Upsala J Med Sci 92: 177-184, 1987 

Cryopreservation of Mouse Pancreatic Islets: Effects of 
Human Serum on Islet Survival 

Stellan Sandler, Bo Nilsson and Arne Anderson 
Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden 

ABSTRACT 

The aim of this study was to compare t h e  survival of cryopreserved mouse pancreatic 
islets frozen in the presence of either a simple salt solution (Hanks' balanced salt solution) 
or a complete tissue culture medium (RPMI 1640). Moreover, the addition of 1 0 %  human 
serum to the freezing solutions was evaluated. Collagenase isolated islets were kept 

in culture for three days, before being cooled at a rate of 5OC/min or 25OC/min t o  -7OoC, 
at which temperature the islets were transferred t o  liquid nitrogen. All freezing media 
were supplemented with 2 M dimethylsulphoxide as cryoprotectant. The islets were 
rapidly thawed a t  37OC and subsequently cultured for another three days. The recovery 
of islets was higher when the more rapid cooling rate was used and the addition of serum 
further improved t h e  recovery. Compared to non-frozen cultured islets there was a 

loss of cells in all groups of cryopreserved islets, as measured by their D N A  content, 
and this was accompanied by a lowered insulin content. All groups of frozen-thawed 
islets responded to a high glucose stimulus in vitro w i t h  a 5-9 fold increase in insdin 
secretion. There was no obvious advantage of using a complete tissue culture medium 
for islet cryopreservation, but the addition of serum had some beneficial effects. Data 
obtained from non-frozen control islets suggest that human serum slightly impairs the 

function of mouse pancreatic B-cells. 

INTRODUCTION 

We have recently demonstrated that cryopreserved mouse pancreatic islets cooled 
at a rapid rate (25OC/min) are able to synthesize (pro)insulin and release insulin, in 
response t o  glucose i n  vitro, at rates not different from those of non-frozen cultured 
control islets (1 5). Such cryopreserved islets significantly reduced alloxan-induced hyper- 
glycemia when transplanted into syngeneic mice. I t  was also found that the function 
of more rapidly cooled islets was better preserved than that of islets cooled at a rate 
of 5OC/min. In two previous studies we observed that a prerequisite for a successful 

177 



cryopreservation of isolated i s l e t s  is t h a t  t h e  i s l e t s  a r e  maintained f o r  a period in c u l t u r e  
b e f o r e  f r e e z i n g  (12,161. 

The aim of t h e  present s t u d y  was to i n v e s t i g a t e  w h e t h e r  t h e  viability of cryopreserved 
p a n c r e a t i c  i s l e t s  could be improved by using a c o m p l e t e  tissue c u l t u r e  medium (RPMI 

1640)(10) instead of a simpler salt solution (Hanks' balanced salt solution)(Z) during t h e  
f r e e z i n g  procedure. W e  have r e c e n t l y  found t h a t  supplementation of t h e  c u l t u r e  medium 
with human serum promotes t h e  development of islet-like s t r u c t u r e s  in v i t r o  (14) when 

using a r e c e n t l y  described method f o r  t h e  c u l t u r e  of human f e t a l  p a n c r e a s  (13). F u t u r e  
a t t e m p t s  t o  cryopreserve human i s l e t s  intended f o r  t r a n s p l a n t a t i o n  to human insulin- 
-dependent diabetics may involve t h e  addition of human serum to media. Therefore, 
we examined t h e  e f f e c t s  of adding human s e r u m  n o t  only t o  t h e  f r e e z i n g  medium but 

also t o  t h e  medium used during c u l t u r e  of t h e  i s l e t s  before and a f t e r  freezing. 

