
cell growth rate. 
Tricarbocyanine dyes (Fox, I. J., et al., Proc. May Clinic, 32: 478-484, 
1957) and Evans-Blue dye (Schad, H., et al., Pfluegers Arch. Eur. J. 
Physiol., 370(2): 139-144, 1977) have been used in vivo to estimate 
cardiac output by a dilution method. Dow (Dow, P., 
Physiol. Rev., 36: 77-102, 1956) describes the method as injection of a 
known amount of some intravascular indicator on the venus side of the 
lungs, and measurement of the time course of arterial concentration of the 
indicator to determine the volume between the points of injection and 
sampling. These dyes are not used to stain cells. 
SUMMARY OF THE INVENTION 
Presently invented are novel methods for measuring cell growth rate. 
According to the present invention, viable cells first are labelled with 
cyanine dyes. Cell growth rate is determined by measuring changes in the 
levels of cyanine dye in the plasma membranes of daughter cells derived 
from cyanine dye-labelled parent cells. The invented methods for measuring 
growth are used, for example, in vivo to monitor healing of corneal 
epithelia, and engraftment of transplanted bone marrow cells. In vitro 
uses of the invented methods include determining sensitivity of tumor 
cells to various chemotherapeutic agents.

DETAILED DESCRIPTION OF THE INVENTION 
In the invented methods for determining cell growth rate, the cells are 
labelled with cyanine dyes. Compounds having the following structure are 
referred to herein as cyanine dyes: 
##STR1## 
in which: Y is oxygen, sulfur, methylene or alkyl-substituted methylene; 
m is 0-3; and 
n is 12-22. As used herein, alkyl-substituted methylene refers to mono- or 
di-substituted methylene having any combination of methyl, ethyl, or 
propyl substituents. 
Compounds of the above structure are referred to by the following generally 
understood shorthand formula: 
EQU DiYC.sub.n (2m+1) 
Sims, P. J., et al., Biochem, 13: 3315 (1974). Thus, for example, the 
compound wherein Y is sulfur and having three carbons bridging the rings 
and two fourteen carbon aliphatic chains is referred to as DiSC.sub.14 
(3). Similarly, DiIC.sub.14 (5) indicates the compound wherein Y is 
isopropyl, and having five carbons bridging the rings and two fourteen 
carbon aliphatic chains. 
Included within compounds referred to herein as cyanine dyes are compounds 
of the above structure having one or more substitutions provided such 
substituted compounds are soluble in a cell labelling media for at least 
as long as needed for labelling and have a sufficiently high membrane 
partition coefficient to remain associated with labelled cell membranes. 
Such compounds also must not significantly affect cell viability in the 
concentrations required for labelling. Solubility in cell labelling media 
is determined as shown below by dispursing a cyanine dye in the labelling 
media and, by standard spectrofluorometric techniques, measuring 
fluorescence intensity over time. Decreasing fluorescence intensity 
indicates dye precipitation and adherence to vessel walls. Whether the 
dyes remain associated with cell membranes is determined, for example, 
using known flow cytometric procedures to monitor fluorescence intensity 
of red blood cells reinjected into the donor animal after labelling. 
Essentially constant fluorescence intensities of the labelled cells after 
reinjection establishes stability of the dye in cell membranes. 
Cyanine dyes used in the present invention can be purchased from various 
sources such as Molecular Probes, Inc., Eugene, Oreg., and can be prepared 
from available starting materials using known synthetic methods. Hamer, F. 
M., The Cyanine Dyes and Related Compounds, Interscience Publishers 
(1964). 
Using the described procedures any viable cell can be labelled with cyanine 
dyes. As used herein, the term cell includes nucleated cells such as white 
blood cells, various tumor cells, other mammalian cells (for example, 
tissue culture cells) yeast, and bacteria. A cell is viable if it is able 
to grow or function essentially as expected for cells of its type. 
Cell labelling is performed in a medium that is non-lethal to cells and 
that provides for reproducible cell labelling. To give the medium the 
necessary characteristics, osmolarity regulating agents in which cyanine 
dyes form stable solutions for at least as long as required for labelling 
are used. Acceptable osmolarity regulating agents include agents such as 
sugars, for example monosaccharides such as glucose, fructose, sorbose, 
xylose, ribose, and disaccharides such as sucrose, sugar-alchols, such as 
mannitol, glycerol, inositol, xylitol, and adonitol, amino acids such as 
glycine and arginine, and certain Good's buffers such as 
N-tris(hydroxymethyl)-methyl-3-aminopropanesulfonic acid and those listed 
in Table II, below. Good, N. E., et al., Biochem. 15, 467-477 (1966), 
Good, N. E. and S. Izawa, Methods Enzymol., 24, Part B, 53 (1968), 
Feguson, W. J., et al., Anal. Biochem. 104: 301-310 (1980). Some cell 
lines, however, may be sensitive to one or more of the osmolarity 
regulating agents, especially sugar-alcohols. Thus, prior to labelling, 
standard tests are conducted to make certain that the cells are viable in 
the intended osmolarity regulating agent. Additionally, small amounts of 
buffering agents may be added to the labelling medium to regulate hydrogen 
ion concentration. 
