Note: Descriptions are shown in the official language in which they were submitted.
B~PIKTNG OF PAII~1CRE.'~TIC EDTDOCRaNE
CELLS F'OR TR~iNBPI~AA3ft°A'fION
10
FIELD OF THE INVENTION
This invention relates to a novel process for banking
pancreatic endocrine cells for transplantation.
BACKGROUND OF THE INVENTION
Successful pancreatic islet transplant in an insulin
dependent diabetic person can potentially achieve physiological
control of the metabolic abnormalities and prevention of long-
term complication experienced in insulin-dependent diabetic
subjects (Sutherland D.E.R., Chinn P.L., Morrow C.W.: Transplan-
tation of pancreas islets, in Gupta S (ed): Immunology of
Clinical Experimental Diabetes. New York, NY, Plenum, 1984, pp
147-246; Tze W.J., Sima A.A.F., Tai J.: Effect of endocrine
pancreas allotransplantation on diabetic nerve dysfunction,
, Metabolism 34: 721-725, 1985). Currently, one major obstacle in
the clinical application of islet transplantation is the
difficulty in collecting enough donor pancreatic islets for
transplantation.
Cryopreservation procedure is a potentially useful way
of banking islet tissues until adequate quantities have been
collected for transplantation. Cryopreserved whole islets and
pancreatic fragments from adult and fetal sources have been shown
to be functional in vitro and in vivo (Taylor M.J., Duffy T.J.,
Hunt C.J., et al.: Transplantation in vitro perfusion of rat
Islets of Langerhans after slow cooling warming in the presence
of either glycerol or dimethyl sulfoxide. Cryobiology 20:185-
204, 1983). However, nearly all studies with cryopreserved whole
islets reported some reduction of islet cell function following
the freezing and thawing process (Toledo-Pereyra L.H., Gordon
D.A., Mackenzie G.H.: Cryopreservation of islets of Langerhans.
Cryobiology 18:2483-2488, 1981). Earlier, the inventors have
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IJ
'~~~r~~~~
demonstrated that dispersed single pancreatic endocrine cells
(PEC) grafts can normalize hyperglycemia in diabetic rats and
monkeys, (Tze W.J., Tai J.: Successful intracerebral allotrans-
plantation of purified pancreatic endocrine cells in diabet rat.
Diabetes 32:1185°1187, 1983; Tze W.J., Tai J.: Intrathecal
allotransplantation of pancreatic endocrine cells in diabetic
rats. Transplantation 41:531-534, 1986; Tze w.J., Tai J.:
Xenotransplantation of rat pancreatic endocrine cells in
spontaneous and streptozotocin-induced diabetic monkeys.
Transplant Proc 2112736°2738, 1989) suggesting cryopreservation
of PEC and islet fragments as an alternative approach to whole
islet preservation. In addition, cryopreservation procedure has
been suggested to reduce immunogenicity of islet tissue, thus
making this approach even more attractive as a means of islet
cell preservation.
SUMMARY OF THE INVENTION
The invention is directed to a method of freezing
pancreatic endocrine cells for storage and thawing the pancreatic
calls for use in transplantation for treatment of diabetes, which
comprises either rapidly or slowly freezing the pancreatic
endocrine cells in the presence of a suitable antifreeze, storing
the frozen cells in a suitable sub-freezing environment, and then
thawing the cells prior to transplantation.
The antifreeze can be dimethyl sulphoxide or may be
to to 20% dimethyl sulphoxide. The antifreeze can be selected
from the group consisting of dimethyl sulphoxide, glycerol,
3o propylene glycol and butane diol.
The cells can be frozen at an ultra rapid rate. They
can be frozen in the presence of liquid nitrogen or using a
vitrification process. The sub-freezing environment can be a
freezer or liquid nitrogen.
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~.ri~~~i~~~
The freezing can be conducted at a temperature drop
rate of between 0.1°C to about 5°C per minute. The cells cart be
frozen at a rats of about 0 .1 ° C to about 5 ° C per minute
from
ambient temperature to about -7°C, and at a freezing rate of
about 0,1°C to about 3°C per minute from about -7°C to
about
-70°C, or they can be frozen at a rate of about 0.1°C to about
0.5°C per minute from ambient temperature to about -40°C, and at
a freezing rate of about 0. 5 ° C to about 5 ° C per minute from
about
-40°C to about -70°C.
