J. Biomedical Science and Engineering, 2008, 1, 163-169
Published Online November 2008 in SciRes. http://www.srpublishing.org/journal/jbise JBiSE
In vitro and in vivo cell tracking of chondrocytes of
different origin by fluorescent PKH 26 and
Weinand Christian1, Timothy S Johnson1, Thomas J Gill1
1Plastic Surgery Laboratory, Department of Plastic Surgery, Massachusetts General Hospital, WACC 453, 15 Parkman Street, Boston, MA, 02114, USA. Corre-
spondence should be addressed to Weinand C, MD (cweinand@partners.org), Tel.: 617 – 726 – 7797.
Received August 17, 2008; revised October 7, 2008; accepted October 7, 2008
Tissue engineering techniques for cartilage re-
pair to heal defects in joint surfaces is a clinical
practice. Harvested autologous chondrocytes are
expanded in culture and delivered in a suitable
carrier medium back into the patient’s joint de-
fect. The defect is then subsequently filled by
new cartilage. Whether the cells in the repair
tissue originate from the engineered tissue of the
host or are derived from the surrounding original
cartilage remains a relevant question for the ap-
plied therapy. To answer this several methods
exist to track cells, such as transfection of cells
with LacZ carrying viruses, radio labeling with
111 IN or 51 Cr or fluorescent labeling with FDA.
However, these techniques have drawbacks such
as they may influence cellular properties, are
radioactive or quickly lose their tracking ability.
New fluorescent probes are easier to handle and
do not to interfere with cells.
PKH 26® is a relatively new cell-labeling agent,
but few data exist on the application of this dye in
chondrocytes in vitro and in vivo. 5-chlorome-
thylfluorescein diacetate - CMFDA (“cell tracker
green”) is an established fluorescent probe for
imaging the dynamic processes of cell prolifera-
tion in vitro and in vivo. Likewise, several studies
exist on different cell types. However, little data
are available for chondrocytes.
The first aim of this study was to evaluate
qualitative differences in fluorescence pattern
after labeling of articular, auricular and costal
chondrocytes. Secondly, we evaluated the influ-
ence of labeling with CMFDA on cellular adhe-
sion properties. The third aim was to compare
the duration of cell labeling of chondrocytes of
different origin with established CMFDA as stan-
dard and PKH 26® for 3 cell generations in vitro
and 12 weeks in vivo. We show that chondro-
cytes from different origin can be labeled effec-
tively with both PKH 26® and CMFDA. The PKH
26® labeled articular chondrocytes maintained
fluorescence longer than CMFDA in vitro and in
vivo. A higher percentage of articular chondro-
cytes remained stained at 63 days than auricular
or costal chondrocytes.
Keywords: Cell tracking, cell generations, inte-
gration, fluorescence, chondrocytes of different
origin, CMFDA, PKH 26, tissue engineering
Applied tissue-engineering techniques are clinical reality
in joint cartilage replacement and defect filling. The clini-
cian harvests chondrocytes from the patient’s joint surface
of a non-weight bearing area. After expansion in culture
the cells are re-implanted into the patient into an existing
defect using a suitable carrier for the cells. The relevant
question to justify the technique remains weather the de-
fect surrounding chondrocytes are migrating into the le-
sion or if the new tissue originates from the implanted
cells. To answer this durable and stable methods for cell
staining are essential in long term follow up studies of
transplanted cells in order to allow identification of the
origin of the daughter cells. Several features are necessary.
The marker should be 1: detectable after a prolonged pe-
riod of time, 2: be easy to handle, and 3: should not inter-
fere with immunocytochemical staining to characterize
the fate and differentiation of grafted cells in the host tis-
sue, such as cartilage.