The effect on cell viability of exposure to a variety of osmolarity 
regulating agents was determined by measuring the doubling time of Yac 
cells after the cells were exposed for thirty minutes to a variety of 
osmolarity regulating agents. Yac cells are a mouse lymphoma tissue 
culture cell line publically available from the American Type Culture 
Collection and is described by Kiessling, R., European J. Immunology 5: 
112-117 (1975). As the data shown in Table 1 demonstrate, when compared to 
phosphate buffered saline, exposure to sucrose, glucose, and the Good's 
buffers: TAPS, CAPS, EPPS, HEPPSO, and DIPSO resulted in negligible 
effects on cell doubling time which indicates the absence of 
exposure-related cellular toxicity. 
TABLE 1 
______________________________________ 
Osmolarity Regulating Agent 
Doubling 
Phosphate Buffered Saline 
31.0 
Sucrose 41.0 
Glucose 34.5 
TAPS 32.7 
CAPS 45.8 
EPPS 32.2 
HEPPSO 23.4 
DIPSO 36.7 
3-Amino-1-propanesulfonic acid 
99.6 
Sodium 3-(N--morpholino)propanesulfonic 
A 
acid (MOPS) 
2-Amino-2-methyl-1,3-propanediol 
B 
2-Amino-2-methyl-1-propanol 
B 
N--tris(hydroxymethyl)methylaminoethane- 
B 
sulfonic acid (TES) 
N,N--bis(2-hydroxyethyl)-2-aminoethane- 
A 
sulfonic acid (BES) 
3-(Cyclohexylamino)-2-hydroxy-1-propane- 
A 
sulfonic acid (CAPSO) 
Triethanolamine B 
Tris(hydroxymethyl)aminoethane (TRIZMA) 
B 
Bis-tris propane B 
2-(N-morpholino)ethanesulfonic acid (MES) 
B 
3-[Dimethyl(hydroxymethyl)methylamino]-2- 
A 
hydroxypropanesulfonic acid (AMPSO) 
N,N-bis(2-hydroxyethyl)glycine (BICINE) 
57.7 
3-[(-3-Cholamidopropyl)dimethylammonio]- 
B 
1-propanesulfonate (CHAPS) 
3-[N-tris(hydroxymethyl)methylamino]- 
63.6 
2-hydroxypropanesulfonic acid (TAPSO) 
3-(N-morpholino)-2-hydroxypropane- 
178.4 
sulfonic acid (MOPSO) 
2-[(2-Amino-2-oxoethyl)amino]ethane 
1038.4 
sulfonic acid (ACES) 
Bis(2-hydroxyethyl)imino-tris- 
A 
(hydroxymethyl)methane (BIS-TRIS) 
2-(N-cyclohexylamino)ethane sulfonic acid 
51.5 
(CHES) 
N-tris-(hydroxymethyl)methylglycine 
A 
(TRICINE) 
Glucosamine 288.4 
Imidazole B 
Glycylglycine 66.9 
______________________________________ 
A No growth or partially cytotoxic 
B Acutely cytotoxic 
Table II shows various osmolarity regulating agents that were examined for 
cyanine dye solubility. All measurements of concentration were made after 
removal of precipitates by centrifugation and dissolving small aliquots of 
osmolarity regulating agents containing cyanine dyes into ethanol for 
spectrofluorometric analysis. The dyes used were DiSC.sub.14 (5) and 
DiOC.sub.14 (3), and the osmolarity regulating agents were at iso-osmotic 
concentrations for mammalian cells. Reductions in fluorescence intensity 
from the ethanol solution standard directly correlate with reductions in 
cyanine dye solubility. 
TABLE II 
______________________________________ 
Relative Fluorescence 
Intensity (CONC) 
Osmolarity Regulating Agent 
DiSC.sub.14 (5) 
DiOC.sub.14 (3) 
______________________________________ 
Ethanol 100 100 
Glucose 31 100 
Fructose 35 100 
Sorbose 40 100 
Sucrose 41 100 
Xylose 36 19-52 
Ribose 24 100 
Lyxose 0.12 1.8 
Glycine 31 93 
Arginine 17 17.2 
Glycerol 39 99.5 
Inositol 42 92 
Xylitol 34 76.4 
Mannitol 29 * 
Adonitol 34 ND 
Tris(hydoxymethyl)- 
18 ND 
methylaminopropane 
sulfonic acid (TAPS) 
3-(Cyclohexylamino)-1- 
40 ND 
propanesulfonic acid (CAPS) 
N-(2-Hydroxyethyl)piperazine- 
18 ND 
N-3-propanesulfonic acid 
(EPPS) 
N-2-hydroxyethylpiperazine- 
20 ND 
N-2-hydroxypropane- 
sulfonic acid (HEPPSO) 
3-[N-N-bis(2-hydroxyethyl) 
43*** ND 
amino]-2-hydroxypropane- 
sulfonic acid (DIPSO) 
NaCl 6 1.7 
Phosphate Buffered Saline 
2.1 6.5 
Na.sub.2 SO.sub.4 7.4 1.6 
NaI 1.1 0.14 
Choline Chloride 11** 6.3 
Choline Iodide 0.16 2.3 
______________________________________ 
*Precipitate in ethanol, no data obtainable. 
