Full text of DOI:10.1007/BF03343821

J. Endocrinol. Invest. 24: 98-103, 2001
Improved yield and functionality of parathyroid cells
separated by using collagenase-digestion with cold
pre-incubation
M. Laudes*, A. Gaumann**, C. Laue***, and J. Schrezenmeir***
*III Department of Internal Medicine-Endocrinology; **Department of Pathology, Johannes
Gutenberg-University; ***Department of Physiology and Biochemistry of Nutrition, Federal Research
Center, Kiel, Germany
ABSTRACT. Preparation of cells from solid or-
gans often induces a functional impairment due
to the proteolytic cell damage by the applied di-
gestive enzyme like collagenase, trypsin or dis-
pase. To preserve the tissue and to enhance the
yield of cells, Laue et al. reported an islet cell iso-
lation with pre-incubation at 4 C permitting the
enzyme to diffuse into the tissue and explicite
activity equally throughout the whole particle.
The aim of this study was to investigate whether
this procedure can be applied to parathyroid
glands. Therefore porcine parathyroid glands
were dissected into 1 mm3 pieces. Subsequently
one group of these pieces was incubated 22 h
at 4 C in 2 mg/ml collagenase before activating
the enzyme by elevating the temperature to 37
C for 30 min. The other group was incubated di-
rectly at 37 C for 30 min. The yield of cells and
their viability was assessed by light-microscopy
and staining with trypan-blue. After the cells
were immobilized in barium-alginate and culti-
vated for 7 days, the function was tested by in-
cubation in different calcium concentrations and
PTH-measurement. Finally, the viability was as-
sessed by histology. Using a cold pre-incubation
with collagenase, a significantly higher number
of isolated cells was retrieved compared with col-
lagenase-digestion without pre-incubation. The
viability was about 100% and did not differ be-
tween both groups. After immobilization and cul-
tivation the viability decreased to less than 30%,
with and without pre-incubation. In contrast to
viability the PTH-secretion of the cells differed
significantly between both procedures. By pre-
incubation with collagenase at 4 C a gentle me-
thod is presented resulting in an enhancement
of yield and function of single cells of parathyroid
glands.
(J. Endocrinol. Invest. 24: 98-103, 2001)
©2001, Editrice Kurtis
INTRODUCTION
trypan-blue staining. Furthermore, a significant stim-
ulation of the PTH-secretion by low calcium concen-
tration was reported. Nevertheless, several authors
described structural and functional cell damage when
using these proteolyitc enzymes (5, 6). In case of islet-
cell-isolation Laue et al. (7) could enhance the yield
and the function of cells by using a pre-incubation in
collagenase-solution at 4 C for 22 hours to permit dif-
fusion throughout the tissue before activating the en-
zyme by elevating the temperature to 37 C. The aim
of this study was to investigate whether the application
of cold pre-incubation of a parathyroid gland is able to
improve the cell viability, function and yield.
For single-cell preparation of parathyroid tissue, solid
organs were first divided mechanically into small
pieces, before incubation in different proteolytic en-
zymes like collagenase (1-3) or trypsin (4). In case of
collagenase-digestion the authors used a concentra-
tion of 2 mg/ml and an incubation time which varied
from 30 min (2) to 70 min (1). In these reports the yield
of cells amounts from 5x105 to 5x106 cells per 100 mg
tissue, showing a viability of 90 to 100% assessed by
Key-words: Collagenase, parathyroid, pre-incubation, alginate, PTH-se-
cretion.
MATERIALS AND METHODS
Study design
Correspondence: Prof. J. Schrezenmeir, Institut für Physiologie und
Biochemie der Ernährung, Bundesforschungsanstalt, Postfach 6069,
24121 Kiel, Germany.
Porcine parathyroid glands were weighed and im-
mediately cut into 3 small pieces of ~1 mm3 each,
E-mail: schrezenmeir@bafm.de
Accepted September 4, 2000.
