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Isolation and culture of chondrocytes from human adult articular cartilage.

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Isolation and Culture of Chondrocytes From Human Adult
Articular Cartilage
By WILLIAM
K. MANNINGAND WALTER
M, BONNER,
JR.
in the concentration of
acid mucopolysaccharides in cartilage
matrix is characteristic of early osteoarthritis, It has been suggested that this might
be due to the degradation of acid mucopolysaccharides by hyaluronidase activity.l
Hyaluronidase activity has been found in
human serum, synovial membrane, and
synovial fluid, but the enzyme has not been
found in extracts of articular cartilage.2
Another possibility is that in osteoarthritis there is an impairment of matrix
production by the chondrocytes. This is
suggested by the fact that the disease
shows a familial tendency in humans and
that inbreeding experiments have resulted
in strains of mice predisposed to osteoarthriti~.~
We have attempted to study matrix production by isolated, viable chondrocytes in
tissue culture. Such an approach will permit the study of matrix production while
the influence of enzymes or other factors
from different tissues is excluded.
This paper describes methods for isolation and culture of viable chondrocytes
from adult human articular cartilage.
A
DIMINUTION
MATERIALS
AND METHODS
Articular cartilage was obtained at autopsy of
31 individuals aged 26 to 84, the time between
death and autopsy not exceeding 8 hours. The
knee was cleansed with alcohoI and opened across
the patellar tendon. Cartilage was taken from the
femoral condyles and the posterior surface of the
patella by excision with a scalpel.
The cartilage was cut into 2 mm. cubes and
500 mg. aliquots were suspended in 5 ml. of Cey’s
From the Department of Medicine, Medical College of South Carolina, Charleston, South Carolina.
Supported by U . S. P . H . S. Grant AM 9899-01.
WILLIAMK. MANNING, AB: Student Research
Balanced Salt Solution ( Microbiological Associates)
containing 60 C units of collagenase (Cal Bio
Chem), 100 units of Penicillin and 100 micrograms
of Streptomycin per milliliter. The mixture was
incubated at 37C. with gentle shaking for 18
hours. At the end of the incubation period, strands
and clumps of undigested cartilage remained.
Chondrocytes were liberated from these clumps by
flushing through the nozzle of a 10 ml. syringe.
The cells were filtered through a double thickness
of lens paper to remove any undigested material,
washed 3 times with balanced salt solution, and
resuspended in a growth medium consisting of
Eagle’s Minimum Essential Medium containing
20 per cent calf serum, L-glutamine, penicillin, and
streptomycin.
Cell counts were made with a Neubauer hemacytometer. Viability was determined with the vital
stains Janus green and neutral red. Freshly liberated chondrocytes were smeared on glass slides,
fixed with alcohol, and stained with toluidine blue.
Monolayer cultures were prepared by placing approximately 1.5 x 106 cells in screw cap culture
tubes and diluting to 4 ml. with additional growth
medium. The tubes were inclined at 5 degrees in
culture racks. Growth medium was changed every
3 days. Colonies of chondrocytes were localized
and observed at different stages of incubation.
Fixed slides of monolayer cultures were prepared
by fixing and removing cells on a collodion membrane by the method of Manos.4 Slides were
stained in 0.1 per cent aqueous toluidine blue,
0.1 per cent toluidine blue in 0.15 M acetate
buffer at pH 3.8, hematoxylin and eosin, and
Alcian blue.
Organ cultures were prepared by centrifuging
chondrocytes into a tight pellet. The pellet was
transferred with a spatula onto a stainless steel
grid in a plastic organ culture dish (Falcon Plastics). The growth medium described above was
also used for organ culture and was changed every
3 days. Pellets were also transferred onto nutrient
agar composed of 1.5 per cent agar in growth
medium.
Assistant, Medical College of South Carolina.
WALTERM. BONNER,JR., M.D.: Assistant Professor
of Medicine, Medical College of South Carolina.
235
ARTHRITIS
AND RHEUMATISM,VOL. 10, No. 3 (June 1967)
236
MANNING AND BONNER
To serve as a control in each experiment, a pellet was fixed prior to incubation in the growth
medium. Pellets were harvested after 7 and 14
day intervals, fixed in 10 per cent formalin containing 0.3 per cent cetylpyridinium chloride, sectioned, stained, and examined for evidence of
matrix production.
