Influence of Mitochondrial Membrane Potential on the
Cryopreservation Survival of Hepatocytes
by
Margaux E. Daly
B.S. Chemical Engineering
Massachusetts Institute of Technology, 2004
SUBMITTED TO THE HARVARD-MIT DIVISION OF HEALTH
SCIENCES AND TECHNOLOGY IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF ENGINEERING IN BIOMEDICAL ENGINEERING
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May 16, 2005
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ARCHIVES
Influence of Mitochondrial Membrane Potential on the
Cryopreservation Survival of Hepatocytes
by
Margaux E. Daly
Submitted to the Harvard-MIT Division of Health Sciences and Technology
on May 16, 2005 in Partial Fulfillment of the Requirements for the
Degree of Master of Engineering in Biomedical Engineering
ABSTRACT
Hepatocytes are widely used in the pharmaceutical and medical fields for drug
metabolism studies, bioartificial liver devices, and repopulation of damaged livers as an
alternative to transplantation. However, these cells are scarce and difficult to maintain in culture
for prolonged periods of time. Banks of cryopreserved liver cells would significantly alleviate
issues of hepatocyte availability, and efforts are being made to improve the viability and
functionality of frozen hepatocytes. Previously, most work on improving post-thaw viability has
hinged on limiting the physical damage of freezing by adding cryoprotective agents and
optimizing cooling rates. Membrane-permeable cryoprotectants, such as dimethyl sulfoxide,
though widely used, can be extremely toxic to the cell. More natural, non-membrane-permeable
cryoprotectants, inspired by freeze-tolerant animals have also been used. A non-metabolizable
glucose analog, 3-0-methyl- glucose (30MG), has shown promise with hepatocytes and was
used in this study. Kinetics of the rGLUT2 cellular transporter used for 30MG uptake were
quantified; Km and Vmaxwere determined to be 27.6 mM and 1.38 mM/s, respectively, by
Lineweaver-Burk analysis and 70.0 mM and 1.82 mM/s, respectively, by Eadie-Hofstee analysis.
This study also aimed to investigate the role of mitochondria in cell death induced by freezing.
In particular, mitochondrial membrane potential (MMP) was investigated as a predictor of a
cell's likelihood to avoid apoptosis from freeze-induced stress. Cells were sorted into high and
low MMP subpopulations, frozen, thawed, and cultured for 24 hours. Cell cultures were
analyzed for attachment yield, viability of attached cells and overall viability, which were 87%,
68% and 59%, respectively for the high MMP subpopulation, and 68%, 53% and 35%,
respectively for the low MMP subpopulation. Morphological differences such as extent of
membrane blebbing were observed as well, verifying that cells with a high MMP are more likely
to survive the cryopreservation process. These results demonstrated that MMP is a determinant
of both frozen hepatocyte adherence efficiency and viability; a high MMP yields a significant
advantage in both. Our understanding of the role of MMP in freeze-thaw death and of the
characteristics of the rGLUT2 transporter will lead to the development of more successful
cryopreservation protocols.
Thesis Supervisor: Mehmet Toner, Ph.D.
Title: Professor of Biomedical Engineering, Harvard Medical School and HST
Thesis Supervisor: Gregory Stephanopoulos, Ph.D.
Title: Professor of Chemical Engineering
2
Table of Contents
1. Introduction .................................................................................................................................
1.1 Cryoprese:rvation Techniques ........................................
.......................................................
6
1.1.1 Cryoprotective agents .................................................................................................... 6
1.1.2 Natural cryoprotectants .................................................................
......... 7...........
7
1.2 Mechanisms of Freeze-Thaw Death ........................................
.............................................
9
1.2.1 Mitochondrial contribution to apoptosis ...................................................................... 10
10
1.3 Hepatocyte Cryopreservation.................................................................
13
1.4 Study Aims .................................................................
2. Materials and Methods.................................................................
2.1 Isolation of Primary Hepatocytes.................................................................
2.2 Kinetic Characterization of rGLUT2 ....................................................................
2.3 Fibroblast Stain and Sort .................................................................
2.4 Fibroblast Cryopreservation and Viability Assessment
2.5 30M G Hepatocyte Cytotoxicity ..................
15
15
......... 15
16
.......
17
. .
...............................................17
2.6 Rh 123 Staining for MMP .................................................................
18
2.7 Creation of High and Low MMP Subpopulations .............................................................. 18
21
2.8 Hepatocyte Cryopreservation Protocol .................................................................
21
2.9 Viability Assessment of Hepatocytes .......................................... .......................
3. Results.......................................................................................................................................
..
25
3.1 Kinetics of rGLUT2 .........................................
...................................................................
25
3.2 Fibroblast MMP Subpopulation Post-thaw Viability ......................................................... 29
29
3.3 30M G Cytotoxicity .................................................................
3.4 Hepatocyte MMP Subpopulation Post-Thaw Morphology and Viability .......................... 33
4. Discussion ................................................................................................................................. 40
4.1 MMP Effects on Cryopreservation Viability ................................................................. 40
4.1.1. Adherence ability v. viability.................................................................
40
4.1.2. Mitochondrial energetics and adherence yield ........................................................... 41
4.1.3. Deviations from expected viability .................................................................
42
4............................
44
4.2 Viability Assessment Methodology .........................................
4.3 rGLUT2 Kinetics .................................................................
44
4.4 Recommendations for Future Study .................................................................
46
5. Acknowledgements .................................................................................................................. 47
6.0 References .............................................................................................................................. 48
APPENDIX A: Raw data.....................................
.......................
.......................... 50
APPENDIX B: Novel cryopreservation solution development and testing ............................ 53
APPENDIX C: Effects of succinate on post-cryopreservation viability ................................. 55
APPENDIX D: Attempted protocols for extraction of hepatocytes from culture ................... 56
APPENDIX E: Effects of heat-shock on viability of cryopreserved fibroblasts ..................... 57
APPENDIX F::Functionality of 30MG-incubated hepatocytes.............................................. 58
APPENDIX G: Calibration protocol for cell concentration assessment by fluorescent
60
microscopy .................................................................
APPENDIX H: Endoxtin pretreatment effects on hepatocyte MMP..................................... 61
3
List of Figures
1. Introduction
1.1: Mitochondrial contribution to apoptotic pathway..............................................................11
2. Materials and Methods
2.1: High and low MMP subpopulation sort histogram ............................................................
2.2: MMP stain and sort protocol.............................................................
2.3: Cryopreservation protocol.............................................................
2.4: Microscope cell concentration calibration.............................................................
19
20
22
23
3. Results
3.1: Lineweaver-Burk plots of rGLUT2 kinetic data............................................................. 26
27
3.2: Eadie-Hofstee plots of rGLUT2 kinetic data ............................................................
3.3: Relative Rh123 fluorescence of high and low MMP fibroblast populations......................30
31
3.4: Post-thaw fibroblast subpopulation viability............................................................
3.5: 24 h culture, cryopreserved hepatocytes.............................................................................34
3.6: Extent of blebbing in high and low MMP subpopulations.................................................35
............................................................36
3.7: Viability of unadhered hepatocyte population.
3.8: Adherence yield of 24-hr, post-freeze/thaw cultured hepatocytes.....................................38
3.9: Viability cf adherent hepatocytes from 24-hr, post-freeze/thaw culture............................38
3.10: Overall 24-hr, post-freeze/thaw viability of hepatocytes..................................................38
List of Tables
3. Results
28
3.1: Kinetic parameters of rGLUT2 ............................................................
32
3.2: 30MG hepatocyte cytotoxicity.............................................................
3.3: Summary of post-thaw, 24-h cultured hepatocyte viabilities.............................................39
4
1. Introduction
Hepatocytes are the major cell of the liver, making up 60% of the organ's cellular
population and 70% of its volume (1). In addition to the essential role they play as
parenchymal liver cells, hepatocytes also play important roles in the pharmaceutical and
medical milieus. Before beginning animal studies, drug companies utilize "metabolically
competent" hepatocytes to analyze how the human body will metabolize and tolerate a
particular compound (2). Patients awaiting liver transplants or suffering from acute liver
failure may soon be hooked up to bioartificial liver (BAL) devices housing cultured
hepatocytes, receiving organ support in much the same way that dialysis replaces certain
kidney function (3). Even direct transplantation of hepatocytes into an ailing liver is
being investigated as a substitute for a complete liver transplant (4). The difficulty in
hepatocyte applications, however, lies in their procurement, which is much more difficult
than for the majority of other cell lines.
One of the greatest challenges with using hepatocytes is their lack of in vitro
proliferation (4). Hepatocyte cells lines cannot be created and as such must be isolated
anew from an organ each time they are needed. The isolation process itself is timeintensive and requires skilled technicians and special equipment. Moreover, once
primary hepatocytes are seeded into culture, the interval of their usefulness may be as
little as a week (depending on culture conditions); after this time, they begin to
dedifferentiate and lose much of the functionality for which they were originally needed
(2, 4, 5). Due to these difficulties, the need for primary hepatocytes often exceeds their
supply and a method for long-term biostabilization of hepatocytes would be greatly
beneficial.