The invention is also directed to a method of freezing
pancreatic endocrine cells for storage and thawing the pancreatic
endocrine cells for use in transplantation for treatment of
diabetes, which comprises: cooling the pancreatic endocrine
cells at a rate of between about 0.1°C to about 5°C per minute
to about -70°C in the presence of between about 10% to about 20%
dimethyl sulphoxide, storing 'the frozen cells in a sub-freezing
environment, and then thawing the cells prior to transplantation.
The sub-freezing environment can be liquid nitrogen.
The cells can be thawed in a water bath of about 37°C tempera-
ture. The pancreatic endocrine cells can be single cells, cell
aggregations, or islet cells.
The invention is also directed to a method of freezing
pancreatic endocrine cells for storage and thawing the pancreatic
endocrine cells for use in transplantation which comprises:
cooling the pancreatic endocrine cells at a rate of about -0.3°C
per minute to about -70°C in the presence of 10% dimethyl
sulphoxide, storing the frozen cells in liquid nitrogen, and then
thawing the cells in an about 37°C water bath prior to transplan-
tation.
The invention is also directed to a method of cryopre-
serving pancreatic endocrine cells which comprises cooling the
cells at a rate of about -5°C per minute to about 4°C, holding
the cells for 3 minutes at about 4°C, subsequently cooling the
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cells at a rate of about -0.3°C per minute to about -7°C,
holding
the cooled cells at about that temperature for about 3 minutes,
then cooling the cells at a rate of about -0.3°C per minute to
about -40°C, and then cooling the cells at a .rate of about -5°C
per minute from about -40°C to about -70°C in about 10% dimethyl
sulphoxide, and finally, transferring the frozen cells to liquid
nitrogen for storage.
The frozen calls can be thawed in a water bath
maintained at about 37°C, and then transplanted into xenogeneic
or allogeneic diabetic recipients to normalize the blood glucose
level in the recipient. The cells can be insulinoma cells. The
cells after being thawed in the water bath can be cultured
overnight at about 26°C, prior to being transplanted into the
diabetic recipient.
DRAWINGS
In drawings which illustrate specific embodiments of
the invention but which should not be construed as restricting
the spirit or scope of the invention in any way:
Figure 1 illustrates the validity of rat insulinoma
cells frozen with five alternative protacols;
Figure 2 illustrates the viability of rat PEC frozen
with five alternative protocols;
Figure 3 illustrates the intrathetical transplantation
of cryopreserved Wi PEC into allogeneic diabetic ACI rats; and
Figuxe 4 illustrates the functional period of cryopre-
served Wi PEC in allogeneic diabetic ACI recipients.
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DETAILED DESCRIFTTON OF SPECIFIC
EMBODIMENTS OF THE INVENTION
To determine optimal freezing and thawing conditions
for pancreatic endocrine cells (PEC) and insulinoma cells taken
from rats, five different cryopreservation protocols were
developed and compared. PEC and insulinoma cells were cooled at
rates of between -0 . 3 ° C/min and -5 ° C/min to -7 0 °
C in the presence
of 100, 15%, or 20% dimethylsulfoxide (DMSO) with a programmable
temperature controller and then transferred to liquid nitrogen
for storage. Frozen cells were thawed by either a rapid (in 37°C
water bath) or a slow (in air) thawing procedure. One hour after
the thawing process, cellular visibility was determined by trypan
blue dye exclusion. The visibility results for PEC and insul-
inoma cells were similar and showed that a slow cooling rate at -
0.3°C/min in combination with a rapid thawing in 37°C water bath
gave the best results, with up to 80o cellular visibility.
