U.S. patent application number 12/831108 was filed with the patent office on 2011-02-03 for methods and systems for identifying and isolating stem cells and for observing mitochondrial structure and distribution in living cells.
Invention is credited to Roy S. Chuck.
Application Number | 20110027787 12/831108 |
Document ID | / |
Family ID | 36407619 |
Filed Date | 2011-02-03 |
United States Patent
Application |
20110027787 |
Kind Code |
A1 |
Chuck; Roy S. |
February 3, 2011 |
METHODS AND SYSTEMS FOR IDENTIFYING AND ISOLATING STEM CELLS AND
FOR OBSERVING MITOCHONDRIAL STRUCTURE AND DISTRIBUTION IN LIVING
CELLS
Abstract
Methods and systems for a) identifying and isolating stem cells,
b) assessing mitochondrial distribution and structure in living
cells and c) performing fluorescence microscopy on living cells
while the cells remain within a condition-controlled cell culture
chamber.
Inventors: |
Chuck; Roy S.; (Towson,
MD) |
Correspondence
Address: |
STOUT, UXA, BUYAN & MULLINS LLP
4 VENTURE, SUITE 300
IRVINE
CA
92618
US
|
Family ID: |
36407619 |
Appl. No.: |
12/831108 |
Filed: |
July 6, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11667280 |
Nov 1, 2007 |
7749726 |
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PCT/US05/40406 |
Nov 8, 2005 |
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12831108 |
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60625597 |
Nov 8, 2004 |
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Current U.S.
Class: |
435/6.16 ;
435/288.7 |
Current CPC
Class: |
G01N 33/5002
20130101 |
Class at
Publication: |
435/6 ;
435/288.7 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12M 1/34 20060101 C12M001/34 |
Goverment Interests
STATEMENT REGARDING GOVERNMENT SUPPORT
[0002] Aspects of this invention were made with government support
through National Institutes of Health grant number EY000412-04.
Accordingly, the government may have certain rights in this
invention.
Claims
1. A fluorescence microscopy system for performing fluorescence
microscopy of living cells, said system comprising: a fluorescence
microscope; a microscope stage; and a cell culture chamber
associated with the microscope stage such that living cells within
the cell culture chamber may be positioned on the microscope stage
and viewed by the microscope without requiring removal of the cells
from the cell culture chamber.
2. A system according to claim 1 further comprising apparatus for
maintaining at least one predetermined condition within the cell
culture chamber.
3. A system according to claim 2 wherein said at least one
condition is selected from the group consisting of: temperature,
humidity, pH, osmolarity, nutrient levels, ion levels and ambient
gas composition, partial pressure or concentration of CO.sub.2,
partial pressure or concentration of O.sub.2 and partial pressure
or concentration of N.sub.2.
4. A system according to claim 2 further comprising a fluid
circulator for circulating cell-containing culture medium over the
microscope stage.
5. A system according to claim 2 further comprising a camera
useable for obtaining photomicrographs through the fluorescence
microscope.
6. A system according to claim 5 further comprising a timer that
causes the camera to obtain photomicrographs at desired time
points.
7. A system according to claim 5 further comprising a programmable
controller for controlling at least the camera so as to obtain
photomicrographs at desired time points.
8. A method for observing structural and/or functional attributes
of mitochondria in living cells, said method comprising the step
of: (a) detecting intrinsic reduced pyridine nucleotides in the
mitochondria.
9. A method according to claim 8 wherein Step (a) comprises causing
mitochondrial pyridine nucleotides to autofluorescence and then
detecting the autofluorescence emitted thereform.
10. A method according to claim 9 wherein Step (a) is carried out
by autofluorescence microscopy.
11. A method according to claim 9 wherein the autofluorescence
distribution is detected.
12. A method according to claim 9 wherein the intensity of
autofluorescence is detected.
13. A method of claim 11 wherein perinuclear concentrations of
autofluorescence are detected.
14. A method according to claim 34 wherein a histogram of
autofluorescence distribution is prepared.
15. A method according to claim 8 wherein Step (a) is performed by
autofluorescence using exitation by light at a wavelength that does
not damage or kill the cells.
16. A method according to claim 15 wherein the excitation
wavelength is about 365 nm.
17. A method according to claim 15 wherein the exitation wavelength
is outside of medium and short UV wavelengths that are capable of
causing cellular damage.
18. A method according to claim 8 wherein Step (a) is performed by
autofluorescence and wherein the method further comprises the step
of: decreasing exposure of the cells to light at wavelengths that
will cause photobleaching.
19. A method according to claim 18 wherein the step of decreasing
exposure of the sample to light at wavelengths that will cause
photobleaching comprises turning off an illumination source during
fluorescence imaging.
20. A method according to claim 18 wherein the step of decreasing
exposure of the sample to light at wavelengths that will cause
photobleaching comprises using an illumination source that emits
light at a wavelength that is not within the excitation or
autofluorescence wavelength detection gates being employed.
21. A method according to claim 18 wherein the step of decreasing
exposure of the sample to light at wavelengths that will cause
photobleaching comprises shielding the sample from light during at
least a portion of the procedure.
22. A method according to claim 18 wherein the cells are maintained
in a cell culture chamber that maintains the living cells under
controlled conditions during performance of the method.
23. A method according to claim 22 wherein the controlled
conditions include at least one condition selected from the group
of: temperature, humidity, pH, osmolarity, nutrient levels, ion
levels and ambient gas composition.
24. A method according to claim 8 wherein the method is repeated at
one or more time points to determine changes in the mitochondrial
distribution and/or structure of the cells over said one or more
time points.
25. A method according to claim 8 wherein the cells are subjected
to a treatment and wherein the method is performed i) prior to the
treatment and ii) after the treatment, to thereby determine a
treatment-effect or a lack of treatment-effect on mitochondrial
distribution and/or structure.
26. A method according to claim 25 wherein the treatment comprises
exposing the cells to a drug, therapeutic agent or other test
substance.
