/









Cell Cultures Preservation

.




! 
'
$&

("#$
)$'(

/)!$
 !
+,

/!$




2005


  

  /
35-34

   

"! 

"! 
   
    


&'( )* #$%

'.-1  21 3 ./0# +, -

 !./ )( + $! $, * )(  $%
(/ 12 (45 4 (89 ) / 67%;* 7

7C4 *
7B A7).4 ?%?= !<7 * 
 $% (% !./  (45 4 <9 69
 +%(9, ) 7%9% (?%* 69 A4
(I=
 (M
J<K! N74(M (?/"! <9 7O
 <9 +%769 Q(M )?= !<7 (C )( 
"!  (S T25 7 J<V/ (?(7 7) %O%
5 ... N74!6
7 (% !./ ! $(? * +76
?
C...
7
, X), 5* .

28/8/2003
C

 
2003/8/28


 
 

 

 
  !"#
 
'& 
$%
. */01
"+,-*)( 
*645  
3

7  
3 -*8 -

) !'9
$%
 
<
) 1= 

) 2*>  0

@AB3C"@ 
1@AD*F E
@A< 2GBA9 
@A< 33I 
4@AD JK C
@A< 5 EL5M>*C
@A< 6 (I
*> &N

3 
 
1*O39F 
2*> 
3>3 
4* Q>F
5*Q*3F Q*35/>B
E

 6*8* Q
7*L F 
8*  3 F
3 9 
 R%

/9>3 
S
* $3 *3*@6 

V
 
1>'! 
 
2
 !N>*9
3*> N
4
 8>*9
5*> &N

*'3 /[ !"WXYZ

 
@\
  


* 6F
*/-> F
*> N

 8>*9
*> &N
*> !N
^&* /]L

 
96*9
*_ R%B
F




 !] 
*_*'

 !] 
*/-> F

 8>*9
*> N
^&* /]L
*> &N



 >L 3-5B

 8>*9

 
96*9
*> N
*_ R%B



*_ 
*/-> F

`b* `a

9 
 
*> &N
^&* /]L
*> N



*_ 4*c

 8>*9
G

*> N
*> &N


*+ 

>9B] 

 3
 

 * 
-


-A* 
-


3*3
Z* 
-

*+* 
-

-d*3
Z"! 
 
A6B 
<
a 
 eA




/ 5-4 . 2003 /
.

.

.

.

.

.

  
2003/8/28

IV

)
 





 / / ( 30-
.
,


Bacillus .

,
, .

,
.

), (Cell viability
.

.

,
.

"










" !




/ / " 47
,
.
1890

Kral

1911 Mycobacterium tuberculosis Dr. Koch
Agrobacterium tumefacience Dr. Smith .

40-25 .

1909
.
1940

.

:
2

mislabeling

.
.

,

. Actinophages
photooxidation
,

Viability .


Biotherapy Probiotics

.

.



.
Escherchia coli


2000-4000% .
Liposomes
) (Drug delivery .
DNA


Sequencing .
Bio electronics
.

: Anabiosis
.
:( Xeropliles) Osmophliles
Hypertonic solution

:Osmoduric
4


Staphlococcus aureus
.
: Halolerant
) ( Osmoduric
: L drying

.
: Stress
,
30 )
( Thermal
. Stress
Compatible Solute
)
(

-1
.
-2

-3

Polyols

.



Tg :

.
Tm :

.
Cosolvents

.
:Anhyrobiotes
.

 :Tc

)
( .
:Osmolytes
.

:

.

48
20

) / /
(49

. :



Uptake Efflux
.
7

 ) Compatible
) solutes
) (Osmotic upshift
) (Zwetterionic organic cosolvents Glycine, Betaine,
Ectoine
).(K+
) (
) (Osmosensory mechanisms
) (Osmosensors
) (Oligomerization

.
) (Nucleoid
.
) (

) ( .
.



.


.


.
10-5

.
) (Integral
Van der Waal
.
) (Peripheral
.

, amphipathic .