Cryoprotectant DMSO used at loo concentration was the most
effective among the three concentrations tested. Later,
.transplantation studies were performed with PEC cryopreserved
with the best protocol , which is -5 ° C/min to 4 ° C, held for
3
minutes, -0.3°C/min to -7°C, held for 3 minutes, -
0.3°C/min to -
40°C, and -5°C/min from -40°C to -70°C in loo DMSO
with a
programmable temperature controller then transferred to liquid
nitrogen for storage. Intraportal transplantation of cryo-
preserved Wistar (Wi) strain PEC into allogeneic ACI diabetic
recipients was discovered to normalize their blood glucose (BG)
for 8.3 ~ 1.9 days (mean ~ SD), which was not significantly
different from that of a noncryopreserved preparation of 6.6 ~
1.5 days. Cytotoxic antibody titers in the ACT recipients of
cryopreserved and noncryopreserved Wi PEC graft were found not
to be significantly different. Intrathecal transplantation of
frozen-thawed Wi PEC into allogeneic ACI diabetic recipients
resulted in prolonged amelioration of diabetic state in l0 of 10
animals, which is similar to that seen with freshly prepared PEC.
This study confirmed that cryopreservation is an 'effective
procedure for the banking of PEC before a transplantation. The
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~~~~3~~~
in vivo survival period of PEC in allogeneic recipients was not
significantly altered by the cryopreservation process used in
this study.
In our investigations, a total of five cryopreservation
protocols were assessed for the cryopreservation of PEC and small
PEC aggregates and the PEC cryopreserved with 'the best protocol
was then further assessed in vivo in allogeneic recipients.
Yokogawa et al. (Yokogawa Y., Takaki R., Ouo J.: Cryopreserva-
Lion of pancreatic islet cells. J Lab Clin Med 103:768-775, 1984)
reported that consistently over 80% cellular viability was
achieved with hamster PEC using a slow freeze and quick thaw
protocol similar to ours. It has been suggested that cryopreser-
vation can be used to decrease islet immunogenicity before
transplantation (Bank H.L.: Cryobiology of isolated islets of
Langerhans circa 1982. Cryobiology 20:119-128, 1983). Coulombe
et al. (Coulombe M.G., Warnock G.L., Rajotte R.V.: Prolongtion
of isleet xenograft survival by cryopreservation. Diabetes
36:1086-1088, 1987) recently reported slight prolongation in
~ islet xenograft (rat-mouse) after freeze thawing process.
Prolonged graft survival was achieved when additional immuno-
suppression was administered to the recipients. Taylor et al.
(Taylor M.J., Bank H.L., Benton M.J.: Selective destruction of
leucocytes by free freezing as a potential means of modulating
tissue immunogeneity. Membrane integrity of lymphocytes and
macrophages. Cryobiology 24:91-102, 1987) observed optimal
survival of both lymphocytes and macrophages after freezing and
thawing with cooling rates in the range of 0.3 to 5°C/min. Only
after cooling at rates greater than 75°C/min was survival of
these cells reduced to a negligible level. Further cryopreser-
vation studies by vitrification would provide useful information
on this issue.
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,i .:..~ l.~ t~ c.~ ~
Materials and Methods
Animals and Islet Call Preparation
Rats of outbred Wistar (Wi) strain weighing 300 to 500
g were selected as donors of islets, and ACI (AgB 4/4) strain as
streptozotocin (SZ) (65 mg/kg intravenously [IV]-induced diabetic
recipients. Pancreatic tissue was digested with collagenase
(type IV, Cooper Biomed, Freehold, NJ). The islets were hand
pinked under a dissection microscope and further purified by the
single layer Hy-paque-Ficoll (H-F) separation technique. Each
batch of 1,000 islets was digested with a combination of 0.04%
EDTA in Ca2*-Mg2* free Hanks' balanced salt solution (HBSS) and
0.020 purified trypsin. Dissociated cells were washed three
times with cold HBSS and resuspended in 3 mL of H-F solution at
a specific gravity of 1.080, and 2 mL of HBSS was layered on top
of the cell suspension. Centrifugation was performed at room
temperature for 8 minutes at 800 g, following which PEC at the
interface were collected. A PEC preparation consisting mainly
~of single cells and some aggregates of fewer than 10 cells,
viability over 900, was resuspended in culture medium (medium
199 containing l0% fetal calf serum, 100 U/mL penicillin, 100
,ug/mL streptomycin) and cultured overnight at 26°C in 5% COZ-
95% air. The PEC were purified with H-F again and calls at the
interface were collected far freezing and transplantation
studies.