**Artifact due to large crystals that did not pellet. 
*** Precipitate in ethanol (data questionable). 
ND Not Determined 
As can be seen from Table II, cyanine dyes are much less soluble in the 
presence of classical salts than in the presence of sugars, except lyxose, 
sugar-alcohols, amino acids, and the Good's buffers, TAPS, HEPPSO, DIPSO, 
CAPS, and EPPS. Additionally, stability of DiSC.sub.14 (5) solutions in 
sugars such as glucose, fructose, ribose, sorbose, sucrose, and xylose, 
sugar-alcohols such as glycerol, inositol, xylitol, and adonitol, and 
amino acids such as glycine and arginine was determined. The cyanine dye 
was stable in the tested solutions for at least twenty minutes, which is 
sufficient time for reproducible labelling, and in many of the agents the 
amount of cyanine dye in solution had not significantly decreased at sixty 
minutes. 
Further, the solubility of cyanine dyes in a medium containing classical 
salts and osmolarity regulators in which the dyes are soluble was 
evaluated. The solubility of DiSC.sub.14 (5) in iso-osmotic glucose 
solution was not significantly affected by dilution with distilled water. 
DiSC.sub.14 (5) solubility in iso-osmotic glucose solution, however, was 
reduced significantly by dilution with only approximately 20% iso-osmotic 
sodium chloride solution. Thus, reproducible cell labelling with cyanine 
dyes can be performed in media containing no more than small amounts of 
classical salts, such as sodium chloride, potassium chloride, calcium 
chloride, sodium acetate, potassium acetate, sodium sulfate, sodium 
iodide, choline chloride, or choline iodide, and preferably is performed 
in a medium in which no classical salts are used to regulate osmolarity. 
Cells cyanine dye labelled using the presently invented procedure were 
analyzed to determine the effect of labelling on cell viability. V79 cells 
which are available from the American Type Culture Collection, Rockville, 
Md., and are described in Prescott, D. M., Ann. New York Acad. Sci., 397: 
101-109 (1982), were labelled with a solution containing DiOC.sub.14 (3) 
at a concentration of 10.sup.-5 or 4.times.10.sup.-5 M and the growth 
kinetics of the stained cells were compared to unstained cells and an 
equal mixture of stained and unstained cells. Cell doubling time was 
unaffected by cyanine dye labelling. Thus, labelling had no effect on cell 
growth. Also, several other standard tests of cell viability such as 
Trypan Blue Exclusion and Propidium Iodide exclusion confirmed an absence 
of effect on cell viability of cyanine dye labelling according to the 
described procedures. 
To test in vivo stability of cells cyanine dye labelled according to the 
presently invented method, rabbit red cells were withdrawn, labelled with 
DiSC.sub.14 (5), and reinfused. Periodically thereafter, blood samples 
were obtained and analyzed for percent labelled cells and fluorescence 
intensity of the labelled cells. The number of circulating red cells 
decreased linearly as a function of time and the measured 52 day lifetime 
of labelled cells correlated closely with the 40 to 60 day average 
reported lifetime of rabbit red cells. Thus, cyanine dye labelling did not 
affect the clearance rate of red blood cells. 
In all but one of the five rabbits tested, fluorescence intensity of the 
stained cells remained essentially unchanged 60 days after labelling and 
reinjection. In the fifth animal, not more than 20% of the cyanine dye had 
migrated from the labelled cells after 60 days in the rabbits circulation. 
These data combined with data from tissue culture showing no transfer of 
dye from labelled to unlabelled cells demonstrates that the cells are 
stably labelled with the dyes. 
Cyanine dye-labelled viable cells are used in the invented methods for 
determining cell growth rate. Growth rate is determined by measuring 
changes in the levels of cyanine dye in the plasma membranes of the cells. 
Each time a cell divides the plasma membrane associated cyanine dye is 
distributed equally between the daughter cells. Thus, serial measurements 
of the plasma membrane cyanine dye levels of labelled, growing cells are 
used to calculate growth rate. 
Flow cytometric methods using standard techniques are preferred for 
measuring plasma membrane cyanine dye levels of non-adherent cells or 
cells that can be removed from their growth substrate and suspended as 
single cells. An adherent cell cytometer (Meridian ACAS 470) is preferred 
for cases where removal from the growth substrate is difficult or not 
feasible. 
Determinations of cell growth rate are used in a variety of applications. 
For example, growth rate of tissue culture cells is measured to optimize 
growth conditions. Sensitivity of tumor cells to chemotherapeutic agents 
is determined by measuring cell growth rate in media containing these 
agents. Similarly, sensitivity of yeast cells to various antifungal agents 
is determined by measuring the growth rate of the yeast cells in media 
containing antifungal agents. 