Description
RELATED APPLICATIONS
[0001] This utility patent application is a division of copending
U.S. patent application Ser. No. 11/677,280 filed on May 7, 2007
and issued on Jul. 6, 2010 as U.S. Pat. No. 7,749,726, which is a
Section 371 national stage of PCT International Patent Application
PCT/US2005/40406 filed on Nov. 8, 2005, which claims priority to
U.S. Provisional Patent Application No. 60/625,597 filed on Nov. 8,
2004, the entirety of each such application being expressly
incorporated herein by reference.
FIELD OF THE INVENTION
[0003] This invention relates broadly to the fields of biology and
medicine and more specifically to methods and systems for
identifying and isolating stem cells, assessing mitochondrial
distribution and structure in living cells and performing
fluorescence microscopy on living cells while the cells remain
within a condition-controlled cell culture chamber.
BACKGROUND OF THE INVENTION
Stem Cells
[0004] Stem cells are undifferentiated primal cells that are
capable of differentiating into cells of various other types.
Because of their ability to form various types of differentiated
cells, stem cells function to replenish other cells throughout the
life of the organism.
[0005] Research efforts are currently aimed at developing methods
wherein stem cells are used to repair damaged tissues or to grow
replacement organs or body parts. In order for stem cells to be
routinely useable for such therapeutic applications, it will be
necessary to develop techniques for efficiently and reliably
identifying and isolating stem cells from populations of other
differentiated cells.
[0006] Stem Cell identification and Isolation
[0007] Techniques to isolate adult and embryonic stem cells for
clinical and experimental use are in development by many workers.
However, although much effort has been expended in searching for
stem cell probes, currently there still exists no reliably specific
marker for stem cells of any type. Additionally, many of the
markers that have been proposed and studied are invasive to the
cell or cell surface and may alter or even kill the cell.
[0008] One of the most accepted properties of stem cells is their
ability to slowly cycle and continuously replenish cell
populations. We hypothesized and found that such slow cycling cells
can be characterized by slower intrinsic metabolism. As such, these
slow cycling populations are detectable via metabolic monitoring.
We have developed a novel fluorescence spectroscopic technique and
device to separate live, unlabeled stem cells from further
differentiated stages of development based on this simple
finding.
[0009] Currently there exists no reliable, non-invasive technique
to specifically detect or screen for the presence of stem cells, or
to reliably isolate stem cells from a matrix that contains stem
cells along with other types of cells. Development of such a
technique, especially a generalizable one, would not only represent
a unique stem cell detection and separation method/device, but
would also provide a viable alternative to the currently popular
search for lineage-specific markers.
[0010] Mitochondria
[0011] Mitochondria are elongate or rod shaped, membrane-enclosed,
organelles located within cells of the body. Mitochondria function
as the major source of a cell's energy by oxidizing the products of
carbohydrate and lipid metabolism. Mitochondria are located outside
the nucleus of the cell but contain some independent DNA and may be
reproduced as needed by the cell within which they reside. Damage
to mitochondria can result in muscular weakness and fatigue and, in
some cases, may lead to life-threatening conditions, such as lactic
acidosis. Nucleoside analogs may cause mitochondrial toxicity.
[0012] In normal healthy cells, mitochondria are diffusely
distributed throughout the cytoplasm. Abnormal clustering of
mitochondria in the perinuclear region of the cell can be
indicative of changes in the metabolic state of the cell. Existing
literature supports two mechanisms to explain how mitochondrial
redistribution occurs. Firstly, these organelles may simply migrate
via a complex microtubular network from the perinuclear region to
the cell periphery. In cells transfected with a mutant member of
dynamin, a family of membrane transport proteins, perinuclear
aggregates of mitochondria were demonstrated by electron
microscopy, whereas cells with normal tubular projections exhibited
diffuse mitochondrial arrangement. Impairment of kinesin-mediated
transport by tumor necrosis factor-related apoptosis inducing
ligand (TRAIL) also leads to these changes in mitochondrial
distribution. Furthermore, treatment with microtubule-active drugs
(taxol, nocodazole and colchicine) results in perinuclear
clustering of mitochondria. The second mechanism involves the
presence of two populations of mitochondria, perinuclear and
peripheral, as observed by confocal fluorescence microscopy of
mitochondria labeled with two potentiometric probes, rhodamine 123
and dimethylaminostyryl-methylpyridiniumiodine, with each
population exhibiting different levels of activity and morphology
depending on cell type. Cultured cancer cells have exhibited
increased perinuclear fluorescence in strains sensitive to
chemotherapeutic drugs and increased peripheral activity and
consequently fluorescence, in the mitochondria of resistant
strains.
[0013] There exists a need for the development of new techniques
for assessing changes or abnormalities of mitochondrial
distribution and structure in living cells without the need for
killing the cell or destroying the tissue within which the cell is
located.
[0014] Reduction-oxidation (Redox) Fluorometry
[0015] Redox Fluorometry is an optical spectroscopic technique
wherein autofluorescence is measured from reduced pyridine
nucleotides (PN) and oxidized flavoproteins (Fp). The ratio of PN
to Fp (PN/Fp ratio) is then calculated. This PN/Fp ratio may be
used as an indicator of tissue metabolic rate.
[0016] In the performance of redox fluorometry, the amount of PN
(i.e., NADH and NADPH) may be estimated by detecting fluorescence
emission in the region of 450 nm after excitation at 366 nm. This
estimate includes both cytoplasmic and mitochondrial NADH and
NADPH, with greater quantum yield from the mitochondrial bound
species. The amount of Fp (i.e., lipoamide dehydrogenase (LipDH)
and electron transfer flavoprotein (ETF)) is then estimated by
detection of fluorescence in the region of 540 nm after excitation
at 460 nm. This measures cellular levels of the flavoproteins which
exist mostly as co-factors for enzymes involved in redox reactions.