Insulinoma Cells
The rat insulinoma line (RIN) was a obtained from Dr.
Oie (National Cancer Ins'ti'tute-New York Medical Oncology Branch,
Bethseda, Md). Single cells were released from culture flasks
with trypsin-EDTA solution. Cell preparation was regularly
greater than 95% viable when assessed by trypan blue dye
exclusion test.
~~~~~~8
Freeze-Thawing. Procedure
Freezing of overnight cultured PEC and insulinoma cells
was performed in a programmable temperature controller (Planer
Dryo ZO Series; Planer Products, Sudbury-On-Thames, England).
Fifty-milliliter sterile centrifuge tubes (Falcon 2070F, Falcon
Plastics, Mississauga, Ontario) containing PEC preparation or
insulinoma cells in culture medium (<3 mL) were placed on crushed
ice. Equal volumes of precooled culture medium with 200, 30%,
and 40% dimethylsulfoxide (DMSO) (Sigma Chemicals, St. Louis, MO)
were added to each tube dropwise over a 15 minute period to
achieve the final DMSO concentrations of 10%, 15% and 20%.
Aliquots of 1 mL each of the cell preparation were then distrib-
uted to cryovials (Cooke Laboratory Products, Alexandria, VA).
The cells were then frozen with one of the following cooling
schedules to -70°C and the vials were then plunged into liquid
nitrogen (LN2) and stored for between 1 week to 6 months:
Schedule 1: -8°C/min to 0°C, hold 20 minutes, -
5°C/min
~ from 0°C to -70°C;
Schedule 2: -5°C/min to 10°C, hold 3 minutes, -
1°C/min
from 10°C to -70°C;
Schedule 3: -1°C/min to -70°C;
Schedule 4: -5°C/min to 10°C, -0.5°C/min from
10°C to
-40°C, and -5°C/min from -40°C to -70°C;
Schedule 5: -5°C/min to 4°C, hold 3 minutes, -
0.3°C/min
from 4 ° C to -7 ° C, hold 3 minutes, -0. 3 ° C/min
from -7 ° C to -4 0 ° C and -5 ° C/min from -4 0 °
to
-70°C.
Preliminary experiments showed that the storage
duration in LN2 did not affect the cellular viability. The
frozen cells were either thawed quickly with constant agitation
_ g -
~'>_~ ~5~~~
in a 37°C water bath or 'thawed slowly in air. Culture medium at
room temperature was added dropwise over a 20 minute period to
the thawed cell suspensions to dilute the DMSO concentration to
less than 1% vol/vol. The cells were then transferred to Petri
dishes and cultured at 37 ° C in a CO~ incubator for 1 hour. Then,
200 cells from each preparation were counted and the percent of
viable cells was determined by trypan blue dye exclusion.
Preliminary experiments indicated that most of the cell death due
to freezing and thawing occurred within the first hour of culture
after thawing.
Transplantation Study
Rat PEC collected daily were frozen according to
schedule 5 (above) and stored in LN2. They were accumulated
until sufficient for transplantation study. Cells were quick-
thawed in a 37°C water bath as described earlier. Frozen-thawed
PEC in culture medium were dispensed into 100 x 20-mm plastic
Petri dishes, and cultured overnight in a humidified 5% COz
incubator. They were centrifuged on H-F gradient for 8 minutes
at 800 g. Viable cells collected at the interface were used for
transplantation study. Two to three x lOb viable PEC were
suspended in a 50 JCL volume in a U-100 insulin syringe (Sherwood
Medical, St. Louis, MO) and injected intrathecally into the
cisterna magna of diabetic ACI recipients. For intraportal
transplantation, cryopreserved or noncryopreserved PEC were
suspended in 200 ESL volume in a monojet U-100 insulin syringe and
injected over a 1-minute period intraportally into diabetic
recipients. Random blood glucose (BG), body weight (BW), and 24-
hour urine volume, were assessed before, and daily for 2 weeks
following transplantation, and at regular intervals thereafter.