MATERIALS AND METHODS 

Islet isolation, c u l t u r e  and cryopreservation. P a n c r e a t i c  islets w e r e  isolated by a collage- 
nase digestion method (5) from male, adult NMRI mice (Anticimex AB, Sollentuna, Swe- 

den). Prior to f r e e z i n g  t h e  i s l e t s  were kept f o r  t h r e e  days free-floating in t i s s u e  c u l t u r e  
medium RPMI 1640 (Flow Laboratories Ltd., Irvine, Scotland) supplemented with 100 

U/ml benzylpenicillin ( A s t r a  Lakemedel, Sodertalje, Sweden), 0.1 mg/ml s t r e p t o m y c i n  

(Glaxo Laboratories, Greenford, England) and 1 0 %  pooled h e a t - i n a c t i v a t e d  human s e r u m  

(v/v) (Blood C e n t e r ,  University Hospital, Uppsala, Sweden). The c u l t u r e s  w e r e  maintained 

a t  37OC in a n  a t m o s p h e r e  of humidified a i r  + 5% C 0 2 .  T h e  c u l t u r e  medium was changed 

a f t e r  t w o  days. 

Cooling was performed using a programmable t e m p e r a t u r e  controller (Planer Mini- 

f r e e z e r ,  Model R 202/200R, Planer Products Ltd., Sunbury-on-ThAmes, England). Groups 

of about 75 i s l e t s  w e r e  t r a n s f e r r e d  at room t e m p e r a t u r e  t o  s t e r i l i z e d  glass ampoules 
containing 0.4 ml f r e e z i n g  medium. The medium was e i t h e r  Hanks' solution or medium 

RPMI 1640 with or without 1 0 %  (v/v) human serum. In all f r e e z i n g  media 2 M dimethyl- 
sulphoxide (Me2SO; Sigma Chemicals, St Louis, MO, USA) was added as c r y o p r o t e c t a n t .  
First, t h e  i s l e t s  w e r e  cool'ed at a r a t e  of 8OC/min from room t e m p e r a t u r e  t o  O°C where 
t h e  t e m p e r a t u r e  was maintained for 20 min t o  allow p e r m e a t i o n  of t h e  c r y o p r o t e c t a n t .  

Cooling was t h e n  resumed at e i t h e r  5OC/rnin or 25OC/min down t o  -7OoC, a f t e r  which 
t h e  ampoules w e r e  immersed in liquid nitrogen. 

A f t e r  s t o r a g e  f o r  60 min at -196OC t h e  i s l e t s  w e r e  rapidly t h a w e d  by s t i r r i n g  t h e  
glass ampoules in a 37'C w a t e r  bath. The resulting warming rate was about 240°C/min. 
When t h e  ice of t h e  f r e e z i n g  medium had just m e l t e d  t h e  islets, with t h e  f r e e z i n g  medium 
w e r e  poured i n t o  c u l t u r e  dishes containing 1 0  ml of t h e  s a m e  c u l t u r e  medium as t h a t  
used before t h e  freeze-thawing experiments. This l e a d  to a rapid dilution of t h e  Me2SO 
t o  less t h a n  0.08M. Finally, 5 m l  of c u l t u r e  medium w a s  removed f r o m  t h e  dishes and 

178 



t h e  i s l e t s  w e r e  c u l t u r e d  f o r  a n o t h e r  3 days before testing. In a previous study w e  have 
shown t h a t  t h i s  Me2SO concentration does not impair t h e  B-cell function of i s l e t s  main- 

t a i n e d  in c u l t u r e  in t h e  presence of Me2SO (11). 

The non-frozen c o n t r o l  islets w e r e  obtained from t h e  s a m e  islet isolations as t h e  

frozen-thawed islets, but maintained in t i s s u e  c u l t u r e  in medium RPMI 1640 + 10% 

human serum f o r  6 days, with medium exchanges every second day. 

Islet recovery. The islet recovery a f t e r  cryopreservation was c a l c u l a t e d  by counting 

t h e  number of islets in a stereomicroscope just before t r a n s f e r  to t h e  f r e e z i n g  ampoules 

and subsequently on day t h r e e  a f t e r  thawing. The recovery of t h e  non-frozen c o n t r o l  

i s l e t s  was c a l c u l a t e d  by counting t h e  i s l e t s  present in t h e  c u l t u r e  dishes on day 3 and 

day 6 of culture. 