The ratio of these fluorescence measurements, which minimizes
interfering factors such as absorption of excitation and emission
light by other intrinsic chromophores, light scattering, and
variations in mitochondrial density and flavoprotein concentration,
has previously been proposed as a non-invasive measure of the organ
cellular metabolic state.
[0017] Initially, most redox fluorometry methods were performed
using one-photon (1P) excitation at near-UV and visible wavelengths
for NAD(P)H and FP fluorescence. However, the use of 1P-redox
fluorometry to determine PN/Fp ratios of cells in situ was found to
be problematic due to photobleaching of intrinsic fluorophores and
other light-induced damage as well as light scattering and
absorption in turbid cell and tissue environments. These problems
with 1 P-redox fluorometry were largely overcome by the use of
multiphoton microscopy coupled with near-infrared (NIR) excitation.
MPM offers several advantages including a) little or no
photobleaching while out of focus, b) three-dimensional resolution,
c) less light scattering and photodamage and the ability to
determine PN/Fp ratio in tissue planes that are below the surface
of an organ or tissue mass. Thus, two-photon (2P) NAD(P)H
fluorescence has become a preferred method for performing redox
fluorometry of in vivo tissues and some other applications. More
recently, the development of two-photon (2P) femtosecond laser
excitation and scanning confocal microscopy has enabled the
three-dimensional mapping of cellular metabolic oxidation/reduction
states in situ with high resolution.
[0018] Redox fluorometry has also been applied to the detection of
cells with deregulated proliferative potential. Using this
non-invasive spectroscopic technique, normal and transformed
fibroblasts have been separated, as have proliferating and
non-proliferating epithelial cells. More recently others have
discovered that intracellular redox state appears to be a necessary
and sufficient modulator of the balance between self-renewal and
differentiation in dividing optic nerve oligodendrocyte-type-2
astrocyte progenitor cells. That is, the intracellular redox state
of freshly isolated progenitors allows prospective isolation of
cells with different self-renewal characteristics.
[0019] The non-invasive microscopic technique of redox fluorometry,
which is based upon stimulated auto fluorescence detection, has
been historically suggested as a viable clinical measure of the
cellular metabolic state. More recently, redox fluorometry has also
been demonstrated to be able to differentiate between self-renewing
and differentiating cells.
SUMMARY OF THE INVENTION
[0020] The present invention provides a method and system for
identifying the presence of stem cells in a sample that contains
differentiated cells of a known cell type, such method comprising
the steps of (a) measuring the PN/Fp ratio of cells present in the
sample and (b) determining whether cells present in the sample
exhibit a PN/Fp ratio that is lower than the PN/Fp ratio that is
known or expected for differentiated cells of the type present in
the sample or within the range of that expected for stem cells of
the type potentially present in the sample. Step (a) may be
performed by i) determining a first fluorescence emission value
indicative of cellular components from reduced pyridine nucleoldes,
ii) determining a second fluorescence emission value indicative of
cellular levels of the flavoproteins lipoamide dehydrogenase
(LipDH) and electron transfer flavoprotein (ETF) and iii)
determining the ratio of the first fluorescence emission value to
the second fluorescence emission value. The first fluorescence
emission value may be determined by measurement of fluorescence in
the region of 450 nm after excitation at 366 nm. The second
fluorescence emission value may be determined by measurement of
fluorescence detected in the region of 540 nm after excitation at
460 nm. Thereafter, Step (b) may be performed by first ascertaining
either a known or expected PN/Fp ratio for differentiated cells of
the type present in the sample and/or ascertaining a known or
expected PN/Fp ratio for stem cells of a type potentially present
within the sample. The actual PN/Fp ratio(s) measured in Step (a)
are compared to the known or expected PN/Fp ratio(s) for
differentiated and/or stem cells and, on that basis, a qualitative
or quantitative determination is made as to whether the sample
contains stem cells.
[0021] Further in accordance with the present invention, to
facilitate the comparison of the measured PN/Fp ratio(s) to the
known or expected PN/Fp ratio(s), the present invention may
optionally comprise a database (e.g., an electronically accessible
database, look-up table, visual key, etc.) that contains known or
expected PN/Fp ratios for various types of human or animal
differentiated cells and/or various types of stem cells. Such
database may then be used for comparison to the measured PN/Fp
ratios to determine whether stem cells are present in the
sample.
[0022] Still further in accordance with the invention, in some
embodiments the sample may comprise cells in a liquid matrix (e.g.,
cell culture medium, umbilical chord blood, etc.) while in other
embodiments the sample may comprise an organ or other mass of solid
tissue. In cases where the sample comprises cells in a liquid
matrix, one-photon (1P) excitation at near-UV and visible
wavelengths may be used for NAD(P)H and FP fluorescence. In cases
where the sample comprises an organ or other mass of solid tissue
two-photon (2P) NAD(P)H fluorescence may be employed to enable the
determination of PN/Fp ratio(s) for cells in vivo, especially for
cells located in tissue planes below the surface of the organ or
tissue mass. Irrespective of whether the sample comprises cells in
a liquid matrix or a solid tissue, the methods of the present
invention may be performed on living cells, without requiring the
cells to be killed or stained for performance of the method.
[0023] Still further in accordance with the present invention, a
method and system of the above-summarized character may optionally
include the additional step of (c) counting, isolating, sorting or
separating stem cells determined to be present within the sample.
This optional step may be accomplished by selectively counting,
isolating, sorting or separating cells that exhibit a PN/Fp ratio
lower than that typical of differentiated cells of the type present
in the sample (or those that match an expected PN/Fp ratio for stem
cells). This may be accomplished by any suitable means, including
but not limited to the use of a fluorescence activated cell sorter
(FACS). Examples of commercially available FACS devices that may be
useable to practice this method of the present invention include
the BD FACSAria.TM., BD FACSVantage.TM. and BD FACSCalibur.TM.
available from BD Biosciences, Inc., 2350 Qume Drive, San Jose,
Calif., USA 95131-1807 and the ALTRA Cell Sorting System available
from Beckman Coulter, Inc., 4300 N. Harbor Boulevard, P.O. Box
3100, Fullerton, Calif. 92834-3100 USA.