Antibody Stud
Sara collected were stored at -20 ° C and heated at 56 ° C
for 30 minutes before antibody determination. Cytotoxic antibody
levels in the ACT recipients of intraportal transplant of fresh
_ g _
.,
or cryopreserved PEC were determined using pooled donor strain
splenocytes as target cells, and rabbit serum (Low-Tox M rabbit
complement cat. no. CL3111, Cedarlane Laboratory, Ontario,
Canada) as complement source. Antibody titer is defined as the
reciprocal of serum dilution that kills 500 of the target cells.
Results
Figure 1 shows the viability of rat insulinoma cells
frozen with the alternative five schedules and thawed at two
rates with 10%, 15%, or 20% DMSO as cryoprotectant. It was found
that a slow freezing rate was more effective than a quick
freezing rate (Schedule 1) in preserving cellular viability. The
best was Schedule 5, with the slowest freezing rate at -0.3°C/
min, which achieved greater than ~30o cellular viability after
quick thawing. It was found that thawing rate also greatly
affected the cellular viability of cryopreserved insulinoma
cells. A11 samples thawed at room temperature in air (slow
thawing) had consistently lower viability than similar prepara-
~ tions thawed in parallel in a 37°C water bath. The concentration
of the cryoprotectant DMSO from 10% to 20% achieved similar
protection of insulinoma cells with 10% and 15% achieving a
slightly higher viability than 20% DMSO.
Figure 2 shows the viability of rat PEC frozen with
five schedules and thawed at two different rates with 10% and 15%
DMSO as cryoprotectant. The results were similar to that of
insulinoma cells, with the highest cellular viability achieved
with slow freezing at -0.3°C/min (Schedule 5) and quick thawing
in a 37°C water bath. The cellular viability of more than 700
achieved for rat PEC was lower than that for insulinoma cells.
For later transplantation studies, rat PEC were cryopreserved
with schedule 5, which was shown to achieve the highest cellular
viability for both insulinoma and rat PEC cells.
Figure 3 indicates that intrathecal transplantation of
cryopreserved Wi PEC into allogeneic diabetic ACI rats resulted
- 10 -
in normalization of random BG in seven of ZO diabetic recipients
within '7 days, while the remainder of the recipient rats achieved
normalization more slowly. All these animals had normal weight
gain and 24-hour urine volume, and became aglycosuric following
transplantation. The metabolic patterns of these animals
following transplantation were similar to those observed
previously in diabetic ACI recipients of noncryopreserved Wi PEC.
Figure 4 shows that the functional period of cryopre-
served Wi PEC (8.3 ~ 1.9 days [mean -!~ SD]N = 7) in allogeneic
diabetic ACI recipients though slightly longer, is not signifi-
cantly different from that of noncryopreserved preparation (6.6
~ 1.5 days, N = 7).
Table 1 demonstrates that the intraportal recipients
of both cryopreserved and noncryopreserved PEC preparations had
cytotoxic antibody formation against donor alloantigens. Table
2 tabulates cytotoxic antibody titers in the ACI Recipients of
Allogeneic Wi PEC Graft. The mean peak antibody titers on days
7 and 10 detected in tine recipients of cryopreserved PEC were not
significantly lower than in the recipients of noncryopreserved
PEC.
TABLE 1
Functional Period of Fresh and Cryopreserved Wi PEC
Transplanted in Alloqeneic Diabetic ACI Recipients
PEC Graft Functional Days Mean ~ SD
Noncryopreserved 4, 6, 6, 6, 8, 8, 8 6.6 ~ 1.5°
Cryopreserved 6, 7, 8, 8, 8, 9, 12 8.3 ~ 1.9
°0.5 > P > .1, nonsignificant
- 11 -
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The major hindrances to islet transplantation in humans
are lack of adequate donor tissue and graft rejection process.
The former problem can partly be solved if isolated islets can
be stored until sufficient quantity is available for transplanta-
tion. Several approaches for storage of islets, including tissue
culture, low temperature storage, and cryopreservation, have been
'tried. For long-term storage, a cryopreservatian procedure would
be the most logical and practical approach. Since single and
small aggregates of PEC can be used for transplantation, the
inventors examined the feasibility of freezing single or small
clumps of PEC instead of whole islets as a means of storage. The
efficacy of several freezing protocols were compared in vitro in
this study using PEC and rat insulinoma cells. Cellular death
ranging from 20% to 90% was observed after the freeze-thaw
process. The best protocol was discovered to be slow freezing
at about -0.3°C in conjunction with a quick subsequent thawing
phase.