5
1.1 Cryopreservation Techniques
One possible biostabilization method for hepatocyte samples is cryopreservation in this way, liver cells could be banked as blood and cell lines are currently stored.
Cryopreservation would thus allow for mass isolations of hepatocytes and subsequent
storage over long periods of time. However, there are problems inherent in the
cryopreservation process as well. Cells that have been frozen and thawed demonstrate
"freeze-thaw injury" in the form of decreased viability, functionality and attachment yield
(6). This freeze-thaw injury, from a physical perspective, can be the result of several
aspects of the freezing process, including intracellular ice nucleation, crystal size and
location, cell dehydration and shrinkage or oxidative stress (7, 8). Optimized rates of
cooling and warming are essential to decrease this damage. Cooling cells too quickly
results in significant intracellular ice formation and thus damage to cellular constituents
and the cell membrane; cooling cells too slowly allows ice formation in the extracellular
milieu, increasing the osmolarity of the remaining liquid solution and resulting in net
efflux of fluid from the cell (9). Unfortunately, optimizing the rate of freezing alone does
not limit freeze-thaw damage enough, and additional techniques must be employed.
1.1.1 Cryoprotective agents
Steps to avoid injury and therefore yield acceptable cell viability after
cryopreservation have been made over the last two decades, particularly through
utilization of cryoprotective compounds. Cryoprotectants are typically permeable to cell
membranes and include solutes such as dimethyl sulfoxide (DMSO), glycerol, ethylene
glycol and propylene glycol. Their effectiveness as cryoprotectants was discovered
6
empirically; although the mechanism of action is not completely known, it is thought to
involve limiting detrimental concentration of electrolytes in the cytosol during
extracellular ice formation as well as reducing the probability of intracellular ice
formation (9). DMSO in particular has been surmised to have membrane-stabilization
characteristics (9).
CPAs are very effective, often yielding as much as 80 to 90 % viability,
depending on cell type; however, there are several major drawbacks. They must be used
in molar quantities to achieve the desired result, often as much as ten to twenty percent
by volume of the freezing solution. At such high concentrations these compounds are
cytotoxic (10). To avoid significant cell death, CPAs must be added at low temperatures
and removed quickly after thawing - exposure for as little as 15 to 20 minutes post-thaw
can have major detrimental effects (11, 12). Very high concentrations of CPAs, along
with slower-than-water diffusion into cells, also results in an initial osmotic gradient that
can cause damaging cell shrinkage and lead to osmotic injury (9). Moreover, CPAs must
be removed from cells or tissues before in vivo use. The procedure to remove the CPAs
from cell suspension can also cause cellular damage, and adds yet another step to an
already complex and time-consuming protocol (10).
1.1.2Natural cryoprotectants
To avoid the problems associated with toxic CPAs, our lab and others have
investigated the feasibility of more "natural" cryoprotectants (NCPs). Inspiration for
candidates for this approach comes directly from freeze-tolerant animals. Carbohydrate
cryoprotectant compounds are used by hatchling C. picta marginata turtles, R. sylvatica
7
frogs and others to withstand freezing temperatures, even in the presence of intracellular
ice formation (10). Taking cues from nature, researchers have utilized sorbitol, glucose,
trehalose, and other small carbohydrates to achieve acceptable post-thaw viability (10,
13). NCPs provide protection to cells through a phenomenon described by Raoult's Law
- solutes small in size are most effective at depressing freezing points to avoid ice
formation. These same small compounds are also best at limiting the extent of ice
formation prior to reaching the glass transition point (8). Diffusion rate analysis of
glucose-water solutions suggests that glucose disrupts water's molecular network to
promote glass transition (14). It is additionally suspected that some NCPs, such as
trehalose, have membrane-stabilization properties (8).
The biggest drawback with NCP utilization is that, unlike their CPA counterparts,
NCPs are impermeable to cell membranes. This translates to a pre-cryopreservation
protocol that can be much more involved than the simple addition of the NCP to solution.
Loading of these compounds has been attempted by thermal membrane permeabilization,
electroporation, and genetic engineering to express a controllable pore (15, 16, 17).
Although effectively introducing sugars into the cytosol, these procedures can be
damaging to the cells, procedurally complex, or both.
Non-carbohydrate NCPs are utilized in nature as well. Anti-freeze proteins found
in freeze-tolerant species prevent ice nucleation and depress the overall rate of ice
formation, but they are present in such small concentrations that they cannot currently be
purified from organisms for commercial use (8). Free amino acids also decrease freezing
points and are suspected to help maintain protein shape in temperature changes and
desiccation; proline in particular has been found to be unusually good at stabilizing
8
membranes (8). However, amino acids by themselves are not sufficient to limit ice
formation to the extent that small carbohydrates do.
1.2 Mechanisms of Freeze-Thaw Death
In order to investigate methods to further improve freeze-thaw viability, one
should first look at the mechanisms underlying cryopreservation death. In fact, both
necrosis and apoptosis play significant roles in the process. Necrosis, cell death due to a
major insult such as injury, infection or membrane disruption, is a direct result of many
of the physical damage problems that cryoprotectants like CPAs and NCPs address.
Apoptosis, on the other hand, has only recently been investigated in the field of
cryopreservation.
Apoptosis, defined as programmed cell death, can be triggered externally by
activation of ''death receptors" on the cell membrane or internally by specific intracellular
events in the absence of physical damage (18). Triggers can include external signaling
molecules, developmental regulators, chemical instigators, and physical stressors such as
radiation and extreme temperatures (19). Moreover, apoptosis has been clearly linked to
the death of frozen cells by Baust et al (20), who referred to the process as
cryopreservation-induced delayed-onset cell death. The exact trigger of apoptosis in this
case is unknown. It may be due to changes in cell volume that directly activate death
receptors or due to internal signaling (from accumulation of free radicals, DNA damage
or injury to the cell that does not directly cause death). Most likely, cryopreservation-
induced apoptosis is caused by a combination of internal and external signals (21). The
9
process has been observed during the cryopreservation of several cell types, including
oocytes, bull sperm and hepatocytes (22, 23, 24).
1.2.1Mitochondrialcontributionto apoptosis
In recent years, mitochondria have been implicated as the central organelle in
apoptosis (Figure 1.1). Members of the Bcl-2 protein family, located in the cytosol,
trigger formation of "pores" within the mitochondrial membrane. These pores allow for
the release of cytochrome c, which subsequently activates enzymes known as caspases,
the mediators of apoptotic death (25). One particular characteristic of mitochondria,
mitochondrial membrane potential (MMP), may itself play a significant role in the
process. MMP can be used to assess the energy metabolism of a cell, where
high MMP translates to a high metabolic state (26). MMP decrease in the initial stages of
apoptosis remodels the intraorganellar matrix, localizing cytochrome c for release (27).
The loss of MMP and subsequent depolarization of the mitochondrial membrane occurs
during the execution of apoptosis and can be observed prior to biochemical and
morphological changes associated with the process (18, 19). Furthermore, a cell's
inherent MMP may contribute to its tendency for apoptosis in a given situation: high
MMP subpopulations of murine hybridoma cells were shown to be much more resistant
to apoptosis as induced chemically by rotenone or staurosporin (18).
1.3 Hepatocyte Cryopreservation
Most work with cryopreservation of hepatocytes has been limited to traditional
CPAs such as DMSO, glycerol and polyethylene glycol. Recently, NCPs have been
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Figure 1.1: Mitochondrial contribution to apoptotic pathway. Stresses such as membrane damage,
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release of cytochrome c, and subsequent cellular apoptosis. Image obtained from:
http://www.sigmaaldrich.com/ (mit_apoptosis.gif)
11
employed as well. One promising candidate for a hepatocyte NCP is glucose, which is
permeable to liver cells by way of the passive, facilitative glucose transporter GLUT2
(found chiefly in liver, kidney and pancreatic ,3-cells(28)). Although glucose provides
the desired physical protection from cryopreservation afforded by NCPs and avoids the
pitfall of membrane-impermeability encountered in most cell types, it cannot be utilized
in hepatocyte cryopreservation - metabolism of higher-than-physiological concentrations
of glucose by the cells yields toxic products which ultimately result in cells death.
However, the glucose analog 3-0-methyl-glucose (30MG) is non-metabolizable but
capable of traversing the membrane via the same GLUT2 transporter; it has shown
promising with hepatocyte cryopreservation. Permeability of 30MG to the hepatocyte
plasma membrane makes loading of the cryoprotectant a simple process of incubation in
an extracellular 30MG medium. Similarly, removing the cryoprotectant after thawing
requires simply establishing the reverse gradient, a process which is extremely short in
time and non-damaging to the cells.
Both the toxic and "natural" versions of cryoprotectants have had limited success
in improving the freeze-thaw viability of hepatocytes. Although samples with these
compounds demonstrate significant survival over controls (<60% vs. <10%), there is still
much room for improvement (20, 29).