Islet DNA c o n t e n t .  On day 3 a f t e r  thawing i.e. on e x p e r i m e n t a l  day 6, frozen-thawed 

and c o n t r o l  i s l e t s  in grou'ps of 30 w e r e  ultrasonically disrupted in 0.2 ml redistilled water. 

The DNA c o n t e n t  of t h e  resulting aqueous homogenates w a s  measured by fluorophoto- 

m e t r y  (4,7) 

Islet insulin c o n t e n t .  A 50 p1 f r a c t i o n  of t h e  islet homogenates was mixed with 125 p1 

of acid e t h a n o l  (0.18 M HC1 in 96% (v/v) ethanol), e x t r a c t e d  overnight at +4OC and 
t h e n  s t o r e d  at -2OOC. T h e  insulin c o n c e n t r a t i o n  in t h e  s a m p l e s  was d e t e r m i n e d  by radio- 
immunoassay (31, using mouse crystalline insulin as s t a n d a r d  (Novo, Copenhagen, Denmark) 

and 1251-labelled insulin as tracer (Novo). 

Islet insulin release. T r i p l i c a t e  groups of 10 i s l e t s  w e r e  incubated f o r  2 consecutive 
hours at 37OC (02:COz; 95:5) in a slowly shaking w a t e r  b a t h  in s e a l e d  glass vials ( 6 )  
containing 0.25 m l  of a Krebs-Ringer bicarbonate buffer (8), supplemented with 2 mg/ml 
bovine albumin (Miles Laboratories, Slough, England) and 10 mM N-2-hydroxyethylpipera- 

zine-N'-2ethanesulphonic acid (Hepes; Sigma), h e r e a f t e r  r e f e r r e d  t o  as KRBH. During 
t h e  first hour t h e  incubation medium contained 1.67 mM glucose. A f t e r  t h i s  incubation 

t h e  medium was c a r e f u l l y  removed and r e p l a c e d  by 0.25 ml of KRBH supplemented 

with 16.7 mM glucose. T h e  insulin c o n c e n t r a t i o n  in t h e  incubation media was d e t e r m i n e d  

by radioimmunoassay as described above. 

S t a t i s t i c a l  analysis. R e s u l t s  a r e  expressed as means t SEM. Groups of d a t a  w e r e  compared 

using Student's unpaired t-test. 

RESULTS 

In g e n e r a l  t h e  recovery of cryopreserved i s l e t s  was higher a f t e r  cooling at a r a t e  
of 25OC/min as compared t o  t h a t  a f t e r  cooling at 5OC/min (Table 1). The highest values 

12-878572 179 



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w e r e  obtained when human serum was added t o  e i t h e r  Hanks' solution or RPMI 1 6 4 0  
used f o r  f r e e z i n g  islets at a r a t e  of 25OC/min. In t h e s e  t w o  cases t h e  r e c o v e r y  figures 
w e r e  very close t o  t h a t  of t h e  non-frozen c o n t r o l  islets. T h e  number of i s l e t s  d e c r e a s e d  
by approximately 13% during c u l t u r e  between day 3 and day 6. In all test situations 
t h e  addition of human serum yielded a higher r e c o v e r i e s  when c o m p a r e d  t o  t h e  s a m e  
group of i s l e t s  f r o z e n  without s e r u m ,  

T h e  DNA c o n t e n t  in all groups of cryopreserved i s l e t s  was reduced by 20-30% 

(Table 1). T h e  addition of human serum to t h e  f r e e z i n g  medium did not a f f e c t  t h e  islet 
DNA content. The insulin c o n t e n t  of t h e  cryopreserved islets was lowered to 50% or 
less of t h e  value found in t h e  non-frozen c u l t u r e d  i s l e t s  (Table 1). T h e  addition of human 
s e r u m  t o  t h e  f r e e z i n g  media slightly increased t h e  insulin c o n t e n t  of t h e  i s l e t s  in t h e  
various groups of f r o z e n  islets, however t h e  d i f f e r e n c e s  did not a t t a i n  s t a t i s t i c a l  signifi- 
cance. 