Different methods have been established to identify
transplanted cells in host tissue such as bromodesoxyu-
ridine – BrdU [7, 15] labeling of cells with [3H] -
thymidine [2, 14, 15] or transfection with the LacZ gene
or beta – galactosidase [6, 12, 22] but each techniques has
some method specific disadvantages, such as involved
radioactivity or special security measures are needed
when handling viruses. Newer labeling techniques use
chemical integration of fluorescent dyes into cellular
structures such as organs or membranes. PKH 26® has
been described as a fluorescent dye integrating irreversi-
bly into the cellular membrane of living cells, and its la-
beling efficiency is more than 90% for many different cell
types [9, 18, 20, 21, 23]. However, little data is available
SciRes Copyright © 2008
164 W. Christian / J. Biomedical Science and Engineering 1 (2008) 163-169
SciRes Copyright © 2008 JBiSE
on its effectiveness with chondrocytes. Chondrocytes
have a slower metabolism and replication rate compared
to liver cells or mesenchymal stem cells [10, 11]. In addi-
tion, chondrocytes produce an extracellular matrix that
encapsulates them and that is different from other matri-
ces produced by e.g. osteocytes or keratinocytes. For
these reasons cell-tracking dyes mentioned before might
not be the best choice for chondrocytes. Several authors
have recommended the use of CMFDA for labeling dif-
ferent cell types for long-term studies, which is easy to
handle. It integrates into the cytoplasm of viable cells,
independent from the cell cycle, and has been used in
bone marrow derived stem cells [8] keratinocytes, myo-
cytes and osteocytes [3], lymphocytes and U937 cells [4].
Despite these advantages, the duration of the labeling is
not known and only few data are available on its use with
chondrocytes. In a previous in vivo experiment, we used
PKH 26® to label transplanted auricular and articular
chondrocytes and transplanted them on a PLGA mesh into
a created meniscus lesion in an in vivo model. Follow up
was done after 12 weeks and staining remained visible of
the transplanted cells [27]. However, no comparison of
cell tracking dyes between chondrocytes of different ori-
gin has been done.
We evaluated the differences in PKH 26® and CMFDA
durability of cell labeling in vitro for 9 weeks for 3
daughter cell generations and in vivo for 12 weeks in a
long-term study. Also, differences in labeling of three
different kinds of chondrocytes and the influence of the
marker on cell replication rate were examined.
2.1. Chondrocyte Preparation
Under sterile operating conditions articular, auricular and
costal cartilage from 6 month-old Yorkshire swine were
harvested. The cartilage was excised, rinsed in phos-
phate-buffered saline (PBS) with 2 % antibiotics, and
minced into 1-mm3 pieces. The cartilage was digested at
37˚C in a 5% CO2 incubator for 12-18 hours in Ham’s
F-12 media with Glutamax-1 (Gibco/BRL, Life Tech-
nologies, Grand Island, NY) containing 0.1% of colla-
genase Type 2 (Worthington Biochemical, Freehold, NJ)
and 1% antibiotics. The solutions were then passed
through a 100-µm filter to remove undigested particles.
The cell suspension of isolated chondrocytes was centri-
fuged at 1500 rpm for 10 minutes, washed 3 times with
PBS containing 1% antibiotic/antimycotics (10.000units
penicillin, 10mg streptomycin and 25 μg amphotericin B,
Sigma Chemical Co., St Louis, MO) and cell viability
assessed using trypan blue exclusion. Only cells with a
viability of >90% were used in this study. Cell counting
was done by a hemocytometer to the nearest 1x106 cells
per ml.
2.2. Chondrocyte Labeling with PKH 26®
PKH 26® (Sigma Chemical Co., St Louis, MO): PKH 26®
is a lipophilic dye with aliphatic tails that binds irreversi-
bly into the lipid regions of the cell membrane. The
amount of dye is then partitioned equally between daugh-
ter cells during mitosis and therefore decreases by half at
each cell division.