Autoradiographs were made by adding 5 microcuries of S35 sodium sulfate (Abbott) to each
milliliter of culture medium. After incubation for
18 hours the pellet was removed, washed with
balanced salt solution, fixed, sectioned, and exposed
to autoradiographic emulsion (Kodak NTB-3) for
one week. The autoradiographs were counterstained with toluidine blue. Pellets were frozen,
but otherwise handled similarly, to serve as
controls.
RESULTS
Treatment of articular cartilage with collagenase, followed by gentle mechanical
disruption of the cartilage remnants, consistently yielded large numbers of chondrocytes. The yield varied between 100,000
and 500,000 cells from each gram of
cartilage, with osteoarthritic specimens giving higher yields. Nearly all cells appeared
to be intact and normal, although a small
number of naked nuclei could be found
along with cells whose nuclei appeared to
be pyknotic. Vital staining with neutral
red and Janus green dyes indicated that at
least 95 per cent of the cells were viable.
Toluidine blue stains of freshly liberated
chondrocytes reveal that the cells vary
somewhat in size, have rounded or slightly
oval nuclei, and have scant cytoplasm containing abundant fine granules (Fig. 1).
Some cells or clumps of cells are surrounded by a halo of weakly-metachromatic material, probably unremoved matrix. We have noted no difference in the
morphology of chondrocytes isolated from
normal and abnormal cartilage, or from
individuals of different ages.
Bacterial contamination was noted in 2,
and fungal contamination in 2 other preparations of isolated cells.
Chondrocytes grown by the monolayer
cell culture technique showed rapid at-
Fig. 1.-Newly isolated chondrocytes. Toluidine blue stain, pH 3.8. x 1000.
tachment to glass. Cytoplasmic spreading
was evident within 48 hours and by 7 days
all cells had become stellate in appearance
(Fig. 2). Rapid proliferation of the stellate
forms occurred after 48 to 72 hours. Monolayer cultures have been maintained for as
long as 90 days before contamination with
bacterial or fungal growth. Staining with
toluidine blue showed no metachromatic
material adjacent to the cells.
Pellets of chondrocytes grown by organ
culture methods for 7 to 14 days showed
evidence of early matrix formation (Figs.
3-5). Many cells appeared to have become
situated within lacunae, as are seen in intact cartilage. Individual cells or clumps of
cells have been found to be surrounded by
abundant metachromatic material (Fig. 6 ) .
No dedifferentiation into stellate cells was
observed and there was no evidence of proliferation of the cells.
The autoradiographic study revealed concentration of the SS5 in and around the
chondrocytes (Fig. 7). Control (frozen)
pellets showed no accumulation of the isotope.
ISOLATION AND CULTURE OF CHONDROCYTES
237
Fig. 2.-Dedifferentiated chondrocytes after
two months in monolayer culture. Toluidine
blue stain. X 100.
Fig. %-Pellet
of newly isolated chondrocytes. Stained with Alcian Blue, pH 2.5. X
250.
Fig. 4.--Pellet
of chondrocytes grown on
stainless grid for Seven days. Alcian blue
stain. There is more abundant Alcian bluepositive extracellular material. X 250.
Fig. 5-Pellet
of chondrocytes grown on
agar medium for fourteen days. Toluidine blue
stain, pH. 3.8 x 250. Granular material beneath the pellet is the agar matrix. The dark
areas around the cells are metachromatic when
seen in color.
238
MANNING AND BONNER
large numbers of viable chondrocytes from
adult human articular cartilage obtained at
autopsy. That the chondrocytes remained
viable after somatic death is not surprising,
in view of the demonstration by Hagerty
et al.5 that at least 98 per cent of the cells
of costal cartilage are viable 6 hours after
death of the individual. In another study,6
10-95 per cent of cells were found viable
after storage of the costal cartilage for up
to 14 days after autopsy.
Digestion with trypsin has been used to
extract viable cells from embryonic cartil a g e ' ~but
~ yielded only a few cells in our
preliminary studies on adult cartilage specimens. The methods described in this paper
are based on the studies of Smith,lo who
used successive digestion with papain,
Fig. 6.-Cells from pellet grown on agar for pronase and collagenase in extraction of
fourteen days. Tohidine blue, pH 3.8. X 400. cells from adult mammals. We have found
The accumulated extracellular material is met- that the use of collagenase alone is sufachromatic.
ficient to release cells from the matrix of
human articular cartilage, when followed
by mild mechanical disruption of the
partially digested fragments. Kawiakll has
described the use of collagenase in extraction of viable cells from calf cartilage.