T h e  basal insulin release of t h e  non-frozen c o n t r o l  i s l e t s  at 1.67 mM glucose, 
was e l e v a t e d  compared t o  all groups of frozen-thawed i s l e t s  (Table 2). When t h e  i s l e t s  
w e r e  challenged with high glucose (16.7 mM) t h e  insulin s e c r e t i o n  was increased in all 
groups of i s l e t s  by 5-9 times. However, in t h e  t w o  groups of i s l e t s  cooled at a rate of 
5'C/min, in t h e  absence of human s e r u m ,  t h e  high-glucose s t i m u l a t e d  insulin release 
was not significantly d i f f e r e n t  from t h e  basal secretion. T h e  r a t e  of insulin release 
at t h e  high glucose c o n c e n t r a t i o n  was lower in t h e  cryopreserved i s l e t s  t h a n  in t h e  con- 
trols, e x c e p t  f o r  t h e  i s l e t s  cooled at a r a t e  of 25OC/min in RPMI 1 6 4 0  plus human serum. 
I t  should be noted t h a t  t h e  stimulation f a c t o r  of insulin r e l e a s e  in response to t h e  high 
glucose challenge in t h e  c o n t r o l  i s l e t s  was fairly low d u e  t o  t h e  high basal insulin release. 

DISCUSSION 

Cryopreservation a p p e a r s  to be t h e  only s u i t a b l e  method f o r  prolonged s t o r a g e  of 
isolated p a n c r e a t i c  i s l e t s  in v i t r o  intended f o r  t r a n s p l a n t a t i o n  t o  insulin-dependent 
diabetics. During t h e  last 5-1 0 y e a r s  a number of d i f f e r e n t  cryopreservation protocols 
have been presented employing various cooling r a t e s ,  modes of addition and r e m o v a l  
of t h e  c r y o p r o t e c t a n t  and warming rates (for a review see Bank, (1)). Although t h e o r e t i c a l  
calculations c a n  be made on how a particular cell preparation should b e  cryopreserved 
(91, when designing t h e  o p t i m a l  freeze-thawing conditions f o r  a given cell population 
t h e  d i f f e r e n t  s t e p s  in t h e  cryopreservation procedure must o f t e n  be t e s t e d  separately. 

This communication i s  s u c h  a s t e p  in t h e  e f f o r t s  to find t h e  best cryopreservation proce- 
d u r e  for isolated mouse p a n c r e a t i c  islets. In t h i s  c o n t e x t  i t  was also of i n t e r e s t  t o  s t u d y  
t h e  e f f e c t  of human s e r u m  addition to t h e  f r e e z i n g  medium, s i n c e  t h i s  biological supple- 
ment will most probably b e  used in conjunction with human islet cryopreservation. 

Overall, t h e  use of RPMI 1640 compared to Hanks' solution as t h e  f r e e z i n g  medium 
a f f o r d e d  of no c l e a r  a d v a n t a g e  f o r  t h e  viability of t h e  frozen-thawed islets. The addition 

182 



of human s e r u m  increased t h e  islet r e c o v e r y  a f t e r  cryopreservation, which i n d i c a t e s  

t h a t  t h e  p r e s e n c e  of s e r u m  h a s  a beneficial e f f e c t  for t h e  survival of t h e  islet cells 
during t h e  freeze-thawing manoeuvres. T h e  reason for t h i s  is obscure. I t  could be specula- 
t e d  t h a t  serum h a s  a supportive role f o r  islet viability a s  during tissue c u l t u r e ,  but it 
cannot be excluded t h a t  f a c t o r s  in t h e  s e r u m  have c r y o p r o t e c t i v e  properties. 