The invented methodology also is used to monitor growth rate of tissue 
cells in vivo. For example, bone marrow transplant engraftment is 
determined by measuring bone marrow cell growth rate following transplant. 
Growth rate of other in vivo cells, such as corneal epithelial cells, is 
measured to determine post-traumatic or post-surgical healing. 
The following examples illustrate the present invention and do not limit 
the scope of the invention defined above and claimed below. 
EXAMPLE 1 
Method for Staining Tissue Culture Cells 
I. Preparation of Cells 
Log phase tissue culture cells are used to obtain best results. Suspension 
cultures are removed from the culture vessel and placed into polypropylene 
centrifuge tubes. 
When using monolayer cultures, supernatants must be removed and the 
adherent cells washed with calcium and magnesium free phosphate buffered 
saline solution to remove serum proteins from the flask. Trypsin-EDTA 
solution (Gibco Laboratories, Grand Island, N.Y., #610-5300) is added to 
cover the bottom of the flask and is allowed to incubate at room 
temperature until the cell monolayer is dislodged and disaggregated. The 
resultant cell suspension is transferred to a polypropylene centrifuge 
tube and an equal volume of culture media containing 10% Fetal Bovine 
Serum (FBS) (Hazelton) is added to arrest the enzymatic action of the 
trypsin. 
Cells are centrifuged at 400xg for ten minutes at room temperature. 
Supernatants are aspirated and an equal volume of iso-osmotic mannitol is 
replaced for resuspension of the cell pellet. This mannitol wash is to 
remove the plasma proteins from the cell suspension and prepare cells for 
staining. Cells are once again centrifuged at 400xg for ten minutes at 
room temperature. The supernatants are aspirated and the resultant cell 
pellet is resuspended in mannitol solution at a concentration of 
2.times.10.sup.6 cells/ml for staining. Some cell lines, however, are 
sensitive to the use of a sugar alcohol (mannitol); in such cases an 
iso-osmotic glucose solution (MW 180.16, 54.05 g/l) may be used. 
II. Preparation of Stock Dye Solutions 
2.times.10.sup.-3 M stock solutions are prepared as follows in absolute 
ethanol. 
______________________________________ 
DiO--C.sub.14 (3) 
MW 800 (1.600 mg/ml) 
DiS--C.sub.14 (5) 
MW 814 (1.628 mg/ml) 
DiO--C.sub.18 (3) 
MW 936 (1.872 mg/ml) 
DiI--C.sub.14 (5) 
MW 850 (1.700 mg/ml) 
______________________________________ 
All dyes are obtained from Molecular Probes, Eugene, Oreg. 
Dye stocks are sonicated to insure complete solubility of the dye and to 
minimize adherence to the tubes. Polystyrene tubes are used for 
preparation of stock solutions so that solubility of the dye can be 
observed. Polypropylene tubes, however, are used to stain cells because 
cyanine dyes in an aqueous environment are much less adherent to 
polypropylene when compared to polystyrene. 
III. Cell Staining 
Cells are adjusted to a concentration of 2.times.10.sup.6 cells/ml in 
iso-osmotic mannitol. To stain cells, 2.times.10.sup.-3 M stock dye 
solution is added to the staining solutions at 5 .mu.l of dye per 1 ml of 
cell suspension giving a final concentration of 10 .mu.M. The sample for 
staining is pipetted or vortexed to thoroughly mix the sample. Cells are 
incubated with the dye for ten minutes, after which a small aliquot is 
removed for examination under a fluorescent microscope to insure that 
intense and uniform staining has occurred. The DiO dye series uses 
microscope filters selective for 488 nm excitation light, while the DiS 
and DiI dye series requires excitation near 575 nm for observation of 
fluorescence. 
After the incubation period, an equal volume of PBS is added to the 
stain-cell suspension. The cells are centrifuged at 400xg for ten minutes 
at 20.degree. C. The supernatant is aspirated and the pellet is 
resuspended in PBS. The centrifugation procedure is repeated and the 
resultant supernatant is observed for the presence of dye. If dye is 
apparent in the supernatant, washing is repeated until the supernatants 
are devoid of free dye as measured by spectofluorometry. After the final 
wash, the supernatant is removed and the pellet resuspended to the desired 
concentration in a suitable culture medium. All procedures are performed 
under sterile conditions. 
EXAMPLE 2 
Measuring Growth Rate of Tissue Culture Cells 
V79 cells were stained with DiOC.sub.14 (3) as described in Example 1. 
Fluoresence intensity was measured using an EPICS 750 Flow Cytometer 
(Coulter Electronics, Inc.). Immediately after staining, fluorescence 
intensity of an aliquot of the stained cells was measured. The remaining 
cells were grown in a humidified air-CO.sub.2 (7.5% CO.sub.2) incubator at 
37.degree. C. in a standard complete growth medium. On days one, two, and 
three after staining, aliquots of cells were removed for fluorescence 
intensity determinations. 