98

Improved method for parathyroid cell separation
before single cell isolation was performed. After
single cell preparation the aliquots of one gland
were collected in one petri dish to permit relation of
the gland from which the cells were derived. Ano-
ther gland was simultaneously prepared after the
same protocol with the aim of testing function.
stadt, Germany). To remove the collagenase the
tubes were centrifuged at 6.7 G for 15 min. The su-
pernatant was then aspirated and the pellet of the
single cells was re-suspended with 2 ml fresh
DMEM tissue culture medium. As shown by light
microscopy some of the isolated cells are coherent
in form of little lobuli.
Isolation of the parathyroid glands
Yield and trypan-blue assessed viability
Parathyroid glands were isolated from slaughterhouse
pigs (German land race). The paired upper thymus
lobe was excised within 15 min after death. The thy-
mus was isolated from surrounding connective tissue
by means of a small sized scalpel and stored imme-
diately on ice in Hanks-solution (Gibco, Germany). In
the laboratory the parathyroid glands were localized
by preparing the effluent arterial vessels and excised
from the surrounding thymus tissue.
The number of viable and dead cells was assessed
by counting the unstained and stained cells in petri
dishes in three microscopic fields (40-fold magnifi-
cation) after addition of 100 μl of trypan-blue solu-
tion (5 g/l physiologic NaCl). Subsequently, a pro-
jection to the whole petri dish was made and the
result was related to 100 mg parathyroid tissue. As
viability the percentage of viable cells in relation to
the whole cell number was defined.
Single-cell preparation
Encapsulation
Parathyroid glands were weighed and dissected
into 3 pieces of ~1 mm3 each, which were placed
into different tubes containing Hanks-medium. The
Hanks-solution was removed and the tissue pieces
of one gland were incubated with 2 ml of 2 mg/ml
collagenase (Serva Pan Plus, Lot. No. 03092) in
NaCl 0.9% with 15% FCS (Seromed Vitromex,
Berlin, Germany), 0.0416 mg/ml D-glucose, 0.25
mg/ml KCl, 0.037 mg/ml NaH2PO4 and 0.021
mg/ml KH2PO4 each at 4 C for 22 hours. FCS was
added to inhibit unspecific proteases known to be
present in commercial collagenase preparations.
Subsequently, most of collagenase was removed
and the tubes were gently shaken in a water bath at
37 C for 30 min. After that, a nearly complete di-
gestion of the parathyroid pieces was found.
The tissue pieces of another gland were directly in-
cubated with the same collagenase solution without
any pre-incubation at 37 C in a water bath. The di-
gestion also finished after 30 min, however it was in-
complete. The intention, therefore, was to achieve
comparable conditions between the two digestion
procedures.
Afterwards in both cases 4 ml PBS (Biochrom Sero-
med, Berlin, Germany) was added containing 5
mg/ml BSA (Boehringer, Mannheim, Germany) and
0.2 mg/ml EDTA (Merck, Darmstadt, Germany).
After 10 minutes incubation the tubes were stirred
for approximately 2 min <1000 U/min. Then the su-
pernatant was suspended in 20 ml DMEM medium
(Biochrom Seromed, Berlin, Germany), containing 2
ml/l Penicillin Streptomycin (Boehringer, Mannheim,
Germany), 2 mg/ml Ciprofloxazin (Bayer, Lever-
kusen, Germany), 25 mmol/l HEPES (Merck, Darm-
stadt, Germany), 10% FCS (Seromed Vitromex, Ber-
lin, Germany) and 0.4 mmol/l CaCl2 (Merck, Darm-
To improve the handling for histological investiga-
tion and function testing, the isolated cells and lob-
uli were immobilized by encapsulation into barium-
alginate microcapsules using an air jet device as
previously described by our group (8). After anoth-
er centrifugation the culture medium was aspirat-
ed and the parathyroid cells were suspended in 4
ml 2.5% sodium-alginate. This suspension was de-
livered by a precision piston pump (Braun, Mel-
sungen) to the nozzle of the encapsulation device.