Little work has been done to improve post-cryopreservation viability of
hepatocytes by decreasing apoptotic death, although apoptosis has been demonstrated to
be a contributing factor. Interestingly, there appears to be promise in this approach: two
groups were able to increase viability of hepatocytes by adding a global caspase inhibitor
during cryopreservation (23, 30). In light of the evidence for the contribution of MMP to
12
apoptotic tendency and the fact that there is a high degree of MMP heterogeneity within
the liver (31), it is possible the high MMP hepatocyte subpopulations will undergo
significantly less apoptosis than their low MMP counterparts when stressed by the
cryopreservation process. This subpopulation selection to limit apoptosis, if effective,
would also translate directly into increased post-thaw viabilities.
1.4 Study Aims
The primary aim of this study is to determine whether hepatocyte MMP is a
contributing factor to the cell's cryopreservation and post-thaw culture survival. To
investigate this, primary rat hepatocytes will be stained with a MMP-sensitive dye and
sorted into high and low MMP subpopulations. Both of these populations, along with an
unsorted control, will be frozen with the NCP 30MG, subsequently thawed, and cultured
to allow for the full extent of apoptosis to occur. Attachment yield and overall viabilities
will be measured and compared.
This study also seeks to characterize the kinetics of the rat GLUT2 transporter.
The GLUT2 transporter is an integral part of the 30MG/hepatocyte cryopreservation
procedure, yet its affinity and maximum uptake velocity have yet to be determined.
Initial 30MG uptake at various extracellular concentrations will be measured by high
performance liquid chromatography (HPLC) and analyzed to calculate Km and Vmax.
It is hoped that a better understanding of the contribution of MMP to
cryopreservation viability, as well as of the cellular loading kinetics of the NCP 30MG,
will translate into improved hepatocyte survival and an optimized freezing protocol.
13
These improvements may themselves pave the way to a feasible and usable approach to
liver cell banking.
14
2. Materials and Methods
2.1 Isolation of Primary Hepatocytes
Primary hepatocytes were isolated from female Lewis rats (Charles River Labs,
Wilmington, MA) according to a standard protocol described in detail elsewhere (32).
2.2 Kinetic Characterization of rGLUT2
To characterize the affinity (Km) and maximum uptake velocity (Vmax)of the
rGLUT2 transporter, hepatocytes were exposed to a glucose analog, 3-O-methyl-Dglucopyranose (30MG; Sigma-Aldrich, St. Louis, MO). 30MG uptake solution was
created by adding 0 - 400 mM 30MG to C+H hepatocyte culture medium (Dulbecco's
Modified Eagle's Medium with 5% fetal bovine serum, 1% penicillin-streptomycin, 0.1%
iletin II insulin, 10 mg/L hydrocortisone sodium succinate, glucagon, and .01 mg/L EGF)
at 37 °C. Aliquots of x10 6 hepatocytes were centrifuged at 350 rpm for 5 min;
supernatant was aspirated. Cells were resuspended in 2.5 ml 30MG uptake solution and
allowed to incubate for 15 s; following this incubation, uptake was immediately
quenched with 25 ml stop solution of 100 gpMphloretin (Sigma-Aldrich, St. Louis, MO)
in free phosphate buffered saline (PBS without calcium and magnesium). Samples were
then centrifuged at 350 rpm for 5 min, washed 3 times with 10 ml stop solution each and
prepared for analysis by high-performance liquid chromatography (HPLC; Agilent
Technologies: 1100 Series, Palo Alto, CA). Total sample protein was measured by
Coomassie Plus® Bradford Assay Kit (Pierce, Rockford, IL). Total protein was
converted to total number of hepatocytes by a previously performed calibration. Total
15
hepatocyte values in turn were converted to cellular volume per sample by estimating an
osmotically active isotonic volume of 2500 pm3 /cell (33). Cellular 30MG concentrations
were calculated by dividing total moles 30MG as determined by HPLC by total volume
of cells in sample as determined above.
2.3 Fibroblast Stain and Sort
J2 3T3 fibroblasts (American Type Culture Collection, Manassas, VA) were
thawed from storage and resuspended in Dulbecco's Modified Eagle Medium (DMEM,
Invitrogen, Carlsbad, CA) high glucose with 10% bovine calf serum (BCS; HyClone,
Anaheim, CA) and 1% penicillin/streptomycin
(Invitrogen, Carlsbad, CA) at 5x10 5
cells/ml. 33.3 pg/ml Rh123 was added to the cell suspension and cells were incubated at
37 C for 10 rin.
Fibroblasts were then centrifuged at 850 rpm for 5 min, stain solution
was aspirated, and pellets were resuspended in PBS at lx10 6 cells/ml. Rh123
fluorescence of individual cells was measured and cells were sorted by a fluorescenceactivated cell sorter (FACS; EPICS ALTRA, Beckman Coulter, Fullerton, CA). High
and low MMP subpopulations were defined as those cells in the upper and lower 20% of
the fluorescence histogram, respectively. Sorted cells were centrifuged at 850 rpm for 5
min, supernatant was aspirated, and pellets were resuspended in DMEM (10% BCS, 1%
penicillin/streptomycin).
Cell suspensions were then seeded into 75 cm2 cell culture
flasks (Corning, Inc., Corning, NY), incubated at 37 C, and passaged as needed. Rh123
fluorescence was measured by aliquoting 2 x 105 Rh123-stained cells into 96-well plate
cells and reading on a fluorescence microplate reader (fmax, Molecular Devices,
Sunnyvale, CA).
16
2.4 Fibroblast Cryopreservation and Viability Assessment
Cells were trypsinized, centrifuged at 850 rpm for 5 min, and resuspended in
KRMD freezing solution (Appendix B) with 5% DMSO (Sigma-Aldrich, St. Louis, MO).
Cryopreservation equipment and procedure paralleled that described below for
hepatocytes. Following liquid nitrogen plunging, fibroblasts were thawed and
resuspended in PBS. 2 x 105 cells (in 100 [l
1 PBS) were aliquoted into 96-well plate
wells. Control wells were aliquoted with 2 x 105 cells (in 100 tl PBS) killed by methanol
exposure. 100 Vlof 5 [tMethidium homodimer (Molecular Probes, Eugene, OR) in PBS
was added to each well, and plates were incubated at room temperature in the dark for 30
min. Fluorescence was read on the fmax reader, and viability was calculated as 1(experimental well fluorescence)/(control
well fluorescence), where control well
fluorescence was representative of complete cell death.
2.5 30MG Hepatocyte Cytotoxicity
To verify that loading of hepatocytes with 30MG is not detrimental to the cells, a
cytotoxicity assay was performed. Aliquots of lxl06 cells were centrifuged at 350 rpm
for 5 min. Supernatant was aspirated and cells were resuspended in 2.5 ml warm C+H
medium with 30MG concentrations of OmMto 250mM. Cell suspensions were
incubated at 37°C for 40 min, then centrifuged again at 350 rpm for 5 min. Medium was
aspirated and the cell pellets were resuspended in 1 ml warm C+H each. Viabilities were
determined by hematocytometer count and trypan blue exclusion.
17
2.6 Rh123 Staining for MMP
Aliquots of cells were centrifuged at 350 rpm for 5 min. Supernatant was
aspirated and cells were resuspended at 0.5x 106 cells/ml in warm stain solution (33.3
tg/ml Rh123 in C+H medium), then incubated at 370 C for 10 min. Following
incubation, cells were centrifuged at 350 rpm for 5 min and stain solution was aspirated;
cells were then resuspended to 4x 106/ml in cold Krebs-Ringer buffered saline (KRB; 154
mM NaCl, 27 mM KC1, 5.5 mM D-glucose, 5.4 mM NaHCO 3 in 20 mM HEPES, pH 7.4)
and stored on ice, in the dark until sorted.
2.7 Creation of High and Low MMP Subpopulations
High and low MMP subpopulations of stained hepatocytes were created in a
similar procedure to that used by Follstad et al (18). Rh123 fluorescence of individual
cells was measured and cells were sorted by a FACS (MoFlo, Cytomation, Fort Collins,
CO). High and low MMP subpopulations were defined as those cells in the upper and
lower 20% of the fluorescence histogram, respectively (Figure 2.1). See Figure 2.2 for
depiction of the MMP stain and subpopulation sort procedures. Control cells were also
run through the FACS, sorting for a population of live, healthy cells; this ensured that
control cells experienced similar shear to sorted hepatocytes and that only cells from the
live population were selected for further manipulation.
18
Figure 2.1: High and low MMP subpopulation sort histogram. Histogram of hepatocyte Rh 123
fluorescence - x-axis is relative fluorescence, y-axis is number of cells. Gates R2 and R3 represent the
top and bottom 20% of the curve, respectively. R2 was sorted as the "high MMP" subpopulation; R3
was sorted as the "low MMP" subpopulation.
19
A
Resuspend in
stainsolution
_
Incubate 10
min, 370 C
Y
Figure 2.2: MMP stain and sort protocol. Ovals represent beginning and end products, rectangles are
active steps and diamonds are passive procedures.
arrows.