FIG. 1 shows the log fluorescence intensity profile of the growting V79 
cells. The numerical value above each peak is the mean log fluorescence 
intensity on each day. As FIG. 1 shows, the mean log fluorescence 
intensity diminishes daily as the cells grow in culture. 
From fluorescence measurements, growth rate is determined from the slope of 
the regression line fit to the linear portion of a plot of log 
fluorescence intensity versus time. 
To make certain that staining the cells did not affect growth rate, growth 
rates of stained and unstained V79 cells were compared. The results shown 
in FIG. 2 demonstrate that unstained cells grew at a rate equivalent to 
cells stained with 10.sup.-5 M dye or 4.times.10.sup.-5 M dye, or an equal 
mixture of stained and unstained cells. 
Since dye transfer from stained to unstained cells would result in 
erroneous growth rate determinations, stained and unstained cells were 
grown together in culture. A human colon carcinoma cell line, HT29, which 
is available from the American Type Culture Collection, Rockville, Md., 
and is described in J. Fogh and G. Tremp, Human Tumor Cells In Vitro, pp. 
115-159, Plenum Press, New York (1975), stained with DiOC.sub.14 (3) and 
unstained human promyelocytic leukemia, HL60, cells, available from the 
American Type Culture Collection, Rockville, Md., and described in 
Collins, S. J., et al., Nature, 270: 347-349 (1977), were grown together 
in a ratio of 1:1 stained to unstained cells. These data are displayed in 
FIG. 3. 
In FIG. 3, the fluorescence intensity of the stained HT29 cells cultured 
alone (diamonds) demonstrated the characteristic reduction in fluorescence 
intensity as a function of time in culture. The unstained HL60 cells had 
very little fluorescence for four (4) days, after which there appeared 
some modest increase in fluorescence intensity. Furthermore, the stained 
HT29 cells which were co-cultured with HL60 cells (squares) lost 
fluorescence intensity exactly as the unmixed HT29 cells until 
approximately day five, where there appeared some additional but minimal 
loss of fluorescence intensity. From these data it is clear that perhaps 
the HL60 cells are picking up some fluorescence after being in culture for 
four (4) days with the HT29 cells. It should be noted that the HL60 cells, 
however, are promyelocytic and have some capability to phagocitize cells 
and debris. The increase in fluorescence intensity experienced after day 4 
in the unstained subpopulation of the mixed culture may in fact be a 
result of phagocytosis of fluorescent debris. More importantly, for four 
days in culture, there was no increase in fluorescence intensity of the 
unstained HL60 cells. Furthermore, the stained HT29 cells in the same 
mixture appear to have the same fluorescence kinetics as the HT29 cells 
which are unmixed. Thus, cell-to-cell dye transfer does not lead to 
incorrect growth rate determinations. 
EXAMPLE 3 
Measuring Growth Rate of Tumor Cells 
If the tumor cells being studied are a tissue culture line adapted to in 
vitro culture conditions, then the cells are stained and evaluated as 
outlined in Examples 1 and 2. If the tumor cells being studied are from 
tumor tissue explants then they are dissaggregated into single cells by 
standard techniques and plated onto Lab-Tech tissue culture chamber 
slides. The cells are stained using the cyanine dyes as outlined in 
Example 1. They are then placed into an humidified air-CO.sub.2 incubator 
at 37.degree. C. and allowed to equilibrate. At periodic intervals the 
slides are placed onto the microscope stage of an adherent cell cytometer 
(i.e., Meridian ACAS 470) and measurements of fluorescence intensity are 
made. The locations of the cells relative to an index mark are determined 
by the computer so that serial measurements of fluorescence intensity can 
be made. The slide is returned to the incubator to allow the cells to 
continue growing. The microscope slide should contain a fluorescence 
standard such as Coulter fullbright polystyrene microspheres (Coulter 
Electronics, Hialeah, Fla. that do not change fluorescence intensity with 
time in culture. This standard is used to make comparison measurements of 
fluorescence intensity. 
Tumor cells are identified from normal stromal cells on the basis of 
morphometric parameters using phase optics on the microscope or 
fluorescent monoclonal antibodies specific to tumor cells. The measurement 
of cell growth is made by monitoring dilution of the cyanine fluorescence 
in the tumor cells as each cell divides and applying the equations of 
Example 9. 
The measurement of fluorescence intensity can also be made using a flow 
cytometer. The cells, however, are removed from the microscope slide 
before flow cytometric analysis is made. This procedure does not permit 
the serial quantification of dye dilution on the same cells day after day. 
In some instances, however, such as leukemia cells, this approach is 
preferred. 
The process of measuring in vitro cell growth is accomplished on cells that 
have been cultured in optimal growth medium or in the presence of various 
levels of agents used to treat tumors. The ability of the therapeutic 
agent to inhibit the tumor cell growth as measured by inhibition of 
fluorescence intensity reduction, is a measure of the effectiveness of 
that agent to kill tumor cells. 
