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Mechanical Dissociation of Swine Liver to Produce Organoid Units for Tissue Engineering and In Vitro Disease Modeling

Center for Regenerative Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.
Artificial Organs (Impact Factor: 2.05). 01/2010; 34(1):75-8. DOI: 10.1111/j.1525-1594.2009.00784.x
Source: PubMed
ABSTRACT
The complex intricate architecture of the liver is crucial to hepatic function. Standard protocols used for enzymatic digestion to isolate hepatocytes destroy tissue structure and result in significant loss of synthetic, metabolic, and detoxification processes. We describe a process using mechanical dissociation to generate hepatic organoids with preserved intrinsic tissue architecture from swine liver. Oxygen-supplemented perfusion culture better preserved organoid viability, morphology, serum protein synthesis, and urea production, compared with standard and oxygen-supplemented static culture. Hepatic organoids offer an alternative source for hepatic assist devices, engineered liver, disease modeling, and xenobiotic testing.

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Available from: Craig M Neville, May 19, 2016
aor_784 75..88
Thoughts and Progress
Mechanical Dissociation of Swine Liver to
Produce Organoid Units for Tissue
Engineering and In Vitro
Disease Modeling
*†‡Katayun Irani, *†Irina Pomerantseva,
*Alison R. Hart, *†Cathryn A. Sundback,
*†Craig M. Neville, and *†§Joseph P. Vacanti
*Center for Regenerative Medicine, Massachusetts
General Hospital; †Harvard Medical School;
§Massachusetts General Hospital for Children,
Boston, MA; and ‡New York University Medical
School, New York, NY, USA
Abstract: The complex intricate architecture of the liver is
crucial to hepatic function. Standard protocols used for
enzymatic digestion to isolate hepatocytes destroy tissue
structure and result in significant loss of synthetic, meta-
bolic, and detoxification processes. We describe a process
using mechanical dissociation to generate hepatic orga-
noids with preserved intrinsic tissue architecture from
swine liver. Oxygen-supplemented perfusion culture
better preserved organoid viability, morphology, serum
protein synthesis, and urea production, compared with
standard and oxygen-supplemented static culture. Hepatic
organoids offer an alternative source for hepatic assist
devices, engineered liver, disease modeling, and xenobiotic
testing. Key Words: Bioartificial liver—Liver slice—
Liver-specific function—Organ culture techniques—
Oxygen-supplemented flow culture.
The principal sources for the biological component
of engineered liver devices and in vitro disease and
toxicology models include isolated primary hepato-
cytes, immortalized and tumor-derived cell lines, and
differentiated stem cells. Freshly isolated liver cells
exhibit the greatest utility but have a finite lifespan,
poor ability to tolerate cryopreservation, limited pro-
liferative capability, and suboptimal function (1). The
availability of human primary hepatocytes is also
limited; consequently, porcine liver is a principal cell
source for hepatic tissue engineering (2). Hepato-
cytes are isolated using a labor-intensive, multi-step
collagenase perfusion method leading to the disrup-
tion of tissue architecture and polarity-dependent
function.
To overcome the limitations of isolated liver cells,
organotypic culture (thin slices of tissue) is used to
create in vitro disease models, study drug metabolism
and toxicity, and as the source of the biological com-
ponent in experimental liver support systems. Prepa-
ration of liver slices does not require proteolytic
enzymes and allows maintenance of native polarity
and differentiation of hepatocytes, and cell–cell inter-
actions (3–5). However, tissue slices are very fragile,
making subsequent utilization of them difficult; we
have found that further dissociation of slices into
cubes allows easier handling of tissue in suspension
and improves diffusion of gases, nutrients, and
metabolites. Here, we describe a method using the
McIlwain tissue chopper for mechanical dissociation
of liver into organoid units for use in tissue engineer-
ing and disease modeling.