Processes were carried out sequentially as indicated by
20
2.8 Hepatocyte Cryopreservation Protocol
MMP subpopulations and control cells sorted as above were centrifuged at 350
rpm for 5 min. Supernatant was aspirated and all conditions were resuspended in warm
incubation solution (200 mM 30MG in C+H), then incubated at 37 C for 40 min. Cell
suspensions were then centrifuged and aspirated as previously and resuspended in I ml
each Hypothermosol (Biolife Solutions Inc., Binghamton, NY) with 200 mM 30MG.
Suspensions were transferred to 1.0 ml cryogenic vials (Nalge Nunc International,
Rochester, NY) and frozen in a controlled-rate freezer (KRYO 10, Planer, Middlesex,
UK) according to the procedure utilized for hepatocyte cryopreservation in Sugimachi et
al (29). The cryogenic vials were then plunged in liquid nitrogen for 15 min and
subsequently stored in liquid nitrogen for one to nine days (Figure 2.3).
2.9 Viability Assessment of Hepatocytes
Frozen cells were thawed in a 37 C water bath and added to 9 ml warm C+H
medium. Suspensions were incubated at 37 C for 10 min to remove intracellular
30MG, then centrifuged and aspirated as previously described. All conditions were
resuspended in 1 ml warm C+H each, seeded onto 6-well, single-layer collagen gel plates
and cultured at 37 C, 10% CO 2 for 24 hrs. Medium from each well was transferred to a
new well for quantifications of floating cells by microscopy. Floating cells were counted
in five representative views per well with a fluorescent microscope (Carl Zeiss Axiovert
200 M, Thornwood, NY) at 32X magnification; total cell counts per well were
21
Centrifuge and
aspirate
supernatant
--
Transfer to
_,,~-' Cool to -6°C"->
cryogenic vials,
"- at -1 °C/min ,insert in freezer I
Is ,.
Resuspend in
Hypothermosol,
200 mM 30MG
I
-
,
I
C
Store frozen
samples in
liquid N2
Figure 2.3: Cryopreservation protocol. Ovals represent starting material, rectangles are active steps and
diamonds are passive procedures. Shapes highlighted in grey indicate those procedures during which
samples are in the controlled-rate
freezer.
22
140
y= 0.0001x+ 1.4845
120
100
E
80
00
60
40
20
0
O.E+00
2.E+05
4.E+05
6.E+05
8.E+05
1.E+06
Microscope Cell Count
Figure 2.4: Microscope Cell Concentration
Calibration.
Cell counts on the x-axis represent total cells
counted in five 30X microscope field views. This calibration curve was used to calculation unadhered, live
and dead cell concentrations in cell viability assays.
23
determined based on a cell count calibration (Figure 2.4, Appendix G) and viability of
these cells was determined by hematocytometer count and trypan blue exclusion. The
remaining adherent cells were washed once with PBS (without calcium and magnesium)
then stained with ml live/dead viability assay solution (Live/Dead Viability Assay,
Molecular Probes, Eugene, OR; 0.5 pM calcein AM/ 30 pM ethidium homodimer in
PBS). Stained cultures were incubated at RT, in foil for 30 min. Live, dead and total
number of cells were counted in five representative views per well with a fluorescent
microscope (Carl Zeiss Axiovert 200 M, Thornwood, NY) at 32X magnification; total
cell counts per well were determined based on a cell count calibration (Figure 2.4). In
addition, membrane blebbing was quantified: for each microscope field, blebs of any size
attached to live cells (as determined by live/dead assay above) were counted. Extent of
blebbing was then expressed as blebs per live cell. Unfrozen cells, seeded immediately
after sorting and cultured for 24 hrs, were also assessed as described above to determine
baseline viabilities of high and low MMP subpopulations.
24
3. Results
3.1 Kinetics of rGLUT2
The activity of a cellular membrane transporter can be treated as that of an
enzyme, with the substrate being the molecule to be transported from the extracellular
environment and the product being the molecule once inside the cell, or vice versa.
Using this approach, kinetics of facilitative transporters such as rat GLUT2 can be
analyzed using the Michaelis-Menton equation:
Km + [S]'
where v is the transporter uptake velocity, Vmaxis the maximal uptake velocity, [S] is the
substrate concentration, and Kmis the Michaelis-Menton constant, which indicates the
substrate concentration at which half the maximal velocity is achieved. As several
different calibration curves were needed to obtain the necessary values for calculation
(cellular 30MG content, protein content and cellular contents of each sample), there was
a great potential for value variability among experiments. Therefore, to obtain the most
accurate results, parameters were extracted from each experiment individually, taking
advantage of internal consistencies. Results were analyzed using Lineweaver-Burk and
Eadie-Hofstee linearized plots (Figures 3.1 and 3.2) to determine the parameters Km and
Vmax
as presented in Table 3.1. It should be noted that the concentration component of
Vmax,mM, refers to cellular volume only. Given an osmotically active volume of 2500
gm
3/cell
as discussed in Materials and Methods, this value can also be reported as 0.194
25
A
32.5 2
1.5 1-
i
',
y = 19.796x+ 0.8139
,~
-0.06
-0.04
-0.02
0
0.02
0.04
0.06
0.08
0.1
0.04
0.06
0.08
0.1
-
1/[30MG] (mM ')
B
K-
0
E
'
r
-0.06
-0.04
-0.02
0
0.02
1/[30MG] (m M-')
C
3
2.5
2
1.5 -
f...2.-0.06
-0.04
-0.02
0
,_--o~
0.02
y = 15.128x+ 0.7196
0.04
0.06
0.08
0.1
1/[30MG] (m M-')
Figure 3.1: Lineweaver-Burk plots of rGLUT2 kinetic data. Plots A, B, and C represent three individual
experiments.
In Lineweaver-Burk linearization, the x-intercept is -I/Km and the y-intercept is /VN,,.
26
A
0.04
y = -0.0197 + 0.0334
in 0.030.02 -
000
. 0.01
2
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
v (m M/s)
B
0.04 -
y = -0.01 09x + 0.0186
O. 0.03
0
0.02
0.01
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
v (m M/s)
C
y = -0.01 48X + 0.0304
0.04
en 0.03
O 0.02
B 0.01
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
v (m M/s)
Figure 3.2: Eadie-Hofstee plots of rGLUT2 kinetic data. Plots A, B, and C represent three individual
experiments.
In Eadie-Hofstee linearization, the slope is -1/Km and the x-intercept is Vmax.
27
Table 3.1: Kinetic parameters of rGLUT2. Values presented are + standard deviation.
Linearization
Lineweaver-Burk
Eadie-Hofstee
Km (mM)
27.6 ± 8.9
Vmax (mM/s)
1.38 ± 0.15
70.0 + 20.6
1.82 + 0.20
28
pmol/min/cell. V,,axobtained was higher than that reported by Johnson et al (34) for
GLUT2 in pancreatic 13islet cells (5.5 and 32 mM/min), but lower than that obtained by
Levitsky et al (92 nmol/min/mg protein) (35). All three sets of values, however, were
measured using different experimental protocols. Kmvalues agree in order of magnitude
with the value of 23 mM obtained by Levitsky et al (35), although experimental
conditions varied substantially.
3.2 Fibroblast MMP Subpopulation Post-thaw Viability
Prior to experiments with the cell type of interest, hepatocytes, a set of proof-ofprincipal experiments was performed with fibroblasts. After a high and low MMP
subpopulation sort and three culture passages, there was still a pronounced difference in
Rh123 fluorescence between the high and low MMP subpopulations (130 and 97 RFU,
respectively), as shown in Figure 3.3. Following cryopreservation and thawing,
fibroblast subpopulations demonstrated markedly different viabilities (p<0.0001, Figure
3.4).
3.3 30MG Cytotoxicity
To verify that exposure to 30MG itself was not detrimental to hepatocytes, a
cytotoxicity test was performed. At all extracellular 30MG concentrations up to and
including that used in the cryopreservation protocol (200 mM), cells did not exhibit
viabilities different from the control (Table 3.2). This was consistent with the viability
value obtained by Sugimachi et al (29) for hepatocyte incubation in 200 mM 30MG. For
29
160
140
T
120
T
100
U.
cc'
80
60 40 20 -
0High MMP
Low MMP
Figure 3.3: Relative Rh123 fluorescence of high and low MMP fibroblast populations. Error bars
represent standard deviations. Fluorescence was measured following three passages.
30
IIn 'No/0
V
/O
90C%'/
I
*
I
80% 70'60'/o0
'Q
50% -
>
40'/o
30%o
20%
0%/O
I~~~~~~~~~~~~~~~~~
I
High MMP
Low MMP
Figure 3.4: Post-thaw fibroblast subpopulation viability. Horizontal bars represent data averages, error
bars denote standard deviations. * p < 0.0001.
31
Table 3.2: 30MG hepatocyte cytotoxicity. * p = 0.00086.
Concentration
0
50
100
150
200
250
(mM)
Viability (%)
90
77
83
82
85
70 *
Std Dev (%)
3
12
11
9
6
5
32
concentrations above 200 mM, however, hepatocytes demonstrated significant toxicity
(70% at 250 mM vs. 90% at 0 mM, p = 0.00086).