EXAMPLE 4 
Measuring White Blood Cell Growth Rate 
Lymphocytes are removed by venipuncture or splenic dissection using 
standard techniques. The cells are labelled with a cyanine dye using the 
protocol listed in Example 1 but substituting glucose or sucrose for 
mannitol as the osmotic support medium. The stained cells then are 
aliquoted into microtiter dishes at a level of 5.times.10.sup.5 cells per 
well (Mazumder, A., Grimm, E. A., Zhang, H. Z., and Rosenberg, S. A., 
Cancer Res. 42, 918 (1982)) and incubated with the appropriate mitogen 
such as Interleukin-2, sodium periodate (IO.sub.4 -), phytohemagglutinin, 
concanavalin A, pokeweed-mitogen and B Cell Growth Factor (BCGF). The 
cells are placed into a 37.degree. C. humidified air-CO.sub.2 incubator 
and allowed to grow. At periodic intervals cells are removed from the 
culture vessel and examined by flow cytometric procedures. The data 
obtained are similar to that obtained by the process of Example 2 and are 
analyzed using the equations of Example 9. 
EXAMPLE 5 
Measuring Bacteria Growth Rate 
The cells are labelled with a cyanine dye using the protocol listed in 
Example 1 but substituting glucose or sucrose for mannitol as the osmotic 
support medium. The stained cells then are aliquoted into microtiter 
dishes at a level of 5.times.10.sup.5 cells per well in a nutrient broth. 
The cells are placed into a 37.degree. C. humidified incubator and allowed 
to grow. At periodic intervals cells are removed from the culture vessel 
and examined by flow cytometric procedures. The data obtained are similar 
to that obtained in the process of Example 2 and are analyzed using the 
equations of Example 9. 
The process of measuring in vitro cell growth is accomplished on cells that 
have been cultured in optimal growth medium or in the presence of various 
levels of antibiotics which are being tested as antibacterial agents. The 
ability of the bactericidal agent to inhibit the bacterial cell growth as 
measured by inibition of cellular fluorescence intensity reduction, is a 
measure of the effectiveness of that agent to kill bacteria. 
EXAMPLE 6 
Measuring Yeast Growth Rate 
The cells are labelled with the cyanine dye using the protocol listed in 
Example 1 but substituting glucose or sucrose for mannitol as the osmotic 
support medium. The stained cells then are aliquoted into microtiter 
dishes at a level of 5.times.10.sup.5 cells per well in a nutrient broth. 
The cells are placed into an incubator and allowed to grow. At periodic 
intervals cells are removed from the culture vessel and examined by flow 
cytometric procedures or by adherent cell cytometric procedures (Meridian 
ACAS 470). The data obtained are similar to that obtained by the process 
of Example 2 and are analyzed using the equations of Example 9. 
The process of measuring in vitro cell growth is accomplished on cells that 
have been cultured in optimal growth medium or in the presence of selected 
levels of compounds which are being tested as antifungal agents. The 
ability of the fungicidal agent to inhibit the fungal cell growth as 
measured by inibition of cellular fluorescence intensity reduction, is a 
measure of the effectiveness of that agent to kill fungi. 
EXAMPLE 7 
Measuring Growth Rate of Bone Marrow Cells 
Bone marrow cells are removed by aspiration (Illinois needle) or by core 
biopsy (Jamshiti needle) from the sternum or iliac crest. The cells are 
labelled with a cyanine dye using the protocol listed in Example 1 but 
substituting glucose or sucrose for mannitol as the osmotic support 
medium. The cells are subjected to flow cytometric analysis to determine 
the level of fluroescence intensity prior to infusion of the bone marrow 
cells into the recipient. Labelled cells are injected intravenously and an 
appropriate time interval is allowed to elapse before a sample of 
peripheral blood and bone marrow is taken. Blood and marrow are taken from 
the recipient, mixed with anticoagulants, and prepared according to 
standard techniques for flow cytometric analysis. These samples contain 
cells which are growing and cells which are in cell cycle arrest. The 
histograms will be complex but are analyzed for cell growth similar to the 
data obtained in the process of Example 2 by applying the equations of 
Example 9. 
Because the tracking dyes are fluorescence green, another dye coupled to 
monoclonal antibodies is used to identify cells from each of the cell 
lineages found in marrow. Using a monoclonal antibody which stains red 
cells and their precursors, the red fluorescence is used to identify this 
lineage and then monitor the reduction in green fluorescence (and 
therefore cell growth) of the cells in the red cell lineage. 
Similar two color approaches are used to evaluate lymphoid, myeloid, and 
monocyte cell growth. 
EXAMPLE 8 
Measuring Corneal Epitheal Growth Rate 
This methodology is used to monitor growth of corneal epithelial cells 
after transplant and uses technology that is not injurious to cell growth 
or painful to the eye when monitoring cell growth. Immediately after 
transplant, the eye is bathed in an opthalmic formulation of the cyanine 
dye which absorbs light at wavelengths greater than 680 nm. The procedure 
also is carried out using dye which absorbed light at wavelengths lower 
than 680 nm, however, the excitation beam causes severe headaches when the 
tissue is examined. 