MATERIALS AND METHODS
Harvest and mechanical dissociation of liver
Livers were obtained under sterile conditions from
six nonfasted Yorkshire swine (38.7 1.6 kg) sacri-
ficed for unrelated experiments. A wedge resection
was performed from the left lateral or left medial
lobe; large vessels were cannulated and perfused
until blanched with sterile ice-cold lactated Ringer’s
solution supplemented with 0.27 M mannitol,
2 U/mL heparin, 25 mM glucose, 10 mg/mL insulin,
5.5 mg/mL transferrin, and 6.7 hg/mL sodium selenite
(Invitrogen, Carlsbad, CA, USA). The liver was
manually cut into 2 ¥ 2 ¥ 4-cm pieces and then into
1.0–1.5-mm thick slices with a scalpel blade. Indi-
vidual slices were further divided into 250 ¥ 250-mm
units using the McIlwain tissue chopper (Campden
Instruments, Lafayette, IN, USA). Resulting orga-
noids were then filtered through a 1-mm nylon mesh
(Small Parts, Miami Lakes, FL, USA), and filtered
pieces were washed repeatedly with lactated Ringer’s
solution using gravity sedimentation, with all remain-
ing supernatant removed by aspiration in the last
wash.Typically, 5–8% of initial tissue by weight would
be retained as organoid units, with a packed den-
sity of ~40 ¥ 10
6
cells/mL (as calculated by DNA
content).
doi:10.1111/j.1525-1594.2009.00784.x
Received April 2008; revised November 2008.
Address correspondence and reprint requests to Dr. Joseph P.
Vacanti, Department of Surgery, Massachusetts General Hospital,
Warren 1151, 55 Fruit Street, Boston, MA 02114, USA. E-mail:
jvacanti@partners.org
Artificial Organs
34(1):75–88, Wiley Periodicals, Inc.
© 2009, Copyright the Authors
Journal compilation © 2009, International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.
75
Page 1
Collagen gel preparation
Hepatic organoids were suspended in a 5 mg/mL
rat collagen I solution (3-D Culture Matrix, Trevigen,
Gaithersburg, MD, USA) on ice at a 1:10 ratio. One
mL of the suspension was placed into each well of
12-well plates or chamber of a perfusion device and
gelled for 30–40 min at 37°C.
Hepatic organoid culture
Organoids were cultured in 12 well plates in 1 mL
(approximately 2.6 mm average depth) of Hepato-
cyte Maintenance Medium with SingleQuot kit
(Lonza, Rockland, ME, USA) in standard 21% O
2
,
5% CO
2
, or oxygen-supplemented vented containers
continuously infused with carbogen gas (95% O
2
and
5% CO
2
); culture medium in both well plates and
devices was replaced daily. Custom perfusion flow
devices (Fig. 1) consisted of interlocking polyether-
sulfone parenchymal and medium flow chambers,
separated by a 0.4-mm pore polycarbonate mem-
brane (Millipore, Bedford, MA, USA). Medium
(20 mL total volume) was continuously pumped with
a Masterflex peristaltic pump (Cole-Parmer Instru-
ment, Vernon Hills, IL, USA) at 2 mL/min through
a closed-flow circuit containing an oxygenator and
a perfusion device housed in a 37°C, 5% CO
2
incubator. The 100-mL Pyrex bottle oxygenator
housed a 200-cm length of 0.9-mm inner diameter
oxygen-permeable silicone rubber tubing, with inlets
and outlets for medium and gas. Medium flowed
inside the silicone tubing (2 mL/min), while carbogen
gas (1 L/min) flowed over the tubing exterior; the
carbogen pressure within the oxygenator was essen-
tially atmospheric as the carbogen outlet was vented
directly to the atmosphere. The perfusion device was
positioned immediately downstream of the oxygen-
ator to minimize diffusional loss of oxygen into the
incubator chamber, and oxygen tension was mea-
sured at the entrance of the perfusion flow device
using an in-line oxygen sensor (Microelectrodes,
Bedford, NH, USA). The sensor was calibrated using
21% and 0% oxygen tension standards, and the cali-
bration was repeated before each measurement to
compensate for the drift of the sensor. The oxygen
partial pressure at the entrance of the perfusion
device was measured to be 686 mm Hg and did not
vary significantly throughout the study. The oxygen
uptake by the organoids could not be accurately mea-
sured because of the large excess of oxygen in the
culture medium.