[0024] Still further in accordance with the present invention, the
sample used in the above-summarized methods may comprise cells in a
liquid substrate or a solid tissue (e.g., an organ). In instances
where the sample comprises solid tissue, it may be difficult to
measure the PN/Fp ratio(s) in vivo or in cells located beneath the
surface of the tissue using standard one-photon (1P) excitation at
near-UV and visible wavelengths for NAD(P)H and FP fluorescence.
Thus, in such instances, the present invention may utilize
two-photon (2P) NAD(P)H fluorescence to enable measurement of cells
located beneath the surface of the tissue or in specific deep
tissue planes. Detailed descriptions of such two-photon (2P)
NAD(P)H fluorescence methods are described in the literature. See,
Yeh A T, Nassif N, Zoumi A, Tromberg B J.; Selective Corneal
Imaging Using Combined Second-Harmonic Generation And Two-Photon
Excited Fluorescence; Optics Letters 27:2082-4 (2002); Zoumi A, Yeh
A, Tromberg B J.; Imaging Cells And Extracellular Matrix In Vivo By
Using Second-Harmonic Generation And Two-Photon Excited
Fluorescence; Proc. Natl. Acad. Sci. USA 99:11014-9 (2002).
[0025] Still further in accordance with the present invention,
there are provided methods for preparing stem cell isolates and/or
preparations for clinical and experimental applications by
identifying and isolating stem cells by way of the above-summarized
methods of the present invention and subsequently expanding (e.g.,
culturing) the population of such stem cells if necessary and/or
combing the stem cells with other materials or apparatus if
necessary, to thereby provide a stem-cell-comprising preparation or
article for clinical or research use. Examples of such
stem-cell-comprising preparations or articles include but are not
limited to: stem cells for subsequent culture and expansion; frozen
or otherwise preserved stem cell isolates obtained from an
individual's umbilical chord blood, bone marrow or other source(s)
for possible use in future stem-cell based therapies of that
individual; stem cells for implantation or transplantation into
specific organs or tissues for therapeutic purposes (e.g., limbal
stem cells for transplantation into or onto an eye to facilitate
regeneration of corneal surface epithelium; stem cell preparations
for implantation on or near the retina of an eye for treatment of
retinal disorders such as retinal degenerations and macular
degeneration; stem cell preparations for direct injection into
organs of interest (e.g. stem cell preparations for implantation
into cardiac tissue for the treatment of cardiac disorders such as
ischemia and arrhythmia and or regeneration of new myocardial
tissue to replace infracted or necrotic tissue; stem cell
preparations for implantation into areas of the brain to give rise
to new functional cells such as dopamine secreting cells for the
treatment of Parkinsons disease; stem cell preparations for
infusion into the blood to give rise to new healthy blood cells and
stem cells disposed on substrates, scaffolds, forms, casts or other
articles for the formation of prosthetic or replacement body
parts.
[0026] Still further in accordance with the present invention,
there are provided methods for assessing the distribution and/or
structure of mitochondria in living cells by autofluorescence. In
this regard, the present invention provides a method wherein
fluorescence emission is measured in the region of 450 nm after
excitation at 366 nm from reduced pyridine nucleotides (NADH and
NADPH) located in the cytoplasm and mitochondria, with far greater
quantum yield from the mitochondrial bound species than from the
cytoplasmic species. The fluorescence from mitochondria as measured
by this non-invasive method is useable to distinguishing different
cellular states.
[0027] Still further in accordance with the present invention,
there is provided a method and system for autofluorescence
monitoring of living cells in a cell culture or other medium. This
system generally comprises an excitation/detection fluorescence
microscope apparatus, a microscopic stage and a cell growth chamber
in which cells are maintained under controlled conditions (e.g.,
controlled temperature, humidity, pH, osmolality, nutrient levels,
ion levels, ambient gas composition(s) (e.g. ambient CO2, O2 and
N2), etc.) The excitation/detection fluorescence microscope
apparatus is useable to continually or periodically obtain
autofluorescence measurements of cells contained in the chamber.
Thus, this system and method can be used to observe the natural
evolution of mitochondrial organization and metabolism. Also, the
cell culture may optionally be exposed to a treatment (e.g., a test
compound, toxin, drug or other challenge or perturbation of a
physical, chemical, thermal, metabolic, nutritional or other sort).
The system and method may then be used to determine the effects of
such treatment on mitochondrial organization and metabolism and/or
other cellular variables measurable by autofluorescence. For
example, this method and system may be used to study the effects of
heating or cooling the cells, hypoxia or hyperoxia, or even the
effects of different substances or drugs such as anti-cancer drugs.
Time laps over minutes, hours, days and weeks may be examined.
[0028] Further aspects, objects, applications, details and
variations of the present invention will be understood by those of
skill in the art upon reading of the detailed description and
examples set forth herebelow.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1A is a histogram of corneal limbal epithelial cells
cultured on plain in glass in 1% CO.sub.2.
[0030] FIG. 1B is a histogram of corneal limbal epithelial cells
cultured on plain glass in 5% CO.sub.2. The inset in FIG. 1B shows
fluorescence images of the cells of this culture stained with
mitochondrial-detecting dye (MitoTracker.RTM. dye) on plain
glass.
[0031] FIG. 1C is a histograms demonstrating autofluorescence
distribution across cells cultured on plain glass in 1%
CO.sub.2.
[0032] FIG. 1D is a histograms demonstrating autofluorescence
distribution across cells cultured on plain glass in 5%
CO.sub.2.
[0033] FIG. 1E is a histogram of corneal limbal epithelial cells
cultured on collagen I-coated glass in 1% CO.sub.2.
[0034] FIG. 1F is a histogram of corneal limbal epithelial cells
cultured on collagen I-coated glass in 5% CO.sub.2. The inset on
FIG. 1F fluorescence images of the cells of this culture stained
with mitochondrial-detecting dye (MitoTracker.RTM. dye).