At time zero, infrared photographs are taken (wavelengths greater than the 
absorbance maximum of dye) while exciting the cyanine dye bound to the 
cornea. The fluorescence intensity level is a measure of cell staining at 
time zero. At subsequent times the eye is photographed and the image is 
compared to the time zero photograph. In areas where there is cell growth, 
the fluorescence intensity of the cells decreases. Quantitative assessment 
of the fluorescence intensity is used to determine the number of cell 
doublings. 
EXAMPLE 9 
Calculating Cell Growth Rate 
The following mathematical formula is used to calculate cell doubling time: 
##EQU1## 
wherein: T.sub.D is the cell doubling time, 
t.sub.2 and t.sub.1 are any times during log phase cell growth, and 
F(t.sub.2) and F(t.sub.1) are the mean cellular fluorescence intensity at 
times t.sub.2 and t.sub.1, respectively, 
ln signifies the natural (base e) logarithm. 
The number of cell doublings occuring during a period of growth is 
determined by the formula: 
##EQU2## 
wherein: N is the number of cell doublings, 
F.sub.o is the initial fluorescence intensity, and F(t) is the fluorescence 
intensity at any time after a period of cell growth. For determination of 
the number of cell doublings, the fluorescence measurements do not have to 
be made during the log phase of cell growth. 
The following derivation demonstrates that the above formulae accurately 
determine cell doubling time and the number of cell doublings, and that 
the behavior of plasma membrane cyanine dye levels in growing cells 
predicted by mathematical modeling parallels that actually measured. 
The derivation is based on the following assumptions: 
1. Cells are set in culture at low density with an initial cell number 
(N.sub.o), and average initial fluorescence intensity (F.sub.o) and a cell 
cycle time (T.sub.2). 
2. Dye distributes evenly to daughter cells on cell division. 
3. There is no lag time in the culture. 
4. Cell death is negligible. 
5. Staining is permanent; there is no cell-cell dye transfer. 
Based on the second assumption above, the average population fluorescence 
should be inversely proportional to the number of cells in the population 
at any time t. This relationship is defined in equation 1, 
EQU F(t)N(t)=K (1) 
where F(t) and N(t) are the average population fluorescence and cell number 
at time t, respectively. K is a proportionality constant, to be defined. 
If we evaluate this equation at time zero, then it follows that K=F.sub.o 
N.sub.o. Substituting this into equation 1 and solving for F(t) gives, 
EQU F(t)=F.sub.o N.sub.o (1/N(t)). (2) 
F.sub.o and N.sub.o were defined in assumption 1. 
It is obvious from equation 2 that the fluorescence kinetics must be 
directly related to the growth kinetics, if the dye being used acts in an 
ideal fashion. The form of the relationship defining the fluorescence 
kinetics will now be determined. The cell number in equation 2 follows 
common growth kinetics defined below, 
EQU dN(t)/dt=AN(t) (3) 
where A is the proportionality constant relating the rate of population 
growth to the number of cells in the population. Equations 4 to 7 show the 
simple solution of equation 3. 
EQU dN(t)/N(t)=d ln N(t)=Adt (4) 
EQU N(t)=exp (At+C) (5) 
EQU N(t)=N.sub.o exp (At) (6) 
EQU N(t)=N.sub.o exp (0.693t/T.sub.2) (7) 
The result in equation 7 can be substituted into equation 2 to solve for 
F(t). 
##EQU3## 
This reduces directly to, 
EQU F(t)=F.sub.o exp (-0.693t/T.sub.2). (9) 
The interpretation of this equation requires careful consideration. First, 
because we have assumed negligible cell death, T.sub.2 represents the cell 
cycle time, not the cell doubling time. This point will be considered more 
carefully below. If there is cell death, this equation is still valid, as 
long as the dye bound to dead cells is not reabsorbed by the live cells. 
The case where reabsorption occurs will be treated below. In the case 
where a fraction of the population is not growing, equation 9 is valid 
only for the growing fraction. Consequently, F.sub.o and N.sub.o apply 
only to the growing fraction. 
The Effect of Cell Death on Fluorescence Kinetics 
Let us now consider the case where cell death is appreciable and dye 
transfer to the live cells occurs rapidly. The kinetics of cell death are 
represented in equation 10. 
EQU (dN(t)/dt).sub.1 =BN(t) (10) 
Above, B is the proportionality constant relating the rate of loss of cells 
from the population to the size of the population. The derivative is 
subscripted; the 1 corresponds to the process of cell death. Let us 
rewrite equation 3 in a similar fashion. 