All steps were performed at 25°C, according to the
manual of the producer. The cells were placed in a conical
polypropylene tube (Falcon, Becton Dickinson Labware,
Lincoln Park, NJ) at concentrations of 20x106 cells per ml
and were washed once with serum free medium. The
chondrocytes were centrifuged for 5 minutes, the super-
natant aspirated and re-suspended in 1ml of Diluent C. A
stock solution of PKH 26® of 4x106M was prepared im-
mediately before staining, added to the Diluent
C/chondrocyte suspension, suspended and incubated at
25°C for 5 minutes. The tube was intermittently inverted
to assure mixing. After adding 2ml of FBS (Sigma
Chemical Co., St. Louis, MO) the cells were incubated for
1 minute and then the suspension was diluted with 4ml of
complete medium, consistent of Ham’s F12 medium with
10% FBS, 1% Glutamine (Mediatech Cellgro, VA), 50mg
L-Ascorbic acid (Sigma Chemical Co., St. Louis, MO)
and 1% antibiotics/antimycotics. The suspension was
centrifuged for 10 min at 25°C, the supernatant aspirated
and the cells were transferred to a new tube for further 3
times of washing with 10ml of complete medium.
2.3. Chondrocyte Labeling with CMFDA
5 chloromethylfluorescein diacetate - CMFDA or “Cell
tracker green” (Molecular Probes Inc., Eugene, OR):
CMFDA passes through the cell membranes into the cell,
where the chloromethyl group reacts with intracellular
thiols, leading to a cell-impermeant fluorescent dye.
CMFDA stained cells have been found to fluoresce
brightly for at least 72 hours after incubation in fresh me-
dium at 37°C and last through at least four cell divisions.
CMFD was warmed to room temperature and dissolved
with anhydrous dimethylsulfoxide (DMSO) to a final con-
centration of 10 mM. This stock solution was diluted to a
final working concentration of 5 and 10 μM in serum free
medium. We found the best labeling concentration to be
10 μM. All cells were spun in a centrifuge to obtain a pel-
let and the supernatant was aspirated and re-suspended in
previously warmed probe containing medium. The cell
medium suspension was then incubated for 45 minutes
under growth conditions. Thereafter the cells were centri-
fuged once more, re-suspended in fresh pre-warmed me-
dium and incubated for 30 minutes. The cells were then
washed again.
2.4. Seeding and Sample Preparation
Successful labeling of the cells was confirmed by exami-
nation under a fluorescence microscope. Unlabeled con-
trols did not fluoresce when evaluated under fluorescent
Cells were obtained for culturing and plating on 25cm2
flasks (Falcon, Becton Dickinson Labware, Lincoln Park,
NJ). We obtained auricular, articular and costal chondro-
cytes from native cartilage by collagenase (0.1%) diges-
W. Christian / J. Biomedical Science and Engineering 1 (2008) 163-169 165
SciRes Copyright © 2008 JBiSE
tion. 400,000 cells were labeled, the rest served as unla-
beled controls. The cell density in the flasks was 16,000
cells per cm2. From each chondrocyte source at least 3
flasks were prepared. Media was changed every other day
for the plated and seeded cells. Photos were taken every 7
days from the flasks. All pictures were digitized to evalu-
ate number of fluorescent cells for each cell type. This
was performed with MetaMorph (Expansion Programs
International, Inc. (Thunderstone), CA, U.S.A.), by 3
times counting 250 cells at each time point for each cell
type, using random counting grids of the fluorescent mi-
croscope at predetermined schedule every 7 days until 63
days (9 weeks). Percentage was calculated and the mean
value determined for each time point of observation (Ta-
ble 3). The chondrocytes were passaged when confluent,
until the third passage was reached at 16,000 cells/cm2,
using 0.25% Trypsin. The counted cells at the fixed
counting schedule every 7 days were then compared to
the cell number achieved at each passage.
For the in vivo part of the experiment chondrocytes of
each source were labeled with PKH 26® and CMFDA.
These cells and unlabeled controls were encapsulated in
fibrin glue to a final concentration of 40 million cells per
ml. Immediately after the gelation of fibrin glue the con-
structs of 0.2 ml volume were inserted into the subcuta-
neous pouch of a nude mouse. The constructs were har-
vested after 3 and 12 weeks time. The constructs were
examined morphologically, snap frozen in liquid nitrogen
and sent for histological examination. Frozen sections of
four micrometers were taken and examined under a fluo-
rescent microscope. The percentage of fluorescent cell
number in the tissue was determined by the Metamorph
(Expansion Programs International, Inc. (Thunderstone),
CA, U.S.A.) program analysis.