[0035] FIG. 2A is a histogram of corneal limbal epithelial cells
cultured on poly-d-lysine coated plates under 1% CO.sub.2.
[0036] FIG. 2B is a histogram of corneal limbal epithelial cells
cultured on poly-d-lysine coated plates under 5% CO.sub.2.
[0037] FIG. 2C is a histogram of corneal limbal epithelial cells
cultured on poly-d-lysine coated plates under 15% CO.sub.2. The
inset in FIG. 2C shows fluorescence images of cells of this culture
stained with mitochondrial-detecting dye (MitoTracker.RTM.
dye).
[0038] FIG. 3A is an autofluorescence images of mesenchymal stem
cell cells cultured in stem cell-maintaining medium.
[0039] FIG. 3B shows a fluorescence image of cells of FIG. 3A later
stained with mitochondrial-detecting dye (MitoTracker.RTM.
dye).
[0040] FIG. 3C is an autofluorescence image of mesenchymal stem
cell cells cultured in osteogenic medium.
[0041] FIG. 3D shows a fluorescence image of cells of FIG. 3C later
stained with mitochondrial-detecting dye (MitoTracker.RTM.
dye).
[0042] FIG. 4 is a schematic diagram of a fluorescence microscope
system that incorporates a controlled condition cell culture
chamber for fluorescence microscopy of living cells in accordance
with the present invention.
DETAILED DESCRIPTION AND EXAMPLES
[0043] The following detailed description and the accompanying
drawings are intended to describe some, but not necessarily all,
examples or embodiments of the invention only. This detailed
description and the accompanying drawings do not limit the scope of
the invention in any way.
Example 1
Redox Fluorometry Imaging As A Non-Invasive Technique for
Distinguishing Mesenchymal Stem Cells (MSCs) From Further
Differentiated Cells
[0044] Adult bone marrow-derived mesenchymal stem cells have been
demonstrated to be pluripotent for differentiation into such
tissues as bone, cartilage and tendon. Standard techniques now
exist to culture these stem cells in vitro for experimental study
and manipulation. It has been shown that differentiation along
different pathways may be controlled by media as well as substrate.
Although much effort has been expended in searching for stem cell
probes, currently there still exists no reliably specific marker
for stem cells of any type. Additionally, many of the markers that
have been proposed and studied are invasive to the cell or cell
surface and may alter or even kill the cell.
[0045] One of the most accepted properties of stem cells is their
ability to slowly cycle and continuously replenish cell
populations. In this example, it is demonstrated that MSCs cultured
in stem cell and osteogenic media have slower intrinsic metabolism
than differentiated cells and, thus, may be detected by the
non-invasive metabolic auto fluorescence spectroscopic technique of
redox fluorometry.
[0046] Materials and Methods
[0047] A. Stem Cell and Osteogenic Media The following were used in
mesenchymal stem cell growth media (Cambrex, East Rutherford N.J.):
MSCBM Basal Medium (2 ml) supplemented with two vials of 25 ml
fetal bovine serum (FBS) to attain an overall concentration of 10%,
L-glutamine 200 mM (10 ml) and penicillin 25 U/streptomycin 25
.mu..g (0.5 ml) (MSCGM SingleQuots Bullet Kit, Cambrex, East
Rutherford N.J.). Subcultures were performed after treatment with
Trysin/EDTA 0.25 mg/ml and trypsin neutralizing solution (TNS).
[0048] Osteogenic media consisted of DMEM without sodium pyruvate
(Invitrogen, Carlsbad, Calif.), 100 nM dexamethasone (Sigma
Chemicals, St. Louis, Mo.), 50 M ascorbic acid-2-phosphate (Sigma),
10 mM-glycerophosphate (Sigma), 10% FBS, 100 U/mL penicillin
(Invitrogen) and 100 g/mL streptomycin (Invitrogen, Carlsbad,
Calif.).
[0049] B. Mesenchymal Stem Cell Harvest and Isolation Bone marrow
samples from goat femurs (Thomas Morrs, Inc, Reisterstown, Md.)
were washed and centrifuged (1000 rpm for 10 minutes) twice in
media (Cambrex, East Rutherford N.J.) and resuspended in fresh
media thereafter. Mononuclear cells were counted using a
hemocytometer and were plated in culture flasks at a density of
120,000 cells/cm2. The culture media was changed after 4 days, then
every 2-3 days thereafter until confluence.
[0050] C. Freezing and Thawing Cells Cells were centrifuged and the
media was aspirated and replaced with an appropriate amount of
freezing media: 40% MSCGM, 50% FBS and 10% dimethyl sulfoxide
(DMSO). The cells were then aliquoted into cryo-vials and frozen at
a controlled rate of -1.degree. C./minute at -80.degree. C. at a
concentration of 2-3 million cells/mL. After 24 hours, the vials
were transferred to liquid nitrogen for long-term storage. To thaw
cells, the frozen vial was placed and gently swirled in a 370 C
water bath.
[0051] D. Plating and Passaging Cells After thawing, the
concentrated cell solution (1-3 million cells/ml) was diluted with
more media to obtain a density of 5000 cells/cm2 for plating. Media
change was performed every 2 to 3 days. Upon reaching confluence
(at approximately 5 days), the media was aspirated and the cells
were rinsed with phosphate buffered saline solution (PBS) to
prepare them for passaging. After rinsing, the PBS was aspirated
and the cells were trypsinized for 5 minutes in accordance with
well known standard technique. Cell release from the culture plate
was confirmed under microscopic visualization and TNS was added to
the solution in order to neutralize the trypsin. The solution was
then placed in a centrifuge tube and spun for 10 minutes at 1000
rpm. Trypsin was aspirated and the cells were re-suspended in fresh
media. The cells were counted and then were either replated for
future passages or used for fluorescence imaging.