3.4 Hepatocyte MMP Subpopulation Post-Thaw Morphology and Viability
Significant morphological differences between high and low MMP conditions
were observed following 24 h culture. Substantially more membrane blebbing was
observed in the low MMP population, indicative of cell damage (Figure 3.5). This
difference in extent of membrane damage was quantified as blebs per live cell (Figure
3.6); high and low MMP subpopulations exhibited significantly different blebbing, with
0.63 and 1.69 average blebs per cell, respectively (p = 0.05). Possible apoptotic bodies
were observed to a greater degree in the low MMP population as well. Moreover, a
greater number of low MMP "adherent" cells appeared rounded, as opposed to the
majority of high MMP adherent cells, which had spread out and often formed cell-cell
contacts.
In addition, high and low MMP hepatocyte subpopulations were assessed for
viability following cryopreservation and thawing. This viability measure consisted of
four parts: attachment yield, viability of attached cells, viability of unattached cells, and
overall culture viability (percent viability of combined floating and attached cells) after
24 h of culture. Regardless of condition or experiment, all floating cells were dead
(Figure 3.7). Adherence yield varied significantly between high and low subpopulations
(87% v. 68%, p = 0.022), although viabilities within these attached cells did not (Figures
3.8 and 3.9). Most striking, however, were differences in overall viabilities among high
MMP, low M:MP, and control conditions (59%, 35%, and 46% respectively).
33
,
. ' ~: ~ - / ~ ,, ~ ~
~: ~
,
~
~ ~ ~ ~ : "x ~ ~' ~:.~"· ¢,
J
.i
-·:.-· 1,·
·.;·-.·':::I
···-:(::.·
·'
..:
--" : ·i
r:::
·r:; -··`r: -
i·:.·
·.
-·*;
,···
i··-··:·:
ai;
:::
· ·-^·
::
i
i
r:~~~~
:~~~~~~~~~~~~~~~~~~~~~~~~~·?~
···
~
~~~r
i.·-fi:
iQ
VV.··
·
' ~ . .
· ·-
~:
·
_~::
~
~
I;i
:
.,,;
- .
t~~~aX''
'''~~~'
;', $'>:'u
:. S Ci"'.'
,t:t..-
t
.' .
- · ,.,·
.
'
h>;. '
7''
*'
--
;
,
;
~.
' 3~,, -;'.-. ,!' ":-- ¥'¢': ~',a.C./:
'.:; ,-.
.
.
.
-: i
, '
-- '-'-",
.
~~,~-.·
.
.
-
t-~~~:
,
''
'
,-r-7)
s~~~~~~~~~~~
'',L.aA
..A
d.
'' -.
.SSX ,.
Figure 3.5: 24 h culture, cryopreserved hepatocytes. A: high MMP subpopulation, B: low MMP
subpopulation. Several examples of membrane blebs are indicated by arrows.
34
4.5
I
4
-
3.5
0
3-
2.5Q
2-
.
1.5-
m
1-
T
0.5 OHigh MMP
I
I
Low MMP
Figure 3.6: Extent of blebbing in high and low MMP subpopulations. Blebbing was assessed as
average number of blebs per live cell; only membrane blebs continuous with the live cell's membrane were
counted. *p = 0.0517.
35
,
..
11-
11
I UU-/cl
90%
-
8
80%-
c
o
*
70% -
C
60%-
zo
50% -
0
40% 30%
m
20%
10%
0% -
---------- ~----~--I-.-High MMP
---I
Low MMP
0
Control
Figure 3.7: Viability of unadhered hepatocyte population. N=4 for all conditions.
36
All three comparisons (high v. low, high v. control, and low v. control) demonstrated
statistical significance, as shown in Figure 3.10. Average viability measurements are
summarized in Table 3.3.
Based on the above results, the MMP contribution to cryopreservation
survivability can be broken down into two segments: effects on cell adherence and effects
on viability of adherent cells. More formally, this can be expressed as:
,u=axv,
where p is overall freeze-thaw viability dependent on MMP, a is adherence yield of
plated cells, and u is viability of adhered cells. This allows the contribution of MMP to
each of the terms to be analyzed. The results in Table 3.3 demonstrate the same
proportional effects, regardless of subpopulation - adherence of cells to the collagen gel
after 24 hours (a) accounts for 56.5 % of survival; viability of the adherent cells (u)
contributes the other 43.5 %.
To verify that the observed differences were a result of differences in
cryopreservation survivability, as opposed to "livelihood" of the populations, high and
low subpopulations were also cultured immediately following the sort. 24 h culture
viability differences between the high and low subpopulations were not significant (72%
and 62% respectively, p = 0.30).
37
100%
90%
8
80%
O
O
O
70%
O
C 60%*s 50%
<
40%I
30%
I
20% 10% 0% 0%
I-II
Control
Low MMP
High MMP
Figure 3.8: Adherence yield of 24-hr, post-freeze/thaw cultured hepatocytes. Diamonds are individual
data points, horizontal bars are means and error bars show standard deviations. * p = 0.022.
-1~o
YU7/o
m, 80%
O
8 70%
0O
O
i 60%
r
=
0~~~
l
50%
IS
B 40%= 30%
20%
!
'
10%
0%
Control
Low MMP
High MMP
Figure 3.9: Viability of adherent hepatocytes from 24-hr, post-freeze/thaw culture. Diamonds are
individual data points, horizontal bars are means and error bars show standard deviations.
**
90%
I
I
80%
l
70%
I
I
60%
$
50%
40%
0
30%
20%
I
10%
0%
_
_
_
_
High MMP
*
_l
I
~
_
_
___
Low MMP
l_~
Control
Figure 3.10: Overall 24-hr, post-freeze/thaw viability of hepatocytes. Diamonds are individual data
points, horizontal bars are means and error bars show standard deviations. * p = 0.00026, ** p = 0.024,
**:*
p = 0.021.
38
Table 3.3. Summary of post-thaw, 24-h cultured hepatocyte viabilities.
Condition
High MMP
Low MMP
Control
Viabilities
(%)
Adherence (%)
87
68
77
Floating
Adherent
Overall
0
0
0
68
53
61
59
35
46
39
4. Discussion
4.1 MMP Effects on Cryopreservation Viability
Significant post-thaw viability differences between high and low MMP
hepatocyte subpopulations suggest that mitochondrial membrane potential does indeed
play a role in how a cell responds to the cryopreservation process. The fact that, for all
three measures (attachment yield, attached cell viability and overall viability), values
obtained for control populations fell between those for the low and high MMP
populations strengthens this interpretation. As the control population represented cells
with a full spectrum of mitochondrial membrane potentials, it would be expected to
demonstrate a.response somewhere between that of the two extremes. Moreover,
observed morphologies of high and low MMP cryopreserved populations suggest an
advantage of the high MMP subpopulation for freeze-thaw survival. Substantially less
membrane blebbing and presence of fewer apoptotic bodies may be indicative of
improved long-term culture survival and functionality of the high MMP subpopulation.
Experiments e xpanded in culture time and tests of hepatocyte function such as urea
production would be needed to verify this interpretation.
4.1.1. Adherence ability v. viability
Interestingly, overall cryopreservation viability values were distributed
approximately evenly between ability of hepatocytes to form connections with the
collagen gel matrix and the viability of these successfully adhered cells. It is possible
that these categories represent separate stages in apoptotic death over the 24 hour culture
period, where loss of cell viability comes first, followed by detachment from the matrix.
40
Alternatively, these two subclasses of dead cells may represent two entirely different
mechanisms of death: one in which cells are unable to attach and therefore are unable to
survive, and those that are able to initially attach but die off by some other mechanism.
The distinction between adhered, dead cells and floating ones may be elucidated by timecourse microscopy to monitor cell cultures after thawing and plating. Following the
behavior of individual cryopreserved cells may demonstrate the likelihood of one theory
over the other and is recommended for future studies.
4.1.2.Mitochondrialenergeticsand adherenceyield
Extensive literature searches have yielded little in the way of associations
between a cell's energy status and the ability of this cell to attach to a matrix. The closest
research has come to investigating this possible association is a study on the effect of
oxygen levels on hepatocyte culture. It has been demonstrated that increasing the oxygen
supply to cultured hepatocytes increases their efficiency of attachment (36); however, the
biological mechanisms behind this effect were not investigated.
It is therefore
remarkable and unexpected that cells from a high MMP subpopulation adhere with
substantially greater efficiency following cryopreservation than those from a low MMP
subpopulation. It is possible that this result is based on differences in membrane integrity
(as visualized by extent of blebbing). Conversely, it may be that mitochondrial
energetics and a cell's metabolic state potentiate a cell's ability to form contacts with a
substrate. In light of the positive dependence of cell adherence on oxygen supply and the
intimate relationship between oxygen and mitochondrial processes, this interpretation
may hold some weight.
41
The fact that high MMP hepatocytes are not only more likely to survive the
freezing process but also more likely to attach post-thaw has far-reaching implications for
tissue engineering. Manipulations to increase cell MMP for banking purposes would
have the added benefit of aiding in their eventual use: cellular adherence is essential for
the proper function of a BAL device and for the eventual development of whole
bioengineered livers.