-:
NH 3

Ethanolamine

CH2
Polar and hydrophilic end

CH3
O
-O- P = O
O
CH3
Glycerol
CH

CH2

C=O C=O
Fatty acids
R

Long, nonpolar, hydrophobic

fatty acid chains

10

 hydrophilic
hydrophobics

-:

.
phospholipids
.


. .

11

 ) (

Hopanoids

-:

12

 -:

.

.
-:

13

 -:

14



)(
.

) (
Van der Waal )
(
.

.
) (
.
.
) (Fatty acyl
.
) (
.



.
40-30
%15 .
15


P=O
) (
.

)
(
P=O ) (

P=O

.




)
( .

.
16

 -:
-1

Water replacement hypothesis

) Trehalose ( %25

) (Anhydrobiotic organisms
) (Macrocysts Dictyostelium
.Active dry yeast


) (
). (Rehydration


.

) (

Van
der Waal
) (Gel phase
). (Liquid crystaline phase

(PC ) Phosphatidyl choline ) Tc (
17


PC
.
PC
) (Protactants
.

Water replacement hypothesis
1973 Crowe .

) . (Hydrated lipids
) (Tc


) ( .
) (IR spectroscopy
) (OH
.


.
-2

Vitrification hypothesis

18






.


.
Stresses


.
K+ . Na+


.


) (Polymerization
.
.

19



.
)( .

-1 High temprature stress

) .(Thermophiles
.

) ( .

(hsps)Heat shock proteins
) .(Chaperones
hsps
. E. coli
30 42 17

Molecular

chaperones Ion protein ) ( proteases

.

.
20

 hsps
RpoH 32 -
) .( 32

- (70-)70 ( 32)32 -
hsps .

.

) (Hyperthrmophiles Pyrodictium occultum
110
%75 108.
2 Low temparture stress


shock response

Cold

)
13-12 ( . E.coli
37 10 4

.

21

 ) (Regrowth
.
-:
-

Cold-shock proteins

.

) ( hsps
.

Csp A
.
-

.
Low-temp. RNA chaperone
RNA . mRNA
RecA IF-2

N-formyl methioine-charged tRNA ) (30S

.
NusA .
Subunit of DNA gyrase
.Topoisomerases

.
ppGpp
p ) ppGppp G (Guanosine

22

 Csps
Cloramphenicol,Erythromycin Tetracyclin

alarmons

. (p)ppGpp
) (

) (Polyunsaturated fatty acids

.

.

) phase

(Stationry .

)
(.
-3 Stringent Stress


. rRNA

23

 ) (alarmons ppGpp
.
-4 Acid stess

Acid-
(ART) tolerance response Acid-
(Asps) shock proteins Acidophiles

. pH Salmonella typhimurium

1.5
8
) (Asps
s Fur-protein
.

.
-5 Oxidation stress


.

24


( O2- ) Superoxide ) ( H2O2 OH
.

) (Oxygen tension
.
.
- -
)(Antioxdant chemicals
.

-:
Glutathione .-

Metallothionein
Cu++ O2- . OH

Thioredoxin ) (disulphides .
polyamines . .
) (
Cu/Zn superoxide dismutase O2-
.

25

 Mn superoxide dismntase .O2 Catalase A

).(Peroxisomes
:Catalase T
: Cytochrome C peroxidas .
: Glutathione reductase
.

.
:Glutathion Thiol Compounds

, Alkylating agents
.Redox balance
Metallothioneins :
.
Cup1 .
:Carotenoids

.



.Adaptive oxidant stress response
26





.



.
-6 Water stress


.
.



.
hyperosmotic
shock Up skock
hyporosmotic shock ,Down skock

. Halobacteria
27


%10

%10
.

%80
.


.






.


0.2g H2O / g dry weight
1 Hydration repulsion
Lateral stress

28

Tc

.

.


) (.



Tm

.



.

29

 Protectants


.
.
Compatible solutes Brown 1974-1972


.


.



.
-1-:

.
.
) (
.

30

 Tc



.


) (Vitrification
.

.
.






.

Tm

.
Tm 22 )To (Tc
.
31


30

.

Tg 36 Tg
To

.


) ( 40.000

Tg 110.