[0052] E. Auto fluorescence Microscopy All images were obtained
using a Zeiss (Thornwood, N.Y. inverted microscope (Axiovert 200M)
with a 100.times. objective (FLUAR 100.times., 1.3 oil). The
microscope was equipped with a mercury lamp (HB 103) and a cooled
CCD camera (Axiocam MRc.sup.5) for taking images. To detect
intrinsic reduced pyridine nucleotides, a Zeiss DAPI filter set
(excitation: G365, emission: bandpass 445/50) was used. Oxidized
flavoproteins were identified using a Zeiss FITC filter set
(excitation: bandpass 450-490, emission bandpass 515-565). To
minimize photobleaching and light stimulation, the illumination
source was turned off during fluorescence imaging. All the images
and fluorescence ratios were processed and analyzed using
AxioVision Softare (Zeiss). Prior to auto fluorescence microscopy
all cells were plated and expanded on glass surfaces (MatTek
Cultureware dishes, Ashland, Mass.). Additionally, cell cultures
were equilibrated in colorless PBS solution before imaging.
[0053] F. Fluorescent Dyes and Subcellular Markers To confirm the
identity of auto fluorescent cellular structures, labeling dyes
were used to stain the specimens. MitoTracker Green PM and
LysoTracker Red (Molecular Probes, Inc., Eugene, Oreg.) were
allowed to warm in room temperature and subsequently diluted with
Dulbecco's Modified Eagle Medium (DMEM) to the desired
concentrations, 30 nM and 75 nM respectively. Cell culture media
was removed and replaced with the appropriate, pre-warmed
(37.degree. C.) probe-containing medium. Incubation was carried out
for 30 minutes with MitoTracker Green FM and 1 hour for LysoTracker
Red. The cells were then washed with buffer solution and imaged
with the appropriate Zeiss FITC (excitation: bandpass 450-490,
emission: bandpass 515-565) and Rhodamine (excitation: bandpass
546/12, emission: bandpass 515-565) filter sets.
Results
[0054] Two dimensional redox fluorometric microscope photos were of
second passage goat mesenchymal stem cells (MSCs) isolated, culture
expanded and examined as detailed in Materials and Methods. These
redox fluorometric microscope photos demonstrated low density and
higher density clustered cells, respectively, excited in the region
of 366 nm and emission detected in the region of 450 nm (channel).
The same cells where then excited around 460 nm and detected around
540 nm (channel2). Unprocessed images of the previous image sets
were then overlaid, with the 450 nm emission (channel 1)
pseudo-colored green and the 540 nm emission (channel 2)
pseudo-colored red. The PN/Fp ratios were calculated for 1) low
density MSCs in stem cell media, 2) high density clustered MSCs in
stem cell media, 3) low density MSCs in osteogenic media and 4)
high density clustered MSCs in osteogenic media. The differences
between the observed PN/Fp ratios were tested for statistical
significance using the as shown in Table 1 below:
TABLE-US-00001 TABLE 1 Autofluoresence (PN/Fp) Ratios Pyridine
(channel 1)/ Cell Type Flavoprotein (channel 2) P Value 1. Low
Density 2.19 .+-. 0.59 MSCs in stem cell media (n = 15) 2. High
Density 1.45 .+-. 0.27 P = O.OOOI clustered MSCs in stem vs. 1 cell
media (n = 15) 3. Low Density 1.38 .+-. 0.57 P = 0.0007 vs. 1 MSCs
in osteogenic media (n = 15) 4. High Density 0.87 .+-. 0.34 P =
0.00002 vs. 2 clustered MSCs in P = 0.007 vs. 3 osteogenic media (n
= 15)
[0055] This example demonstrates that redox fluorometry imaging of
MSCs expanded in culture is feasible. Moreover, besides providing
simple metabolic information, reasonably detailed cytoarchitecture
is also visible. Based on that which was known in the prior art, it
could reasonably have been predicted that nearly all of the
stimulated autofluorescence observed would arise from superimposed
mitochondrial compartments. However, the overlayed images showed
separation of the two pseudo-colored compartments (green and red),
especially in the non-clustered cell image, with less blended
(yellow) overlap than expected. The reduced pyridine species
stimulated at 366 nm appeared to fluoresce in a mitochondral
pattern (green), whereas the oxidized flavoproteins stimulated at
460 nm appear to be comparentalized into a combination of
mitochondra and lysosomes/peroxisomes (red). This was not
completely unexpected, though, as a significant traction of
flavin-associated autofluorescence is non-redox responsive,
although most of this fluorescence is quenched when bound as
protein co-factors. The remaining observed non-redox responsive
fluorescence has been found to not co-localize with mitochondral
sub-cellular markers, but rather with lysosomal markers. To confirm
these subcellular anatomic locations, secondary staining with
Mito-tracker and Lyso-tracker dyes (Molecule Probes, Inc., Eugene,
Oreg.) was performed.
Results and Discussion
[0056] As expected, the pyridine/flavoprotein ratio decreased upon
transitioning from the stem cell to the differentiated state. When
compared to differentiated cells, the lower cellular respiration in
the stem cells resulted in a higher reduced pyridine nucleotide
fluorescence signal (channel 1) and a lower oxidized flavoprotein
fluorescence signal (channel2). Thus, the stem cells demonstrated a
significantly higher channel 1/channel 2 ratio (i.e., the PN/Fp
Ratio). Also, significant differences are noted between low density
and high density clustered cell cultures.
[0057] Commitment of stem cells to separate lineages appears to be
regulated by multiple cues in the local tissue environment
including mechanical ones which appear to be integral to the
commitment of their fate. Altering the redox state of embryonic
cells through enzymes has been shown to affect transcription
factors and modify gene-expression patterns to influence
totipotentiality and ultimate cellineage. Metabolism, including the
parameter of redox potential, in cultured cells is also known to
depend on cell density, especially mechanical cell-cell contact,
and thus cell density must be considered in maintenance of cell
cultures. Work comparing immortalized to non-immortalized
fibroblasts demonstrated that as soon as either type of cell came
into contact with one another, the total redox potential dropped.