T h e  islet insulin c o n t e n t  was markedly reduced a f t e r  cryopreservation, which confirms 

our previous findings (12,15). However , when comparing t h e  insulin c o n t e n t  of t h e  c o n t r o l  
i s l e t s  cultured with 1 0 %  human serum with t h a t  of i s l e t s  c u l t u r e d  in t h e  presence of 

10% calf serum (12,151, i t  is obvious t h a t  human serum r e d u c e s  t h e  insulin c o n t e n t s  

of mouse p a n c r e a t i c  islets. I t  may b e  t h a t  human serum is slightly t o x i c  to mouse islets, 
leading t o  a passive insulin s e c r e t i o n  or a l t e r n a t i v e l y  a n  impaired insulin biosynthesis. 
The finding of a n  increased insulin s e c r e t i o n  by t h e  c o n t r o l  i s l e t s  at t h e  low glucose 
c o n c e n t r a t i o n  speaks in favour of a n  e f f e c t  on t h e  s e c r e t o r y  apparatus. If t h e  addition 

of 10% human serum t o  i s l e t s  in c u l t u r e  slightly impairs t h e i r  viability, i t  c a n  be e x p e c t e d  

t h a t  t h e i r  functional c a p a c i t y  will b e  f u r t h e r  reduced by cryopreservation, s i n c e  t h e  

prefreezing s t a t u s  of t h e  islets is c r u c i a l  f o r  t h e i r  ability t o  r e s i s t  t h e  stresses of t h e  
freeze-thawing process. 

In conclusion t h e  d a t a  of t h e  present s t u d y  suggest t h a t  t h e  use of a c o m p l e t e  t i s s u e  
c u l t u r e  medium (RPMI 1640) as f r e e z i n g  medium does not i n c r e a s e  t h e  viability of cryo- 
preserved p a n c r e a t i c  islets. On t h e  o t h e r  hand, t h e  addition of 10% human serum t o  

t h e  f r e e z i n g  medium supported t h e  survival of t h e  cryopreserved islets, but human s e r u m  

may, to s o m e  degree, impair t h e  viability of mouse p a n c r e a t i c  i s l e t s  in culture. W e  
have, however, found t h a t  t h i s  supplement may s t i m u l a t e  t h e  g e n e r a t i o n  of p a n c r e a t i c  

B-cells from human f e t a l  p a n c r e a t i c  glands (14). 

ACKNOWLEDGEMENTS 

W e  t h a n k  Eva Forsbeck, Astrid Nordin and Margareta Engkvist f o r  e x c e l l e n t  t e c h n i c a l  
assistance and Agneta Snellman for c a r e f u l  preparation of t h e  manuscript. Financial 
support from t h e  Swedish Medical R e s e a r c h  Council (12X-109; 17X-7501; 12P-7680), 
t h e  Swedish Diabetes Association, t h e  Nordic Insulin Fund, t h e  Juvenile Diabetes Founda- 
t i o n  International, t h e  Novo Company, t h e  O.E. and Edla Johansson Fund, t h e  Ake Wiberg 
Fund, Ernfors Diabetes Foundation, t h e  C l a s  Groschinsky Memorial Fund, t h e  Swedish 
Society of Medicine and t h e  Syskonen Svenssons Fond, is g r a t e f u l l y  acknowledged. 

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of t i s s u e  by refrigeration. Proc.Soc.Exp.Bio1.Med. 71:196-200, 1949. 

3. Heding, L.G.: Determination of t o t a l  s e r u m  insulin (ILI) in insulin-treated patients. 
Diabet ologia 8: 26 0 -266, 1 9  7 2. 

4. Hinegardner, R.T.: An improved f l u o r o m e t r i c  assay for DNA. Anal.Biochem. 39:197- 

5 .  Howell, S.L. & Taylor, K.W.: Potassium ions and t h e  s e c r e t i o n  of insulin by i s l e t s  
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20:119-128, 1983 

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I83 



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A d d r e s s  f o r  c o r r e s p o n d e n c e :  
D r  S t e l l a n  S a n d l e r  
D e p a r t m e n t  of Medical C e l l  Biology 
Biomedicum, P.O.Box 571 
S-751 2 3  Uppsala, Sweden 

184