Histological evaluation and detection of apoptosis
On days 0, 1, 3, 5, and 7, organoids were fixed in
10% formalin. Paraffin sections were stained with
hematoxylin and eosin (H&E) and with periodic
acid Schiff (PAS) stain to detect glycogen. Terminal
deoxynucleotidyl transferase biotin-dUTP nick end
labeling (TUNEL) assay (In Situ Cell Death Detec-
tion Kit, Roche Diagnostics, Indianapolis, IN, USA)
was used to detect apoptotic cells.
Urea production and albumin secretion
Urea production by organoids was determined by
culturing for 2 h in medium supplemented with 1 mM
ammonium chloride using the QuantiChrom Urea
Assay Kit (BioAssay Systems, Hayward, CA, USA).
FIG. 1. Liver organoid processing and
perfusion culture. (A) Perfusion device. (B)
Schematic showing cross-section of the
perfusion device. (C) Medium in the perfu-
sion bioreactor flow system was oxygen-
ated with carbogen by the oxygenator.
THOUGHTS AND PROGRESS76
Artif Organs, Vol. 34, No. 1, 2010
Page 2
Albumin secretion was measured with the Quan-
tiChrom BCG Albumin Kit (BioAssay Systems). All
sample measurements were performed in duplicate
with three samples/culture method; total DNA
content (DNeasy Blood & Tissue Kit, QIAGEN,
Valencia, CA, USA) was quantified by fluorimeter for
determination of relative cell number. All values are
expressed as the mean +/- SD. Analysis of variance
was used to calculate statistical significance.
RESULTS AND DISCUSSION
Freshly isolated organoids had intact liver micro-
architecture and viable cells (Fig. 2A1, 11); mechani-
cal processing caused only minimal damage that was
restricted to the surface. In vitro incubation con-
ditions had significant impact on hepatic organoid
viability and function. Oxygen-supplemented condi-
tions better maintained organoid cultures than stan-
dard CO
2
supplementation (Fig. 2A), consistent with
prior observations for isolated hepatocytes and liver
slice cultures (5,6). The benefit of oxygen supplemen-
tation was most apparent for hepatic organoids cul-
tured longer than 24 h. At 5 days, organoids cultured
without oxygen supplementation had few viable cells
(Fig. 2A2); many of the remaining nuclei showed
signs of apoptosis (Fig. 2A12), while organoids cul-
tured with oxygen supplementation had better
viability. Currently, there is no consensus regarding
the effects of oxygen tension on the viability and
function of isolated hepatocytes and liver slices in
vitro (5–7). Oxygen levels ranging from 21 to 95%
have been utilized and the effects have been species-
dependent (4,5). Standard incubator conditions that
provide 21% oxygen result in low oxygen tension in
cultured tissues, adversely affecting the cells as indi-
cated by morphological integrity. On the other hand,
excessive oxygen supplementation can produce
detrimental reactive oxygen species (5). Perfusion
culture further improved the viability, consistent with
reports on isolated hepatocytes and precision-cut
liver slices (6,8). In our experimental conditions,
FIG. 2. Assessment of hepatic organoids
in three culture conditions. (A) H&E stain-
ing was performed on paraffin sections of
organoids that had been: freshly isolated
(1), cultured in static conditions for 5 days
with 5% CO
2
(2) or carbogen (3), cultured
in flow with carbogen for 5 (4) and 7 (5)
days. PAS staining was used to evaluate
glycogen stores in organoids that were
freshly isolated (6), 3 days static culture
with CO
2
(7) or carbogen (8), or perfusion
for 3 (9) and 5 (10) days. Likewise, orga-
noids from identical conditions were
assessed for apoptosis by TUNEL (11–15).