In this drop generator a central cannula with a di-
ameter of 0.5 mm was adjusted radially and axially
with thin screws in a nozzle. A continuous air flow to
the tip was provided via a tube connected with the
nozzle to tear the drops by the coaxial air stream.
The flow was regulated by a precision valve (Ber-
noulli tube) and controlled by precision flow meter.
The air pressure was maintained at 250 mbar using
a buffer volume to muffle possible fluctuations. The
temperature during all operations was below 25 C.
The drops were precipitated in a 5 mmol/l BaCl2
solution which was stirred at 100 rpm. After incu-
bation for less than three minutes the capsules were
transferred into a tissue culture bottle filled with 5
ml culture medium (RPMI 1640, Biochrom Sero-
med, Berlin, Germany) containing 100 mg/dl gluco-
se and 10% FCS. The average diameter of the mi-
crocapsules was 700-900 μm.
Function testing
Before function testing the immobilized cells were
cultured for 7 days in DMEM after which PTH leak-
age due to cell lysis by the previous digestion is ne-
glectable. The medium was changed daily. To test
the functionality of the parathyroid cells we used a
99

M. Laudes, A. Gaumann, C. Laue, et al.
Table 1 - Yield of cells isolated by collagenase-digestion with and without pre-incubation at 4 C.
Digestion without pre-incubation
10.0x106 1.3x106
Digestion with pre-incubation
32.4x106 2.9x106
Mann-Whitney test
p<0.001
Total yield
Yield of single cells
Yield of conglomerates
Mean SE; no.=15.
4.1x106 2.6x105
8.9x106 2.3x105
p<0.001
14.1x104 1.8x103
50.8x104 6.9x103
p<0.001
culated via the average diameter which was 72.4
μm after digestion with pre-incubation and 85.8 μm
after digestion without pre-incubation. Assuming a
cell diameter of 10 μm the conglomerates con-
tained 380 vs 630 cells. The number of conglom-
erates, however, was significantly higher when pre-
incubation was used (Table 1). According to this the
calculated total number of isolated cells was sig-
nificantly higher after collagenase-digestion using
cold pre-incubation (Table 1).
medium which contained 45% DMEM and 64% RP-
MI 1640 (Biochrom Seromed, Berlin, Germany),
containing 2 ml/l Penicillin Streptomycin (Boeh-
ringer, Mannheim, Germany), 5ml/l Ciprofloxacin
(Bayer, Leverkusen, Germany), 10% FCS (Seromed
Vitromex, Berlin, Germany), 1% L-Glutamin (ICN
Biomedicals). This mixture resulted in a concentra-
tion of 1 mM calcium and permitted elevation to 3
mM calcium without precipitation of calciumphos-
phate. About 60 microcapsules were incubated in 2
ml 1 mM and 3 mM calcium containing medium
mixture each during 24 h at 37 C. The PTH-secre-
tion was measured by RIA [Incstar human PTH-
MM™-II-Iod-125, sensitivity: 9.8 pmol/l, specifici-
ty: 100% (As 44-68), cross-reaction with porcine
PTH: 70%].
Viability
The viability of the isolated cells, assessed by stain-
ing with trypan-blue directly after digestion, was
next to 100% and did not differ significantly be-
tween both methods. The viability of the cells his-
tologically assessed after immobilization, cultiva-
tion and testing function amount to 30-35% and
showed also no significant difference between the
two digestion procedures (Fig. 1). In both cases the
single cells showed a higher viability compared to
Histological assessment of viability
After function testing the microcapsules were fixed in
Bouin solution, embedded in paraffin and stained
with hematoxylin-eosin. For quantification a score
was used, which considered the grade of cell dam-
age. Cells with intact chromatin structure were clas-
sified as 100% viable, cells with pycnotic nuclei 50%
viable and necrotic cells with lacking nuclei 0% vi-
able. Thus the “viability score” was expressed by the
following formula: p1*100% + p2*50% +p3*0%=to-
tal viability % where p1, p2, p3 are the portions of
the total area presenting nuclei with intact chromatin
(p1), pycnosis (p2) and necrosis (p3).