.
)(Tg

. polyols
) (Tg .
Tg
) (

.
32



)
( .


) Polylys (
%20

Inositol
.
-2 Betain


) (N-trimethyldervitative of glycine
.


) (.



.
33

Pseudomonas

Ps. halophila

Ps. halosaccharolytica
.
-3 Mannitol
Pseudomonas
.Salt-stress


.
Ps.putida

) (Glass matrix . Ps.
putida

.
88g /109 CFU
. 32g / 109 CFU

) 2.7g/109CFU
(.

34

-4 Carnitine

) (L .


MRS
22 /
.
-5

Choline & Acetylcholine

Succinylcholine
) (Neurotransmitters
.
K+
.
Acetylcholine Succinylcholine
Betaine
.
-6 Proline


) (Proline

35

 ) Cytosol amino acids

(pool .
-7 Glycerol

2/5
) ( Tc 2/1.
) (Tc
) (Fully hydrated
.


) (Fusion-producing properties
.


Van der Waal
.

) (Protein inactivation

) ( Hydroxylated compounds
) (Intersite bonds
.

36

-8 Glutamate


NaCl .K+
.
E.coli 0.5 M NaCl
K+
) 30 (

)(.
) (

K+.
-9 -:
Pseudomonas Ps. aeuroginosa
Carboxy amide glutamine
(NAGGN ) N - acetyl glutaminyl glntamine amide
Ps. memdonica
Ps. pseudoalcaligenes O- -glyucopyronosyl - (1,2)glycerol
. taurine
-alanine
Trimethylamine oxide Sarcosine

37

 .
.Tetrahydro pyrimidines

-:
) (de novo synthesis

-:

.
. (Proton motive force )pmf


.


.
) (Coctail
.

38

 Chaperones

) ( Stresses

.
.
.
.
GroEs GroEL Dnaj Dank E.coli
) Grp E . ( E.coli

Heat shock
( hsps) proteins . Stress proteins
.


) (Stationary phase proteins

)(Quorum sensing
.
Trehalose

- D-glucopyranosyl - D-gluranoside
39

) (Stress metabolites

) (Anti-desiccatant ) (Cryoprotectant
) (Thermoprotectant )(Chemical detoxicant
.


.
) (

% 20-10 .

) (Liposomes
) .( Drug delivery

.
) 2001(

.

.
200-50

40

 100 .

.

) ( Tm

.
.
E.coli
7105.6 /
E.coli
.

E.coli
) (Log phase
230g/109CFU 150g
)
NaCl

.(0.6M

. 0.5g /109CFU


) . ( Anhydrobiotes

41

 100

.
)
(

) Tg (115 C ) (High glass-transition temp.

Glass trehalose
.



.

.
) (

Trehalase .

42



.
.

Cryptobiosis

Ps.aeruginosaS.typhimurium E.coli


.

Tap water agar
30.
) (
.
Freezing

.
Freez drying


.
43

 )52( .

.
) (Old culture
.

) ( Aceton dry ice
)80-(
Cryopreservation
196
273- .

)20-( .


) . (Polyunsaturated
) (Cryoprotective agents
.


.
) (Antifreezing agents
(Dimethyl sulphoxide DMSO) .
44


.


.
.


.


) ( Freeze-resistant
) (Cyyopreservation selection
.
) ( Cyoprotectants

) (Hypertonic stress
.
-:
.-

).(Thawing

.
45

 .
DMSO

.

) (
.
-:

.
-:
.-

) .( Formulation

.

.
-:
L- drying .

46

 Connective drying Air drying

). (Active dry yeast
-

Spray drying .

. Fluidized-bed drying

. Spray granulation drying

.


.

:
.
-

47

.
.

Cells preconditioning

.

.
-:

0.6-0.3 .
-

48


.
.
-

.
-

Oleic acid

.

.

) (Cold hardening
4

.

49




. % 80


) . (Early Stationary phase
.

) ( .

-:
-:


. 1

.
Lyophilization

-:
-1

.
50

-2

-3

.


) (Electrolytes
DNA
.



.


%1
%3-2 .



10

10

/ .

mllitorrs 10

.High frequency tester

51



.
.
4


.