3. Results
3.1. Cultured Chondrocytes in Flasks
Chondrocytes in culture were monitored until stain was
no longer observed. PKH 26® labeled chondrocytes re-
flected brightly under the fluorescent microscope. La-
Table 1. PKH 26® labeled chondrocytes – articular cells showed
the longest lasting staining.
Labeling method PKH 26®
Chondrocyte type Articular Auricular Costal
Day Cells fluo-
Cells fluo-
Cells fluo-
7 87.9% 91.1% 84.7%
14 73.4% 79.2% 76.7%
21 70.4% 73.2% 71.0%
28 63.2% 50.3% 52.5%
35 50.3% 47.3% 48.2%
42 44.1% 42.0% 43.3%
49 41.0% 36.2% 37.1%
56 33.1% 26.6% 24.3%
63 22.1% 8.3% 10.4%
beled cells with PKH 26® of all three sources were clearly
separable from the background noise for the first 48 days.
The chondrocytes were followed up until day 63, when
fluorescence was no longer observed (Tables 1,
2).Weekly observation of the labeling of the chondrocytes
showed less labeled chondrocytes after 14 days of culture.
However, cells were still clearly distinguishable from
background noise. After 28 days, fluorescence was less
clear on fewer cells in each flask. Until day 63 the number
of fluorescent chondrocytes decreased gradually. Only
single cells were fluorescent, the articular chondrocytes
revealed the clearest fluorescence and the highest number
of labeled cells from day 49 (Tables 1, 2, Figure 1).
Chondrocytes labeled with CMFDA showed fluores-
cence only until day 14. After that all three types of the
chondrocytes did not reveal any further fluorescence ca-
pacity. CMFDA labeled auricular chondrocytes and con-
trols were confluent after 12 days.
Both, PKH 26® labeled auricular chondrocytes and
auricular controls were passaged first after 10 days of
culturing. Each flask contained 1 million cells, i.e. 2.5
times the original cell number. More than 79% of the cells
were fluorescent. At the second passage after 15 days in
culture, more than 75% of the chondrocytes were labeled.
The flasks contained 1 million cells, equaling 2.5 times
the plated cell number. The third passage of PKH 26®
labeled auricular chondrocytes was at 25 days, showing
more than 50% of the cells labeled; controls were conflu-
ent after 29 days of culture. Labeled and unlabeled costal
chondrocytes were confluent after 16 days. Flasks con-
tained 1.5 million cells, 4 times the cell number of origi-
nal plated cells. More than 71% of the labeled cells were
fluorescent. At 28 days at the second passage, more than
52% costal chondrocytes were fluorescent. The flasks
contained 2.5 times the original plated cell number. The
third passage was performed after 42 days with 43% la-
beled chondrocytes. We counted 1 million stained and 1
million control chondrocytes in each flask equaling a 2.5
times passaged number of costal chondrocytes. PKH 26®
labeled articular chondrocytes and controls were first
passaged after 20 days. More than 70% of the chondro-
cytes were labeled. Flasks held 1 million chondrocytes
each, which is a 2.5 times doubling rate. Articular chon-
drocytes were second time passaged at 35 days. 1 million
chondrocytes per flask were counted, equaling 2.5 times
the passaged cell number. The third passage at 42 days
showed 44% of the cells fluorescent. Flasks contained
2.5times the original plated cell number, i.e. 1 million
Table 2. CMFDA labeled cells – no fluorescence was detectable
after 14 days in all three chondrocyte types.