EQU (dN(t)/dt).sub.2 =AN(t) (3) 
Here, the 2 corresponds to the process of cell growth. Combining the growth 
and death processes gives, 
EQU dN(t)/dt=(dN(t)/dt).sub.1 +(dN(t)/dt).sub.2. (11) 
This equation is simply the result of the superposition of two independent 
processes. By substitution, it follows that, 
EQU dN(t)/dt=(A+B)N(t) (12) 
By analogy to equations 4 to 6, this equation reduces to, 
EQU N(t)=N.sub.o exp ((A+B)t). (13) 
A was determined in equation 7 to be 0.693/T.sub.2. In a similar fashion, B 
can be shown to be equal to -0.693/T.sub.1/2. The negative sign 
corresponds to the fact that cell death is a decay process. Likewise, 
T.sub.1/2 now represents a decay constant which we will call the average 
cell half-life in the population. By substitution, equation 13 becomes, 
EQU N(t)=N.sub.o exp ((0.693/T.sub.2 -0.693/T.sub.1/2)t) (14) 
By combining equations 3 and 14, we can solve for the fluorescence 
kinetics. 
EQU F(t)=F.sub.o exp ((0.693/T.sub.1/2 -0.693/T.sub.2)t) (15) 
In the above equation, we can combine the constants T.sub.2 and T.sub.1/2 
as shown below. 
EQU 1/T.sub.D =1/T.sub.2 -1/T.sub.1/2 (16) 
Incorporating the relationship in equation 16 into equations 14 and 15 
gives the cell and fluorescent kinetic equation listed below. 
EQU N(t)=N.sub.o exp (0.693t/T.sub.D) (17a) 
EQU F(t)=F.sub.o exp (-0.693t/T.sub.D) (17b) 
In equations 17a and 17b, the parameter, T.sub.D, has the units of time and 
represents the actual cell doubling time rather than the cell cycle time. 
Equation 17b will define the fluorescence distribution in a culture where 
cell death is significant and dye reabsorption is rapid. If either of 
these conditions is not met, then equation 9 applies. A comparison of 
equations 17a and 17b shows that the cell and fluorescence kinetics are 
inversely related, such that a plot of the cell growth curve and the 
inverse of the fluorescence kinetics curve can be superimposed. This fact 
was clearly demonstrated in FIG. 4. 
Incorporation of a Cell Growth Lag Time 
Let us now assume that there is a lag time when the cells are put into 
culture. Let us also simplify the treatment by further assuming that the 
population will go from a growth rate of zero to its maximum rate in one 
step. Under these conditions, it can readily be shown that the 
relationship defining cell growth is, 
##EQU4## 
Here, t.sub.L is defined as the lag time. Combining equation 3 with the 
above relationships gives, 
##EQU5## 
Using these equations, both the doubling time and the lag time can be 
determined by knowing F.sub.o and by determining F(t) at three or four 
times during exponential growth. 
Determination of the Growing Fraction 
Let us consider the case where a fraction of the population is not growing. 
Initially let us assume there is no lag time. We will add back a lag time 
later. For the purpose of this derivation a definition is needed. In 
equation 21, 
EQU N.sub.o =(N.sub.o).sub.g +(N.sub.o).sub.n (20) 
The subscripts g and n refer to the growing and non-growing fractions, 
respectively. At any time, t, the number of cell in the population will be 
the sum of the growing and non-growing populations. 
EQU N(t)=(N.sub.o).sub.n +(N.sub.o).sub.g exp (0.693t/T.sub.D) (21) 
However, this equation cannot be substituted into equation 3 as we have 
previously done. In this case, the growing and non-growing fractions must 
be handled separately. For this purpose, equation 3 must be rewritten, as 
shown. 
EQU F(t)=(F.sub.o).sub.n (N.sub.o).sub.n (1/N(t)).sub.n +(F.sub.o).sub.g 
(N.sub.o).sub.g (1/N(t)).sub.g (22) 
In equation 22, (1/N(t)).sub.n is equal to (1/(N.sub.o).sub.n). Therefore, 
the equation reduces as shown below. 
EQU F(t)=(F.sub.o).sub.n +(F.sub.o).sub.g (N.sub.o).sub.g (1/N(t)).sub.g (23) 
EQU F(t)=(F.sub.o).sub.n +(F.sub.o).sub.g exp (-0.693t/T.sub.D) (24) 
We can also introduce a lag time by analogy to equation 19b. 
ti F(t)=(F.sub.o).sub.n +(F.sub.o).sub.g exp (-0.693(t-t.sub.L)/T.sub.D) 
(25) 
The fraction of growing cells in the population can be determined using 
equation 25. The determination is more complex than in previous cases 
because sub-populations are involved. To determine the growing fraction, 
one must wait until the sub-populations are distinguishable. At this 
point, the fluorescence intensity of the non-growing fraction can be 
determined directly. An extrapolation of the change in fluorescence 
intensity over time of the growing fraction to time zero allows one to 
calculate the fluorescence intensity of the growing fraction at zero time. 
The growing fraction is then simply the ratio of the fluorescence 
intensity of the growing fraction at zero time to the total population 
fluorescence intensity. 
The preferred embodiments of the invnention are illustrated by the above, 
however, the invention is not limited to the instructions disclosed 
herein, and all rights to all modifications within the scope of the 
following claims is reserved.