4.1.3. Deviationsfrom expected viability
One observation on overall freeze-thaw viability values should be noted: although
high MMP population viability was significantly higher than that of the control, the high
MMP condition did not yield survival markedly higher than that obtained from unsorted
cells in previous studies (>50% each) (29). In fact, unsorted, cryopreserved cells in this
case demonstrated poorer viability than previously. The major contribution to this
discrepancy is probably time from isolation to freeze. In previous studies, this time was
minimal; in the study reported here, this time interval was often as long as six hours due
to the stain procedure, transport to sort facilities, actual sort time, and transport back.
Such a long time "in transit" could be detrimental for several reasons. First, hepatocytes
are adherent cells both in vitro and in vivo - long periods of time in suspension, without
forming cell-surface connections, may lead some cells to undergo anoikis. Second, much
of the time spent "in transit" for these hepatocytes was spent settled as the bottom of 3 ml
of buffer. This liquid layer, as well as the layering of the cells themselves, present
barriers to oxygen diffusion that are not present in the culture condition. As hepatocytes
have an oxygen uptake rate of approximately 8.4 nmol 0 2 /min/mg dry cell weight, this
42
diffusion barrier could easily have led to hypoxia and death of a significant number of
cells. Other factors, such as fluid shear imposed by the FACS or jostling of cells during
transport may also have contributed to lower than expected viabilities.
The ideal way to avoid the problems discussed above would be to eliminate the
cell sorting step completely. This could be achieved by increasing average cell
population MMP through more active manipulation. Two recent methods purported to
increase MMP appear promising.
In one study, average MMP of rat hepatocytes was
increased prior to isolation by treating the rats with a low dose of endotoxin in vivo (37).
48 hours after treatment, hepatocytes were isolated and demonstrated significantly higher
MMP as measured by transmembrane electrical potential and by Rh123 fluorescence. If
these results could be repeated, such an endotoxin pretreatment could be administered to
rats whose isolated hepatocytes were destined for cryopreservation. Unfortunately, this
method to increase MMP could not be utilized in human hepatocyte cryopreservation.
Besides the extreme morbidity associated with endotoxin sickness, liver donors are most
often victims of accidents or other unexpected death, who most likely could be not
identified 48 hours before the accident to begin the necessary pretreatment!
Another way with which to avoid cell sorting to obtain populations with high
MMP might be to increase the MMP of hepatocytes in vitro. A second study looking at
the effects of interleukin- 1 (IL-1 ) and interleukin-6 (IL-6) on hepatocyte mitochondria
found that exposure to IL-6 led to emphasis of MMP generating processes (38). This
effect could be utilized to increase MMP of cultured hepatocytes prior to
cryopreservation; however, a protocol must also be developed to freeze hepatocytes in
43
culture, rather than in suspension. This alternate cryopreservation platform has not yet
been addressed by our lab.
4.2 Viability Assessment Methodology
It has been observed previously (12) that most freeze-thaw viability assessments
base their values on live:total ratios of only those cells which come out of the
cryopreservation process intact rather than on live:total ratios based on the overall
number of cells placed in the tube for freezing. Such a methodology ignores possibly
substantial cell losses from lysis over the course of the freezing process. This study was
no exception to these criticisms. However, the viability assessment utilized here is
justified in two respects. Cellular lysis occurring during cryopreservation is usually the
result of membrane destruction by ice crystals by osmotic injury - these are both physical
causes of death, and thus should not have an influence on the biochemical triggers of
apoptosis. As this study focused on differences in apoptotic death between high and low
MMP populations, death from physical effects could be neglected. Additionally, due to
FACS operation, cell sort counts reported by the FACS counter were often inconsistent
with those obtained by hematocytometer counts. Without a consistent and reliable
method to quantify the number of cells being frozen per tube, the former method of
viability determination was more feasible.
4.3 rGLUT2 Kinetics
It is interesting to note that the Michaelis-Menton parameters, Km and Vmax,
obtained for rGLUT2 in this study, when used to calculate the 30MG uptake velocity at
44
200 mM and plotted as a time course, do not yield the empirical curve obtained by
Sugimachi et al (29). Values published by others similarly do not yield the appropriate
curve. Perhaps the strongest reason for this is that GLUT2 is not a unidirectional
transporter - although Vmaxfound in this study may very well be an accurate
representation of Vmax, forward, Vmax, reversehas not been assessed. As some of the 30MG
entering the hepatocyte invariably exits it via the same transporter, it is likely that
equilibration of intracellular 30MG concentrations takes significantly longer than
implied by the Michaelis-Menton parameters obtained here. It is possible that transport
problems associated with cell settling in incubation tubes may be a factor as well.
Another important point to make is that, while the majority of glucose transporters
expressed by adult hepatocytes are GLUT2, a minor presence of GLUT1 has also been
observed. This may have led to slightly skewed GLUT2 kinetics values.
Although Km and Vmaxare not beneficial in creating time-course curves for
30MG uptake, it is still important that they are characterized in the setting of the
cryopreservation protocol. Based on comparison to values obtained in different
experimental conditions, it can be deduced that Km is relatively unaffected by variations
in cellular milieu (medium v. buffer), incubation temperature, or measurement in
suspension versus culture. In contrast, it appears that Vmaxis very sensitive to these
parameters, although more research is needed to determine whether this is a true effect or
simply an artifact of sensitivities in linearization and parameter extrapolation.
45
4.4 Recommendations for Future Study
The results obtained in this study are extremely promising in terms of potential to
increase viability of frozen, banked hepatocytes. However, several further studies are
recommended to bolster these findings and extend their impact. A global caspase assay
of freeze-thawed hepatocytes of high and low MMP subpopulations is advised. Caspases
are the major effectors of the apoptotic pathway, thus variability in their levels between
conditions would verify that viability differences were indeed the result of changes in rate
of apoptosis a.lsopposed to necrosis. It is also advised that high and low MMP freezethawed cells are cultured long-term to measure any potential benefits of a high MMP on
the functionality of these cells. Finally, it is highly recommended that alternative
methods to increase intrinsic MMP, as discussed above, should be investigated to offer a
more practical approach to the field of cell banking.
46
5. Acknowledgements
Many thanks to my advisors, Dr. Mehmet Toner and Dr. Gregory
Stephanopoulos, for both inspiration for my thesis and support during my research.
Thank you as well to the Harvard-MIT Division of Health Sciences and Technology for
funding my research. I would also like to express my appreciation to Ken Roach, for his
constant guidance and for allowing me to use him as a "sounding board" for my ideas and
difficulties. None of my research would have been possible without Avrum Leeder, Noor
Ahmad and Chris Chen, who were responsible for all of the hepatocyte isolations, nor
without Vasilis Toxavidis, who ran the FACS for my cell sorts. And of course, the
consistent emotional support of my family and friends was always welcome and
appreciated.
47
6.0 References
1. Blouin, A., Bolender, R.P., Weibel, E.R. Distribution of organelles and membranes
between hepatocytes and nonhepatocytes in the rat liver parenchyma. A
stereological study. J Cell Biol 72 (1977): 441 - 455.
2. Engl, T. et al. Phosphorylation of hepatocyte growth factor receptor and epidermal
growth factor receptor of human hepatocytes can be maintained in a (3D)
collagen sandwich culture system. Toxicol in Vitro 18 (2004): 527 - 532.
3. Strain, A. J., Neuberger, J. M. A bioartificial liver - state of the art. Science 295
(2002): 1005 - 1009.
4. Mitry, R.R., Hughes, R.D., Dhawan, A. Progress in human hepatocytes: isolation,
culture & cryopreservation. Cell Dev Biol 13 (2002): 463 - 467.
5. Baker, T. et al. Temporal gene expression analysis of monolayer cultured rat
hepatocytes. Chem Res Toxicol 14 (2001): 1218 - 1231.
6. Rialland, L..et al. Viability and drug metabolism capacity of alginate-entrapped
hepatocytes after cryopreservation. Cell Biol Toxicol 16 (2000): 105 - 116.
7. Odani, M. et al. Screening of genes that respond to cryopreservation stress using
yeast DNA microarray. Cryobiology 47 (2003): 155 - 164.
8. Lillford, P.J., Holt, C.B. In vitro use of biological cryoprotectants. Phil Trans R Soc
Lond 357 (2002): 945 - 951.
9. Karlsson, J.O.M., Toner, M. Long-term storage of tissues by cryopreservation: critical
issues. Biomaterials 17 (1996): 243 - 256.
10. Storey, K.B. Biochemistry of natural freeze tolerance in animals: molecular
adaptations and applications to cryopreservation. Biochem Cell Biol 68 (1990):
687 - 698.
11. Pegg, D.E. The history and principles of cryopreservation. Sem Reprod Med 20
(2002): 5- 13.
12. Lloyd, T.:).R. et al. Cryopreservation of hepatocytes: a review of current methods for
Banking. Cell Tiss Bank 4 (2003): 3 - 15.