(B) Albumin and urea production in three
culture conditions over 5 days. Rates of
albumin secretion into the medium (graph
1) increased during the culture period and
were greatest in the oxygen-supplemented
perfusion system. Hepatic organoids cul-
tured in oxygen-supplemented perfusion
devices maintained more ureagenesis
(graph 2) than in well plates, *P < 0.05,
**P < 0.001. There was no statistically sig-
nificant difference in albumin secretion or
urea production between standard and
oxygen-supplemented well plates.
THOUGHTS AND PROGRESS 77
Artif Organs, Vol. 34, No. 1, 2010
Page 3
hepatic organoids cultured in oxygen-supplemented
perfusion culture retained their viability and function
for 7 days (Fig. 2A5, 2B1, 2), and had the best viabil-
ity at 5 days as indicated by the results of H&E stain-
ing, TUNEL assay (Fig. 2A4, 14), and glycogen store
maintenance (Fig. 2A9). Relative to conventional
static and dynamic culture methods, perfusion culture
ensures continuous supply of both oxygen and nutri-
ents and prevents the buildup of metabolic waste
products and cytokines that rapidly lead to hepato-
cyte apoptosis (8,9).
Environmental conditions (i.e., oxygen tension,
culture medium supplements, and additional cell
types) can greatly affect specific hepatocellular
functions. Serum protein synthesis and ureagenesis,
two of the vital functions of the liver, are often
used as markers to assess hepatic function (1,6,8,9).
Oxygen supplementation resulted in increased
albumin synthesis and ureagenesis in both static well
plates and perfusion devices (Fig. 2B1, 2); without
oxygen supplementation, synthetic functions in well
plates remained constant. Enhanced metabolic activ-
ity in the remaining viable cells has been suggested as
a possible explanation of the discrepancy between
the morphological appearance and maintenance of
synthetic functions (5,7). In our experiment, albumin
secretion and ureagenesis were significantly lower
in static culture conditions, both with and without
oxygen supplementation, indicating that oxygen
supplementation alone is insufficient to prevent the
trauma-induced crisis associated with hepatic orga-
noid isolation.
Apoptosis and necrosis after enzymatic dissocia-
tion result in significant cell death in isolated hepa-
tocyte cultures (1,10). Characteristic apoptotic and
necrotic morphological changes and positive TUNEL
staining appeared earlier and were more apparent in
hepatic organoid preparations maintained in stan-
dard well plates relative to the oxygen-supplemented
perfusion system. Even cells that appear viable may
be stressed, causing them to conserve energy by
decreasing enzymatic function and protein synthesis
(1), consistent with our urea and albumin results. In
standard and oxygen-supplemented well plates, hepa-
tocytes have poorer recovery after isolation and tend
to undergo apoptosis; conditions within the perfused
device lead to better functional recovery with a sig-
nificant increase in albumin production and ureagen-
esis over the course of the experiment.
Enzymatic dissociation of liver disrupts intercellu-
lar connections responsible for maintaining both
organ architecture and cellular morphology essential
for hepatic function. In particular, tight junctions
between adjacent hepatocytes form physical barriers
defining basolateral and apical domains and func-
tionally segregate receptors, channels, and other
restricted components, and are targeted by collage-
nase dissociation protocols. Processing the liver into
organoid units avoids the loss of cellular polarization
and maintains much of the tissue architecture and
minimizes anoikis. Cytokine-induced apoptosis can
result from factors released by cells either damaged
during mechanical processing or stressed from enzy-
matic dissociation. Continuous perfusion of orga-
noids in the devices may prevent the buildup of
stress-induced cytokines that can trigger apoptosis in
static cultures.
CONCLUSION
Our isolation protocol generates hepatic organoids
with viable cells and intact native tissue architecture
without using proteolytic digestion techniques.
Oxygen-supplemented perfusion culture system pre-
served viability and function of the hepatic organoids
in vitro, making them a promising potential cell
source for hepatic assist devices, engineered liver,
disease modeling, and xenobiotic testing.
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