In order to compare metabolic parameters among different cell types
one should take into account the density dependence of these
factors, especially low density single cell vs. higher density and
more confluent. Thus there is a distinct possibility that stem
cells may not possess an entirely consistent redox fluorometric
signature under different culture conditions and densities. If so,
as metabolic rates and sub-cellular organization may change under
varying conditions and it may be necessary to perform a larger
number of examinations under different conditions (e.g. differing
media, substrates and densities) in order to more easily separate
stem cells from non-stem cells.
[0058] Although, theoretically at least, fluorescence
photobleaching of intrinsic fluorophores may be a potential problem
in assays of this type, reliable signals may be obtained if
experimental technique is optimized, such as by maintenance of low
ambient light conditions, decreased exposure times, turning off an
illumination source during fluorescence imaging, using an
illumination source that emits light at a wavelength that is not
within the excitation or autofluorescence wavelength detection
gates being employed, shielding the sample from light during at
least a portion of the procedure, etc.
Example 2
Non-Invasive Mitochondrial Imaging In Live Cell Culture
[0059] In this example, autofluorescence microscopy is used to
image mitochondria in live cell culture and to detect changes in
the intracellular distribution and/or structure of mitochondria
under varying conditions.
Materials and Methods
[0060] Limbal epithelial cells from fresh human donor tissue not
suitable for transplantation (Central Florida Lions Eye and Tissue
Bank, Tampa, Fla.) were cultured within 4 days of death on uncoated
and coated glass surfaces [MatTek Cultureware dishes, Ashland,
Mass., EpiLife Medium, Human Corneal Growth Supplement and PSA
Solution (penicillin, streptomycin, and amphotericin B), Cascade
Biologics, Portland, Oreg.]. Different substrate coatings
(uncoated, poly-d-lysine or collagen I) were used and cells
cultured under differing CO.sub.2 incubator tensions (1%, 5% and
15%). Media was changed every three days until near confluence.
[0061] MSCs were harvested from goat femurs and isolated after
washing, centrifugation and suspension. The cells were plated on
uncoated glass surfaces (Matek) in either mesenchymal stem cell
growth media (MSGM) made up of several components [MSCBM Basal
Medium, Fetal bovine serum (FBS), L-glutamine, penicillin 25
U/streptomycin, Cambrex, East Rutherford N.J.], or osteogenic media
consisted of DMEM without sodium pyruvate (Invitrogen, Carlsbad,
Calif.), dexamethasone (Sigma Chemicals, St. Louis, Mo.), ascorbic
acid-2-phosphate (Sigma), glycerophosphate (Sigma), FBS, 100 U/mL
penicillin (Invitrogen) and streptomycin (Invitrogen, Carlsbad,
Calif.). Media was initially changed after 4 days, then every 2-3
days thereafter until near confluence.
[0062] Autofluorescence microscopy images were obtained using a
Zeiss (Thornwood, N.Y.) inverted microscope (Axiovert 200M) with a
100.times. objective (FLUAR 100.times., 1.3 oil). The microscope
was equipped with a mercury lamp (HB 103) and a cooled CCD camera
(Axiocam MRc5) for taking images. To detect intrinsic reduced
pyridine nucleotides, a Zeiss DAPI filter set (excitation: G365,
emission: bandpass 445/50) was used. To minimize photobleaching and
light stimulation, the illumination source was turned off during
fluorescence imaging. All the images were processed and analyzed
using AxioVision Software (Zeiss). Prior to autofluorescence
microscopy imaging all cells were equilibrated in balanced salt
solution (BSS, Alcon, Forth Worth, Tex.), and then imaged at room
temperature under room air.
[0063] Total cellular mitochondrial distribution was determined by
staining with MitoTracker.RTM. Green FM (Molecular Probes, Eugene,
Oreg.). A 30 nM probe solution was prepared in DMEM (GIBCO, Grand
Island, N.Y.) and incubated with the culture for 30 minutes at
37.degree. C. after samples were rinsed twice with PBS. After
incubation, each sample was again rinsed with PBS and imaged with a
Zeiss FITC filter set (excitation: bandpass 450-490, emission
bandpass 515-565).
[0064] Intensity histograms of autofluorescence distribution were
obtained using AxioVision 4.3 (Carl Zeiss, Incorporated, Thornwood,
N.Y.). Line profiles were generated to plot the intensity of gray
values over the course of the perpendicular axes of the cell.
Results And Discussion
[0065] The non-invasive mitochondrial imaging technique of the
present invention was used on limbal epithelial cells grown on
plain glass (FIG. 1A) or collagen I (FIG. 1E). In the 1% CO.sub.2
environment, mitochondrial autofluorescence appears crowded around
the nucleus. Histograms of autofluorescence distribution were
obtained by measuring the intensity of gray values over the course
of the perpendicular axes of the cell. The histogram in FIG. 1C
demonstrates distinct peaks of autofluorescence at the nuclear
border, supporting a perinuclear description. In the 5% CO.sub.2
environment, as shown in FIGS. 1B and 1F, especially on the
collagen I-coated surface, in the majority of cells
autofluorescence is observed to redistribute more diffusely
throughout the cytoplasm where mitochondria display characteristic
polymorphic structures, with spherical, tubular and bean-like
shapes. A representative histogram seen in FIG. 1D has a wider
distribution of broader peaks. These multiple peaks demonstrate a
more diffuse distribution of autofluorescence across the ordinary
and extraordinary axes of the cell.
[0066] FIGS. 2A-2C show autofluorescence from limbal epithelial
cells expanded on poly-d-lysine-coated glass under varying CO.sub.2
incubator tensions of 1% (FIG. 2A), 5% (FIG. 2B) and 15% (FIG. 2C).