13. Son, J.H. et al. Optimization of cryoprotectants for cryopreservation of rat hepatocyte.
Biotechnol Lett 26 (2004): 829 - 833.
14. Smith, L.J. et al. Dynamics of glucose solutions.
Http://www.ncnr.nist.gov/AnuualReport/FY2003_html/RH8/
(May 24, 2005).
15. Beattie, G.M. et al. Trehalose: a cryoprotectant that enhances recovery and preserves
function of human pancreatic islets after long-term storage. Diabetes 46 (1997):
519 - 523.
16. Shirakashi, R. et al. Intracellular delivery of trehalose into mammalian cells by
electropermeabilization. J Membrane Bio 189 (2002): 45 - 54.
17. Eroglu, A. et al. Intracellular trehalose improves the survival of cryopreserved
mammalian cells. Nature Biotech 18 (2000): 163 - 167.
18. Follstad, B.D., Wang, D.I.C., Stephanopoulos, G. Mitochondrial membrane potential
differentiates cells resistant to apoptosis in hybridoma cultures. Eur J Biochem
267 (2000): 6534 - 6540.
19. Coppola, S., Ghibelli, L. GSH extrusion and the mitochondrial pathway of apoptotic
signaling. Biochem Soc Trans 28 (2000): 56 - 61.
48
20. Baust, J.M., Buskirk, R.V., Baust, J.G. Modulation of the cryopreservation cap:
elevated survival with reduced dimethyl sulfoxide concentration. Cryobiology 45
(2002): 97 - 108.
21. Baust, J.M., VanBuskirk, R., Baust, J.G. Cell viability improves following inhibition
of cryopreservation-induced apoptosis. In Vitro Cell Dev Biol - Animal 36 (2000):
262 - 270.
22. Men, H., et al. Degeneration of cryopreserved bovine oocytes via apoptosis during
subsequent culture. Cryobiology 47 (2003): 73 - 81.
23. Matsushita, T. et al. Apoptotic cell death and function of cryopreserved porcine
hepatocytes in a bioartificial liver. Cell Transplant 12 (2003): 109 - 121.
24. Martin, G. et al. Cryopreservation induces an apoptosis-like mechanism in bull
sperm. Bio Reprod 71 (2004): 28 - 37.
25. Esposti, M.D. Mitochondria in apoptosis: past, present and future. Biochem Soc Trans
32 (2004): 493 - 495.
26. Juan, G. et al. A fast kinetic method for assessing mitochondrial membrane potential
in isolated hepatocytes with rhodamine 123 and flow cytometry. Cytometry 15
(1994): 335 - 342.
27. Gottlieb, E. et al. Mitochondrial membrane potential regulates matrix configuration
and cytochrome c release during apoptosis. Cell Death Diff 10 (2003): 709 - 717.
28. Brown, G.K. Glucose transporters: structure, function and consequences of
deficiency. J Inherit Metab Dis 23 (2000): 237 - 246.
29. Sugimachi, K et al. Non-metabolizable glucose compounds impart cryotolerance to
primary rat hepatocytes. Submitted: Tissue Eng.
30. Yagi, T. et al. Caspase inhibition reduces apoptotic death of cryopreserved porcine
hepatocytes. Hepatology 33 (2001): 1432 - 1440.
31. Cossarizza, A., Ceccarelli, D., Masini, A. Functional heterogeneity of an isolated
mitochondrial population revealed by cytofluorometric analysis at the single
organelle level. Exp Cell Res 222 (1996): 84 - 94.
32. Berthiaume, F., Tompkins, R.G., Yarmush, M.L. Isolation and long-term
maintenance of adult rat hepatocytes in culture. Methods in Molecular Medicine
18: 447 - 456.
33. Toner, M., Tompkins, R.G., Cravalho, E.G., Yarmush, M.L. Transport phenomena
during freezing of isolated hepatocytes. AIChE J 38 (1992): 1512 - 1522.
34. Johnson, J.H. et al. The high Km glucose transporter of islets of Langerhans is
functionally similar to the low affinity transporter of liver and has an identical
primary sequence. J Biol Chem 265 (1990): 6548 - 6551.
35. Levitsky, L.L. et al. GLUT-1 and GLUT-2 mRNA, protein, and glucose transporter
activity in cultured fetal and adult hepatocytes. Am JPhysiol 267 (1994): E88 94.
36. Rotem, A. et al. Oxygen is a factor determining in vitro tissue assembly: effects on
attachment and spreading of hepatocytes. Biotech Bioeng 43 (1994): 654 - 660.
37. Guidot, D.M. Endotoxin pretreatment in vivo increases the mitochondrial respiratory
capacity in rat hepatocytes. Arch Biochem Biophys 354 (1998): 9 - 17.
38. Berthiaume, F. et al. Control analysis of mitochondrial metabolism in intact
hepatocytes: effect of interleukin-1l3and interleukin-6. Metab Eng 5 (2003): 108 123.
49
APPENDIX A: Raw Data.
Table Al: Hepatocyte 30MG toxicity. Data utilized in Table 3.2.
30MG
Concentration
(mM)
Live
Dead
Total
Viability (%)
0
77
49
50
6
6
4
83
55
54
93
89
93
59
9
68
87
64
28
7
18
71
46
90
61
54
27
15
7
69
34
78
79
59
28
38
3
4
11
62
32
49
95
88
78
51
21
72
71
57
18
75
76
58
7
65
89
34
18
4
7
38
25
89
72
43
43
27
5
7
8
48
50
35
90
86
77
45
6
51
88
40
31
34
23
14
11
63
45
45
63
69
76
49
20'
69
71
50
100
150
200
250
._.....
1
50
o
r
I
__
>1·
>1
0
(Z
o
, -
c~
o
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h-
o0 - -
N
CD C) C)
c -
-
(
(0O
01'
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0
O
CO) O
CO LOU CJ
(D IC CO C) C)
0
L) (0
1 CO
-
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LO
co
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rI
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C
(OO
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C)
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C 00
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a00 0 r- Cf) CO rN D It LO t LO
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t
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C
C
a
c-C
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r
IC
(0')
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c6u ac
O or
C)
Ct
Q
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.M
c
a
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.,
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.
:
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cm
t C c) a C( 0
1
'1
11
UI
C
r
s- C
1
C,
C
C
(1
H
r:
C
C
-
0-
5
2.
,
C
..C
W.
..
0
-j
C
C
c
CL
0
C
0
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I
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ct
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IC-
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tv
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N-C
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(1)
a
C\
Table A4: Bleb and live cell counts for extent-of-blebbing measurement. Live cells were assessed by
calcein AM fluorescence, blebs were any membrane protrusions still attached to the cell body. All counts
are summations of four 30X field counts. Summarized data presented in section 3.4.
Blebs
Live Cells
Blebs/Cell
Low MMP
24
10
15
1
30
9
15
4
5
18
2.67
0.67
3.75
0.20
1.67
High MMP
14
38
0.37
24
40
0.60
5
25
27
9
66
29
0.56
0.38
0.93
52
APPENDIX B: Novel Cryopreservation Solution Development and Testing.
In an effort to avoid use of the commercial product Hypothermosol ® and to
develop a solution with even greater protective characteristics, a novel cryopreservation
solution was developed with Ken Roach. This solution, KRMD, consists of the following
components:
Table B1: KRMD freezing solution recipe.
Compound
Concentration (mM)
Choline hydroxide
100
Lactobionic acid
HEPES
100
25
KH 2PO
10
4
KHCO 3
KOH
CaCl12
MgC12
NaCl
5
30
0.05
5
10
Trehalose
20
Glucose
5
Dextran
60 g/L
Adenosine
Glutathione
2
3
Proline
5
Glycine
Taurine
Glutamine
5
5
5
Cryoprotection capability of KRMD was compared to that of Hypothermosol using the
200 mM 30MG freeze/thaw protocol discussed in Materials and Methods; viability was
assessed by trypan blue exclusion immediately following thawing of hepatocytes. Results
were as follows:
53
Table B2: Post-freeze/thaw
viabilities with Hypothermosol or KRMD as freezing solution.
Hypothernmosol+ 30MG (200 mM)
KRMD + 30MG (200 mM)
Live
Dead
Total
Viability (%)
Live
Dead
Total
Viability (%)
98
127
78
126
120
98
224
247
176
43.8
51.4
44.3
119
101
67
98
56
63
217
157
130
54.8
64.3
51.5
46.5
TOTAL
TOTAL
56.9
Despite differences in average viabilities, Student's t-test assuming unequal
variances showed viability differences were not statistically significant (p = 0.107). This
demonstrated that KRMD did not perform better as a cryoprotective solution, but also
showed that the two solutions were comparable. KRMD can effectively be substituted
for Hypothermosol in the cryopreservation protocol. Although the results of hepatocyte
cryopreservation survival with KRMD were promising, Hypothermosol was utilized in
the experiments presented in the body of this report due to time constraints and
availability of materials.
54
APPENDIX C: Effects of Succinate on Post-cryopreservation Viability.