Labeling method CMFDA
Chondrocyte typeArticular Auricular Costal
Day Cells fluoresc-
Cells fluo-
Cells fluo-
7 85.8% 83.4% 89.1%
14 43.2% 38.2% 37.2%
21 Not detectable Not de-
Not detect-
166 W. Christian / J. Biomedical Science and Engineering 1 (2008) 163-169
SciRes Copyright © 2008 JBiSE
Table 3. Number of cells and daughter cell generations in flasks at time points of passage. The CMFDA labeled articular and costal chon-
drocytes were not confluent when no labeling could be observed any longer. Controls represent unlabeled costal chondrocytes. The repli-
cation of the cells is not inhibited by the dye.
method PKH 26®
type Articular Auricular Costal Controls
Passage # Cell # Genera-
tions Cell # GenerationsCell # GenerationsCell # (aver-
age) Generations
1 0.4x10^6 0.4x10^6 0.4x10^6 0.4x10^6
2 1x10^6 2 1x10^6 2 1.5x10^63 1.5x10^6 3
3 1x10^6 2 1x10^6 2 1x10^6 2 1x10^6 2
method CMFDA
type Articular Auricular Costal Controls
Passage # Cell # Genera-
tions Cell # GenerationsCell # GenerationsCell # (aver-
age) Generations
1 0.4x10^6 0.4x10^6 0.4x10^6 0.4x10^6
2 -- -- 1.5x10^6 3 -- -- 2x10^6 4
3 -- -- -- -- -- -- -- --
Figure 1. Seeded articular chondrocytes in flasks chondrocytes, labeled by PKH 26® and CMFDA show a loss of fluores-
cence over time. Time in days (time x d) Controls show no staining. Fluorescence microscope, 40x magnification.
chondrocytes (Table 3, Figure 1).
3.2. Transplanted Chondrocytes in Vivo
The constructs of PKH 26® and CMFDA stained chon
drocytes in fibrin glue were harvested after 3 and 12
weeks. The samples were examined morphologically and
histologically under a fluorescent microscope. The sam-
ples showed cartilage-like gross macroscopical appear-
ance and had the consistency of cartilage. Under micro-
scopic examination the fluorescence of PKH 26® labeled
chondrocytes was bright and clearly distinguishable from
the background (Figures 2, 3, 4, 5), even after 12 weeks
in vivo after implantation. All three types of chondrocytes
revealed the same pattern of fluorescence. Neo cartilage
was formed and the daughter cells were tracked due to
The constructs containing CMFDA labeled chondro-
cytes of all three sources did not reveal fluorescence after
W. Christian / J. Biomedical Science and Engineering 1 (2008) 163-169 167
SciRes Copyright © 2008 JBiSE
Figures 2, 3, 4. PKH 26® labeled articular, auricular and costal chondrocytes after 12 weeks in vivo. Fluorescence is clear distin-
guishable. Single cells are bright red fluorescent within the pale red fibrin glue hydrogel. Fluorescent microscope, 200x magnifica-
Figure 5. Controls with no fluorescent cells. Fluorescent
microscope, 200x magnification.
3 and 12 weeks. None of the controls showed fluores-
cence (Figure 5).
In vitro and in vivo cell tracking is important to distin-
guish the origin of newly formed tissue. Data have been
collected on different labeling techniques, such as BrdU
– bromodesoxyuridine [7, 15], labeling of cells with [3H]
- thymidine prior to transplantation [2, 14, 15], or trans-
fection with the LacZ gene or green fluorescent for the
expression of beta – galactosidase [6, 12, 22]. These
techniques have some method specific disadvantages.
BrdU is a relatively expensive agent that depends on the
synthesis phase of the cell cycle for integration and an
additional secondary antibody is needed to detect labeled
cells. In addition there is little information about the lon-
gevity of the labeling in cells. 3H – thymidine is also cell
cycle dependant and labeled cells have to be incubated
for 2 months in order to detect the labeling [2, 14, 15].
The use of viral vectors to transport LacZ as a marker
gene into cells requires specific timing for use and might
cause unknown effects onto cells, such as immune reac-
tions. Also, extra safety methods are required when using
Adenovirus as a vector and the cell staining might last
through only a few generations of daughter cells [6].
Other methods offered on the market are lipofection,
which comes as a complete kit, but the transfection rate
is low and the kits are rather expensive [16].
Thus, our goal was to identify labeling techniques that
are stable with time, easy to use, and highly effective in
cell labeling and cell staining for several generations
culturing. They should also not be dependant on cell cy-
cles for labeling chondrocytes for a long-term study.