In cells grown in the low CO.sub.2 environment (FIG. 2A), the
majority of cells are observed to have higher relative
mitochondrial fluorescence in the perinuclear region. In contrast,
at the higher CO.sub.2 concentrations (FIGS. 2B and 2C), an
increasing number of larger cells are observed with structured
mitochondrial fluorescence diffusing away from the perinucleus and
spreading towards the cell membrane. Under all 3 environmental
CO.sub.2 conditions, a population of small cells appears to be
resistant to the effects of the changing incubator gas mix in these
heterogeneous expansions. It is possible that these small cells
with marked perinuclear clustering of mitochondria belong to the
stem cell pool as flow cytometry and in vivo confocal microscopy
studies have demonstrated that the smallest cells in a mixed cell
population originate from the limbal basal epithelium, the location
of corneal epithelial stem cells.
[0067] FIG. 3 demonstrates fluorescence from MSCs grown in either
stem cell maintaining (FIGS. 3A and 3B) or differentiating
osteogenic (FIGS. 2C and 3D) media. Both autofluorescence in live
culture (FIGS. 3A and 3C), and MitoTracker.RTM. fluorescence after
fixation (FIGS. 3B and 3D) are shown. In both MitoTracker.RTM.
images, it is apparent that mitochondria are actually distributed
diffusely throughout the cytoplasm. In contrast, in stem cell
maintaining medium unlike in osteogenic medium, there appears
significant relative enhancement of autofluorescence in the
perinuclear region in the majority of cells.
[0068] In this example, two techniques for imaging mitochondrial
populations where compared. Traditional mitochondrial staining
(MitoTracker.RTM.) is invasive and does not result in fluorescence
until the stain has accumulated in the lipid environment of
mitochondria. The invasive nature of this stain prohibits tracking
live cells in culture serially in time. The non-invasive
mitochondrial imaging methods of the present invention utilize an
excitation wavelength (365 nm) away from medium and short wave UV
light avoiding cellular damage and the large number of species that
can autofluoresce following excitation at these short wavelengths.
The major advantage of our technique is its non-invasive nature, as
the cells can be imaged serially in live culture without
perturbation by exogenous stains which can result in not only cell
disturbance, but also death. Furthermore, because we detect
metabolically active endogenous fluorophores, this also serves as a
functional assay at the sub-cellular level.
[0069] Previous studies have demonstrated the co-existence of
mitochondrial populations with distinct distribution patterns;
diffuse mitochondria throughout the periphery of the cell and a
perinuclear sequestration. The difference between these populations
is the observable change in membrane potential of the active
mitochondria. Each technique used in this study uses a different
characteristic of the mitochondria to elucidate its distribution.
The traditional MitoTracker.RTM. stain targets the lipophilic
nature of the mitochondria leading to accumulation and fluorescence
typically extending throughout the cytoplasm. Our autofluorescence
technique relies on the metabolic activity of the mitochondria to
demonstrate the sub-distribution of active mitochondria within the
larger mitochondrial pool. Our findings show that mitochondrial
autofluorescence distribution and appearance in living cells can
change when substrates, media or ambient CO.sub.2 conditions are
altered.
[0070] Thus, this example demonstrates that autofluorescence
imaging of mixed cell culture expansion is feasible. Additionally,
reasonably detailed cytoarchitecture is visible allowing the
revelation of different mitochondrial autofluorescence distribution
patterns.
[0071] It is to be appreciated that, in at least some embodiments,
the methods of the present invention may be practiced on living
cells. In the regard, the present invention includes an
fluorescence microscopy system that incorporates a controlled
condition cell culture chamber that enables fluorescence microscopy
to be performed on living cells in culture without requiring
removal of the cells from the cell culture chamber. FIG. 4 is a
schematic diagram showing an example of one such fluorescence
microscopy system 10 of the present invention. As shown, this
system 10 comprises a focusing lens 12, an excitation light source
14, a microscope stage 16, an objective 18 having an oil droplet 20
disposed thereon, a glass cube or prism 19, a cell culture chamber
22 and a control apparatus 28 for controlling one or more
conditions within the cell culture chamber 22, such as temperature,
humidity, pH, osmolarity, nutrient levels, ion levels and ambient
gas composition (e.g., partial pressure or concentration of
CO.sub.2, partial pressure or concentration of O.sub.2 and partial
pressure or concentration of N.sub.2, etc.). Optionally, the cell
culture chamber 22 may have relatively wide regions 24 located
lateral to the center of the objective 18 and a thin region 26
located directly above the center of the objective 18, thereby
providing a thin film of cell-containing culture medium within the
thin region 26 directly above the objective 18 while allowing
greater volumes of cell-containing culture medium to be present in
the wide regions 24 located away from the area where the
microscopic imaging occurs. Optionally, the control apparatus 28
may alternatively or additionally include a pump or motion
imparting apparatus for continuously or periodically circulating
cell containing culture medium through the portion of the culture
chamber 22 located immediately above the objective (e.g., the thin
region 26) thereby providing a turn-over of cells being imaged by
the microscope. Also, optionally, the microscope may include a
camera with a timer or other controller that causes images (e.g,
histograms or other photomicrographs) of autofluorescence to be
obtained at desired time points, thereby providing for observation
of changes in certain parameters (e.g., mitochondrial distribution
and/or structure) over a desired period of time.
[0072] Although the schematic diagram of FIG. 4 shows a fixed stage
microscope, those of skill in the art will appreciate that the
controlled condition cell culture chamber 22 and control apparatus
28 may also be useable in moving stage microscopes used for
fluorescence microscopy.
[0073] It is to be appreciated that the invention has been
described hereabove with reference to certain examples or
embodiments of the invention but that various additions, deletions,
alterations and modifications may be made to those examples and
embodiments without departing from the intended spirit and scope of
the invention. For example, any element or attribute of one
embodiment or example may be incorporated into or used with another
embodiment or example, unless to do so would render the embodiment
or example unsuitable for its intended use. Also, where the steps
of a method are described or recited in a particular order, such
ordering of the steps may be changed unless doing so would render
the method non-useable for its intended purpose. All reasonable
additions, deletions, modifications and alterations are to be
considered equivalents of the described examples and embodiments
and are to be included within the scope of the following
claims.
* * * * *