Compounds that increase hepatocyte MMP chemically (and therefore eliminate
the need for sorting) were investigated for their impact on post-thaw viability. In this
experiment, 7 mM or 0.7 mM succinate (a metabolic intermediate known to increase
MMP) was added to the incubation solution, wash solution or both during the
cryopreservation protocol (200 mM 30MG was used in all incubation solution
conditions). Freezing procedure was identical to that in Material and Methods. Viability
of thawed hepatocytes was then determined by trypan blue exclusion and
hematocytoimleter count. Results were as follows:
Table C1. Post-thaw viability with addition of succinate. Experimental conditions were as follows: A 7 mM succinate in incubation and wash solutions; B - 7 mM succinate in incubation solution; C - 7 mM
succinate in wash solution; D - 0.7 mM succinate in incubation solution; E - 0.7 mM succinate in wash
solution.
Condition
Live
Dead
Total
Viability
Control
218
225
265
210
150
172
428
375
437
51%
60%
61%
210
116
326
64%
198
175
373
191
184
178
180
369
364
53%
58%
52%
51%
225
160
385
58%
B
144
122
93
121
237
243
C
137
193
176
150
313
343
A
54%
61%
50%
55%
44%
56%
50%
D
E
196
151
215
127
411
278
48%
54%
51%
199
201
400
50%
175
187
362
48%
49%
Except for condition E, all viabilities were not statistically different from that of the
control. Instead of having a higher viability as hypothesized, condition E yielded a
significantly lower viability than control (p = 0.02). Based on these results, succinate
was no longer pursued as a compound to improve freeze-thaw viability by increasing
MMP.
55
APPENDIX D: Attempted protocols for extraction of hepatocytes from culture.
As an alternative to fluorescent microscopy and manual counting, it was hoped
that flow cytometry could be used to quantify hepatocyte viability following thaw and
24-hr culture. This necessitated extracting the cells from their collagen wells and
creating suspensions. The following protocols were attempted to achieve this; none
yielded sufficient number of undamaged cells to be viable options.
Extraction Protocol 1:
1. Aspirate medium from wells.
2. Add 2 ml mM cold EDTA solution (in PBS) to each well; incubate 10 min at
37 °C.
3. Transfer cell suspension gently to tubes.
Extraction Protocol 2:
1. Aspirate medium from cells.
2. Wash once with 1 ml PBS.
3. Add 1 ml mM cold EDTA solution (in PBS) plus 1 ml 0.05% trypsin;
incubate 15 min at 37 °C.
4. Aspirate EDTA + trypsin, wash with 1 ml PBS and aspirate.
5. Add 1 ml collagenase solution (1 mg/ml PBS); use sterile pipette tip to
separate gel from edges of well.
6. Incubate 30 min at 37 °C.
7. Transfer cell suspension to tube, add 9 ml PBS and centrifuge at 350 rpm
for 5 min.
8. Aspirate supernatant, resuspend in 1 ml KRB.
Extraction Protocol 3:
1. Place culture plate on ice.
2. Aspirate wells, add 1 ml 1 mM cold EDTA solution (in PBS) to each well;
incubate 10 min on ice.
3. Dislodge cells gently with I ml pipette.
4. Transfer cell suspension to tube.
Extraction Protocol 4 (modified from Martin et al (24)):
1. Aspirate wells, wash with 1 ml cold PBS.
2. Aspirate wells, add 1 ml 0.05% trypsin; incubate 5 min at 37 °C.
3. Add 1 ml C+H medium; collect cells by pipeting or scraping.
56
APPENDIX E: Effects of heat-shock on viability of cryopreserved fibroblasts.
Heat-shock was investigated as another manipulation to increase the post-
cryopreservation viability of cells, in this case fibroblasts. Inspiration for this
investigation came from experiments by Odani et al (8).
Aliquots of 4 x 106 fibroblasts were either stored on ice or exposed to a 50 °C
water bath for 20 sec. Both conditions were centrifuged at 2000 rpm for 5 min,
supernatant was aspirated, and cell pellets were resuspended in KRMD (see Appendix B)
+ 5% volume DMSO. Suspensions were incubated for 10 min at room temperature to
acclimate to the DMSO solution. Suspensions were then transferred to 1 ml cryovials
and frozen in a controlled-rate freezer according to protocol in Materials and Methods.
Cell suspensions were subsequently thawed and viability of fibroblasts was determined
by trypan blue exclusion and hematocytometer count. Results of this preliminary
experiment were as follows:
Table El. Post-thaw viability of heat-shocked fibroblasts.
Condition
Live
Dead
Total
Viability
Control
86
16
102
84%
79
22
101
78%
81%
Heat-shocked
86
121
12
12
98
133
88%
91%
89%
Although statistical analysis did not reveal a true difference in viability between these
two conditions (p = 0.14), it is recommended that this experiment be repeated to increase
the data pool and either confirm or disconfirm this finding.
57
APPENDIX F: Functionality of 30MG-incubated hepatocytes
Along, with cytotoxicity tests, 30MG was assessed for its affect on hepatocyte
function in culture. Hepatocytes were incubated for 40 min at 37 °C in C+H and 0 - 500
mM 30MG. Cells were then centrifuged at 350 rpm for 5 min, supernatant was
aspirated, and cells were resuspended in warm C+H. Hepatocytes were seeded at lx106
cells/ml onto 6-well collagen gel plates and cultured at 37 °C overnight. The following
day, medium was removed for urea assessment and fresh C+H was added to the culture.
Hepatocyte cultures were returned to the 37 °C incubator, and the process was repeated.
Medium samples taken were assessed for urea nitrogen content as a measure of
hepatocyte function using a urea nitrogen test kit (Stanbio Laboratory, Boerne, TX).
Results were as follows:
Table Fl: Urea production of hepatocytes incubated transiently in 30MG. All urea concentration
values are in [tg/ml.
30MG Concentration
0 mM
100 mM
200 mM
Day 1
104.37
140.95
115.28
127.07
95.12
125.42
115.10
140.52
99.45
104.32
110.27
118.20
Ave
122.66
121.18
110.27
127.81
101.88
114.23
Std Dev
25.87
8.33
21.43
17.97
3.44
5.61
Day 2
60.60
84.93
75.65
82.12
70.58
77.70
92.43
104.55
68.15
61.60
70.65
73.67
Ave
Std Dev
72.77
17.21
78.88
4.57
74.14
5.03
98.49
8.57
64.88
4.63
72.16
2.13
Day 3.5
77.82
107.02
80.70
96.67
69.55
90.77
86.12
96.37
61.00
74.53
88.40
82.50
Ave
Std Dev
92.42
20.65
88.68
11.29
80.16
15.00
91.24
7.25
67.77
9.57
85.45
4.17
300 rnmM 400 rnmM 500 mM
58
160.00 140.00 E 120.00 -
T
I I I
I
L
v7 I
IT
E0 mM
TT--II
m 100.00 C
0
ir
E
i mM
l100
T
T,_
80.00-
Il
I
-,
I
-
* 400 mM
60.00-
a
E500 mM
0
o
0
~~"
o 200 mM
o 300 mM
40.00 20.00 n--- on ."
l
Day 1
I
Day 2
Day 3.5
_
Figure Fl: Time-course urea production of 30MG-incubated hepatocytes.
The above results demonstrate that incubated in 30MG did not have a significant
detrimental effect on hepatocyte culture function at any concentration. The assessment
verifies that the experiments performed in the body of this work can be extended to
longer-term culture without adverse effects on cell functionality from the cryoprotectant
used.
59
APPENDIX G: Calibration protocol for cell concentration assessment by
fluorescent microscopy.
To create a calibration curve of the number of cells counted in a 30X fluorescent
microscope field to the total number of cells in the culture well, known concentrations of
hepatocytes were added to 6-well plates. Concentrations ranged from lx 104 to 1x106
cells per well. Under 30X magnification, cells were counted in a total of five fields (one
from each quadrant and one from the center) and summed. A calibration curve was
developed from these results as shown in Figure 2.4 in the body of this work.
60
APPENDIX H: Endoxtin pretreatment effects on hepatocyte MMP.
Low-dose endotoxin pretreatment of rats to increase intrinsic hepatocyte MMP
was investigated. As a first step, we attempted to repeat those results obtained by Guidot
(37), following the protocol published by him. Following hepatocyte isolation, cells were
stained with Rh 123 to assess MMP as outlined in Materials and Methods. 200 [tl aliquots
of stained and washed hepatocytes in PBS were placed in a 96-well plate; fluorescence
was read with a fluorescent microplate reader (Molecular Devices, Sunnyvale, CA).
These results were compared to the fluorescence of stained hepatocytes from a nonpretreated rat.
500 -
450 -
T
400 T1
:350 '300 U. 250 -
200 150
100 50 -
0-
I
L
Control
Endotoxin Pretreatment
Figure H1: RhI.23 fluorescence of hepatocytes following endotoxin pretreatment.
Rh 123 fluorescence
was utilized as a measure of MMP and is reported here in relative fluorescence units.
Preliminary results were unsuccessful at repeating the significant hepatocyte MMP
increase achieved by Guidot. Further experiments are recommended to establish this
protocol for use in cryopreservation.
61