80
NS
70
60
NS
NS
(p<0.01)
50
40
30
20
10
0
Statistics
Cell-number, viability, basal and stimulatory values
were expressed as mean SE. The obtained data
were compared using the Mann-Whitney U test for
unpaired and the Wilcoxon test for paired samples.
Fig. 1 - Viability of immobilized parathyroid cells, separated by
collagenase-digestion with and without pre-incubation at 4 C.
ꢀ represents viability of immobilized parathyroid single cells
and ꢁ represents cells in conglomerates (no.=25), separated
RESULTS
Yield
by digestion without pre-incubation.
represents viability of
After collagenase-digestion with pre-incubation at
4 C the yield of viable single cells was significantly
higher compared with digestion without pre-incu-
bation (Table 1). To assess the total yield of cells
the number of cells contributed by lobuli was cal-
immobilized parathyroid single cells and ꢁ represents cells in
conglomerates (no.=25), separated by digestion with pre-incu-
bation. Viability was histologically assessed after 7 days of cul-
ture and 2 days of function testing. Statistical significance was
tested by Mann-Whitney test.
100

Improved method for parathyroid cell separation
contrast, after collagenase-digestion using pre-in-
cubation the PTH-secretion amount to 46.79 6.19
pmol/l in medium containing 1 mmol/l calcium and
33.04 5.85 pmol/l in medium containing 3 mmol/l
calcium (Fig. 2). Thus, the PTH-secretion was sig-
nificantly (p<0.05) stimulated by low calcium con-
centrations only in case of collagenase-digestion
with cold pre-incubation.
80
70
60
50
40
30
20
10
0
p<0.05
NS
DISCUSSION
In case of collagenase-digestion without pre-incu-
bation at 4 C for 22 hours the total yield of cells
amount to 10.0x106/100mg, which is comparable
with the results reported from other authors (9-11).
Using cold pre-incubation, the yield of cells in-
creased to 32.4x106/100mg, although we applied
the same collagenase concentration and the same
duration of incubation. This effect may be explai-
ned by diffusion of collagenase into the parathyroid
pieces during the pre-incubation. When activating
the enzyme by elevating the temperature to 37 C
the collagenase digested throughout the whole
pieces. In case of digestion without pre-incubation
the collagenase only separates the superficial cells,
while the inner part of the pieces remains un-
touched. After both methods not only single cells
but also lobuli were found. This may be explained
by an unequal distribution of collagenase concen-
tration within the tissue pieces during digestion.
Cold pre-incubation with collagenase resulted in
smaller particles and higher yield indicating a more
homogenous and more efficient digestion. The vi-
ability of the isolated cells did not differ significantly
when assessed directly after digestion, as well as
Fig. 2 - Function of immobilized parathyroid cells, separated by
collagenase-digestion with and without pre-incubation at 4 C.
ꢀ represents PTH secretion in 1 mM and ꢁ represents 3 mM
calcium containing medium in pmol/l (no.=50) of immobilized
parathyroid cells, separated by digestion without pre-incuba-
tion.
represents PTH secretion in 1 mM and ꢁ represents 3
mM calcium containing medium in pmol/l (no.=50) of immobi-
lized parathyroid cells, separated by digestion with pre-incu-
bation. The function was tested after cultivation for 7 days. Time
of incubation for each medium: 24 h. Statistical significance was
tested by Wilcoxon test.
the cells in lobuli, however, this difference was on-
ly significant (p<0.01) in case of collagenase-di-
gestion with cold pre-incubation.