In this long term in vitro and in vivo study we evalu-
ated the durability and staining differences of two newer
labeling methods in chondrocytes: PKH 26® and
CMFDA. We labeled three different sources of cells and
showed that there was no qualitative difference in label-
ing between articular, auricular and costal chondrocytes,
using either PKH 26® or CMFDA. However, PKH 26®
proved to be more durable in labeling than CMFDA over
63 days. This result was also found in our previous in
vivo experiment, tracking transplanted chondrocytes in a
meniscus [27]. All three types of chondrocytes were la-
beled under the same conditions at the same time. We did
not observe any difference in the fluorescence of differ-
ent chondrocytes at the beginning of the study, although
a higher cell number of fluorescent articular chondro-
cytes was observed at day 63 in comparison to auricular
or costal cells. The fluorescence of PKH 26® labeled
chondrocytes was stable in vitro until day 63, when only
single cells showed fluorescence and the third passage
was achieved. The longest lasting period for PKH 26®
labeled cells in vivo was reported to be 4 months for
neural cells post transplantation into the caudate putamen
[9]. Other groups tracked labeled lymphocytes or pe-
ripheral blood mononuclear cells for shorter periods of
time [3, 8, 10, 19], as well as neuronal precursors and
neuronal cells [9, 23], endothelial cells [18], lympho-
cytes [20], L9292 cells [21] and hematopoetic stem cells
In contrast to PKH 26® CMFDA labeled chondrocytes
revealed fluorescence only until day 14, consistent with
findings for myocytes, keratinocytes and osteocytes [3].
After that, no further fluorescence could be detected.
168 W. Christian / J. Biomedical Science and Engineering 1 (2008) 163-169
SciRes Copyright © 2008 JBiSE
Yang et al. labeled osteoprogenitor cells with CMFDA
and fluorescence was visible until 4-6 weeks of in vitro
culture [27]. This might indicate that lasting of fluores-
cence depends on the cell source labeled. The manufac-
turer states bright labeling will last for at least 72 hours
(Molecular Probes, Product information 17-June-2002).
We found similar results in our experiment.
Under high intensity illumination used for fluores-
cence microscopy irreversible destruction of the excited
fluorophore often becomes the limiting factor for a long
term follow up. This “photo bleaching” has been de-
scribed by Song et al. [25] as photochemical reaction
pathways; some involving reactions between adjacent
dye molecules. Although some effective “antifade” re-
agents are available the majority of them are incompati-
ble with living cells [11]. Photo bleaching might explain
the quick fading of the CMFDA labeled chondrocytes.
PKH 26® were more stable when exposed to high inten-
sity fluorescent light.
Tracking of different cells in vivo is described to after
PKH 26® i.v. infusion [1, 17, 19, 24]. Direct in vivo
staining of lymphocytes with PKH 26® is possible by
intramuscular injection [19. For our in vivo experiments
we chose to label the chondrocytes in vitro and then im-
plant the cells suspended in hydrogel, according to the
technique described by Yang et al. [27] for osteopro-
genitor cells and Lee-MacAry et al. for splenocyte ef-
fector cells [13]. Fluorescence was observed in the neo
cartilage after 3 and 12 weeks in vivo. Fluorescence of
the cells was clear and distinguishable at both time points,
confirming the passage of the fluorescence dye by cell
division to daughter cells. Thereby durable tracking of
transplanted cells is provided and origin of the newly
formed tissue could be defined to be from the trans-
planted chondrocytes. All three types of chondrocytes
revealed stable fluorescence within the newly formed
tissue. We did not observe any qualitative differences in
fluorescence between the three cell types.
So far, only few data exist on the use of CMFDA or
PKH 26® for labeling chondrocytes in vitro or in vivo.
Further studies are necessary to evaluate the effects of
this long lasting dye on chondrocytes on the cellular
level. This study demonstrated that PKH 26® is the pref-
erable marker for chondrocytes in a long-term study
when tracking daughter cells. It can reliably label up to
63 days or 3 passages of cells, and serve as an indicator
for the source of neo-cartilage in tissue-engineered con-
structs in vivo.
This study was generously funded by the Plastic Surgery Education
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