Moscona7 has shown that isolated embryonic cells grown in a dispersed monolayer culture lose their histotypical features
and histoformative tendencies while reaggregated cells retain their features and ability to reconstruct tissues. Studies by
Holtzers confirm that embryonic chondrocytes behave in this manner. Embryonic
chondrocytes grown in pellets form metachromatic capsules and incorporate S35sulfate into chondroitin sulfate. However,
the same cells grown in monolayers lose
their ability to synthesize matrix. The
progeny of such cells fail to differentiate
into cartilage cells when reconstituted into
Fig. 7.-Autoradiograph, counterstainedwith
pellets and are said to have become dedifaqueous toluidine blue. X 1000.
f erentiated.
The results reported herein show that
DISCUSSION
chondrocytes from adult human cartilage
The development of an enzymatic behave like embryonic chondrocytes in
method has made possible the isolation of monolayer and organ cultures. Matrix pro-
239
ISOLATION AND CULTURE OF CHONDROCYTES
duction has been detected i n pellets cultured for 14 days, a n d it is anticipated that
this procedure wilI permit histochemical or
chemical assay of the polysaccharides pro-
duced. These methods of isolation and
culture should prove helpful in studying
the functions of chondrocytes from normal
and abnormal tissues.
SUMMARY
Viable chondrocytes have been isolated from human adult cartilage by digestion with
collagenase followed by mild mechanical disruption.
Monolayer cultures of chondrocytes showed dediff erentiation of the cells with faiIure
to synthesize matrix. Aggregations of chondrocytes in organ cultures retained their capacity to produce matrix.
The methods described should prove helpful in studying functions of chondrocytes
from normal and abnormal cartilage.
SUMMARIO
IN INTERLINGUA
Viabile chondrocytos esseva isolate a b cartilagine de humanos adulte per digestion
con collagenase sequite de leve disruption mechanic.
Culturas monostratal de chondrocytos monstrava disdifferentiation cellular con
absentia de synthese matrical. Aggragatos de chondrocytos in culturas de organos
reteneva lor capacitate de producer matrice.
Le methodos describite promitte esser utile in studiar le functiones d e chondrocytos
a b cartilagine normal e anormal.
REFERENCES
1. Bollet, A. J., Handy, J. R., and Sturgill, B. C.:
Chondroitin sulfate concentration and protein-polysaccharide composition of articular
cartilage in osteoarthritis. J. CIin. Invest.
42:853, 1963.
2. Bollet, A. J., Bonner, W. M., Jr., and Nance,
J. L.: The presence of hyaluronidase in
various mammalian tissues. J. Biol. Chem.
238:3522, 1963.
3. Sokoloff, L., Crittenden, L. R., Yamamoto,
R. S., and Jay, G. E., Jr.: The genetics
of degenerative joint disease in mice. Arthritis Rheum. 5:531, 1962.
4. Manos, J. P.: Rapid removal of tissue culture
monolayers by collodion for subsequent
staining. Stain Techn. 40:75, 1965.
5. Hagerty, R. F., Calhoon, T. B., Lee, W. H.,
Jr., and Cuttino, J. T.: Characteristics of
fresh human cartilage. Surg. Gynec. Obstet.
110:3, 1960.
6. Lee, W. H., Jr., Hagerty, R. F., and Braid,
H. L.: Measurements of cellular viability;
7.
8.
9.
10.
11.
A comparative study of neutral red and
radioactive sulfate in the examination of
the viability of cartilage. Plast. and Reconstr. Surg. 26:280, 1960.
Moscona, A. : Rotation-method histogenetic
aggregation of dissociated cells. Exp. Cell
Res. 22:455, 1961.
Holtzer, H., Abbott, J., Lash, J., and Holtzer,
S.: The loss of phenotypic traits by differentiated cells in vitro, I. DedifFerentiation of cartilage cells. Proc. Nat. Acad.
Sci. U.S.A. 46:1533, 1960.
Thorp, F. K., and Dorfman, A,: The occurence of intracellular chondroitin sulfate. J.
Cell Biol. 18:13, 1963.
Smith, A. U.: Survival of frozen chondrocytes
isolated from cartilage of adult mammals.
Nature 205:782, 1965.
Kawiak, J., Moskalewski, S., and Darzynkiewicz, Z.: Isolation of chondrocytes from
calf cartilage. Exp. Cell Res., 39:59, 1965.
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