With PEC and small aggregates of fewer than 10 cells,
freer temperature and medium exchange can be achieved than is
possible for whole islets. Another factor is that mammalian
islets are not of uniform size. They can vary from 100 ~Cm to 200
~m in diameter. A freeze thawing protocol that is ideal for one
particular islet size may not be so for others in the same
preparation. Therefore, it is a simpler task to formulate a
freeze thawing protocol specially for PEC or clumps of relatively
uniform size.
The concentration of dimethylsulfoxide DMSO used in
this study ranged from 10% to 200. Since the viability counts
of freeze-thawed cells indicate 'that 10% DMSO was adequate for
the protection of PEC during freezing and DMSO is known to have
soma toxic effects on cells, a loo DMSO concentration would be
preferable.
- 13 -
Our investigations tested the in vivo function of
cryopreserved PEC by transplantation into diabetic recipients.
Rat PEC frozen with Schedule 5 and guick thawed in a 37° water
bath followed by overnight culture at 26°C were transplanted
intrathecally into diabetic recipients. ~fhe rates of BG decline
in seven of 10 diabetic recipients were similar to that seen in
the recipients of intrathecal fresh PEC grafts as observed in a
previous study. The remaining three had a slower decline in BG,
but once their BG were normalized, the metabolic parameters and
BW gain were comparable to the other seven animals arid similar
to those recipients of fresh PEC and normal controls. The slower
response in the last three rats would likely have resulted from
a transient subnormal functional state of cryopreserved PEC or
a fewer number of PEC being transplanted. The results suggest
to the inventors that cryopreserved PEC were effective in the
amelioration of the diabetic state in the recipients.
The prolonged survival of intrathecally implanted
cryopreserved allogeneic PEC that we have observed was likely due
to the protection by the immunoprivileged nature of subarachnoid
space, rather than to the decreased immunogenieity of PEC after
the freeze thawing process. To further assess the possibility
of reduction of graft immunogenieity by cryopreservation, PEC
were transplanted intraportally into allogeneic diabetic
recipients. The similar rejection period of the cryopreserved
and noncryopreserved PEC allograft following intraportal
transplantation and comparable levels of antidonor antibody
attained in both groups of recipients observed would suggest that
cryopreservation with the present pratocol has an insignificant
effeet on the immunogenieity of PEC graft.
The cryopreservation protocol with slow freezing and
rapid thawing to achieve high PEC viability used in this study
was similar to that used for the cryopreservation of lymphoid
cells. Therefore, it is not surprising that no significant
reduction of islet cell immunogeneity was achieved. The marginal
prolongation of the survival of cryopreserved PEC and the
- 14 -
marginal reduction in antidonor antibody titers in the allogeneic
recipient compared with noncryopreserved PEC observed could be
due to the additional washing steps with the PEC following the
freeze thawing process. Although the results of this study did
not detect any significant reduction of immunogenieity of PEC
following cryopreservation, it is still possible that reduction
of islet cell immunogenieity can be achieved by cryopreservation
process if optimal differential cooling and thawing rates can be
found for the PEC and the contaminating immunogenic cells.
The results of our investigations show that cryopreser-
vation procedure with slow freezing and quick thawing is an
effective procedure for the banking of single or small aggregates
of PEC. Cryopreserved PEC were functional in vivo in diabetic
rat recipients. However, the cryopreservation protocol used in
our investigations that resulted with high cellular viability
following the freeze thawing process did not achieve significant
reduction of PEC immunogenieity when assessed by in vivo
allotransplantation.
As will be apparent to those skilled in the art in the
light of the foregoing disclosure, many alterations and modifica-
tions are possible in the practice of this invention without
departing from the spirit or scope thereof. Accordingly, the
scope of the invention is to be construed in accordance with the
substance defined by the following claims.
- 15 -