Function
After cell preparation without pre-incubation we
found a PTH-secretion of 21.29 5.10 pmol/l in
medium containing 1 mmol/l calcium and 22.19
4.69 pmol/l in medium containing 3 mmol/l calci-
um, which was not significantly different (Fig. 2). In
Fig. 3 - Parathyroid cells separated by collagenase-digestion
with pre-incubation at 4 C, stained by trypan-blue. Some of the
cells are coherent in form of little conglomerates (40x magnifi-
cation). Viability: 100%.
Fig. 4 - Parathyroid cells separated by collagenase-digestion
with pre-incubation at 4 C. The cells were histologically pre-
pared and stained with HE after a cultivation period of 7 days
and 2 days of function testing (50x magnification).
101

M. Laudes, A. Gaumann, C. Laue, et al.
3. Xiao W.F., Sun A.M.
after cultivation for 9 days. In both cases, however,
the viability decreased from near 100% to less than
30% during cultivation. Directly after digestion try-
pan-blue staining was used which detects cytotox-
ic damage of cell membranes. By histology, ap-
plied 9 days later, nuclear damage was assessed,
which could not be discovered directly after di-
gestion. According to this the different viability may
be explained by induction of apoptosis by ischemia
and digestion. The lower viability of cells in lobuli
when compared with single cells is probably cau-
sed by an insufficient supply of oxygen and nutri-
ents due to the higher diffusion distance in greater
particles (12).
In function testing after cultivation for 7 days, only
the cells separated by collagenase-digestion using
cold pre-incubation showed a significant stimula-
tory response of PTH-secretion to low calcium con-
centrations. In contrast, there are some authors who
reported significant stimulation using collagenase-
digestion without pre-incubation (11, 13-14). The
authors, however, tested function directly after di-
gestion. Nevertheless, it is reported that cell cul-
ture systems do not permit the maintenance of
functionality active parathyroid cells (15, 16).
Furthermore, Mithal et al. (17) described a reduced
responsiveness of cultured bovine parathyroid cells
to calcium associated with a lower expression of
calcium receptor mRNA. According to that, colla-
genase-digestion using cold pre-incubation is able
to improve functionality of separated parathyroid
cells even in culture for 7 days compared to colla-
genase- digestion without pre-incubation.
Microencapsulated parathyroid cells as a bioartificial
parathyroid.
Transplantation 1989, 47: 432-435.
4. Fitzpatrick L.A., Leong D.A.
Individual parathyroid cells are more sensitive to cal-
cium then parathyroid cell population.
Endocrinology 1990, 126: 1720-1727.
5. Schrezenmeir J., Laue C., Sternheim E.T., Wölbert
K., Darquy S., Chicheportiche D., Kirchgessner J.,
Reach G.
Long-term function of single-cell preparations of
piscine principal islets in hollow fibers.
Transplant. Proc. 1992, 24: 2941-2945.
6. Pipeleers D.G., Pipeleers-Marichal M.A.
A method for the purification of single A, B and D
cells and for the isolation of coupled cells from rat
islets.
Diabetologia 1981, 20: 654-663.
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Macromol. Chem. Phys. 1994, 195: 1557-1567.
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Secretion of intact PTH by dispersed human hyper-
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Compared with direct digestion, cold pre-incuba-
tion with collagenase resulted in similar viability but
higher yield and better function, which is compa-
rable to the results reported from Laue et al. when
isolating islet cells. This may be explained by more
homogenous distribution of the digestive enzyme
throughout the tissue before activation.
10. Brown E.M., Brennan M.F., Hurwitz S., Windeck R.,
Marx S.J., Spiegel A.M., Koehler I.O., Gardner D.G.,
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Lack of a direct effect of 1,25-Dihydroxycholecal-
ciferol on parathyroid hormone secretion by normal
bovine parathyroid glands.
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