.

3-2

2-1

.
Cryopreservation

20 273 .
196.


52






.
) (Electrolytes


.

.

.


.
10 /
.

) (Cryoprotectants .
.
53

 . .
. DMSO


130 .

,
DMSO %5
30
3-1
100
.

40-37 60-40.
Desiccation

.

54

 Desiccator .
.
Freezing

Cryopreservation

20
70
.
L-Drying


.
Soil cultures


) (
.


) (Stabbing .

55


) (aw
.

.


) (Lysosomes

.

.


. G1

.
.

. Petit strains

56

 .
.

.




.

). (Stationary phase


(High Osmolarity glycerol) HOG

Protein kinases
) Stress response elements(STREs
FPS1


Ca++

) (Anhydrobiosis
57


.





.

) (Trehalose carriers

) (de novo synthesis

.

) (.



.

) (Slants
58


4 .
.
Cryopreservation

20 80 .
) (
.
196
%20 -5
.
Lyophilization



.
. Saccharomyces boulardii

59


 


 

11 13 15

3 5
7 9
 ) 
(


    

110
100
90
80
70
60
50
40
30
20
10
0
1

Saccharomyces
boulardii








10

5 6 7
 )
(


    

110
100
90
80
70
60
50
40
30
20
10
0
1

Saccharomyces
boulardii Mg-stearate
60









9 10

5 6 7
 )
(


    

110
100
90
80
70
60
50
40
30
20
10
0
1

Saccharomyces
boulardii

 

 

15

13

11

7
9
 )
(


    

110
100
90
80
70
60
50
40
30
20
10
0
1

Saccharomyces
boulardii

61

 L-Drying


) (Xerophiles
Desiccation


.
Actinomycetes


.Actinophages


.
.

-:
-1 .
-2 .
-3 .
-4 .
62

 -5 .
-6
.
-:
Cryopreservation

14-10
%10 ) (
Borosilicate glass .Teflon lined screw

%20
20 80
196 .
.
37 .
72 .
Soil preservation

.

.

63

 6
150
.

.
) (Vials
) 14-13
100 ( 60
.

Loop ful

45 37 28 24
) . ( Sterility test


.
) (1
)25-24(
.

64

 ) (Loop


:

100

2.5

:
10:

3-2 .
25
.
Lyophilization
)
14-10( %15 ) (Ampules
Borosilicate glass .
) (

) 4( .

.
65

 . .
70.



.




) (Heterokaryon )(Parasexual cycle

.

.
.

) (Sclerotia
) (Mites .
:
66

 :


.

.

.

300 200 .

.

.

.

.


.


.
67


.

) (
.
.
) (Dermophytes
.
7-3
. 4-2
4-2 .
6-4 7-4 .
7 24
5-4
.
:
) (
) (
10
30 . ) (Mineral oil
) ( Specific gravity 0.89 0.83
.

68


.

.
30-12 .
:Cryoptobiosis


.

McCartney bottles
) 25 ( .
:

:
-1 :
180
.
.
-2 .

10-5
69

 ,
7-4
.
-3 :
Whatman #4
%5
Desiccator 4 .
3-2
)(Haploid
) (Diploid
.
.
:L-drying
%20
) ( Inositol
0.5 ) (Ampules
) (Desiccator
.
%1
. DNA

.

70

%1 . )
( .

115-114 .
1 /

.
4
)25( .

.


.

:
71

 -1

.

.
-2 ) (Slides

310

25 )
(
20-10 ) (X 400
.


.

.Pellets

.

.
.

72

 :
Cryopreservation

.
. . )(Freeze-resistant DMSO .
) (Old .:


80

%5
:
.
Protozoa

73


.

130 . 70
100 .
) ( Cysts
.

DMSO

5 .%10 2

Entamoeba %3

%20 .
5 %7
.

70

DMSO
Ca++
40 90. 70 .

74



%1 .

610x 5-1 %10-5

DMSO ) 1 ( 1 /

50
)196-( )135-( )140-(
37
.

. ) ( Orthodox seeds
.
% 7-5
5 20- .

.

.
75


DNA .

.


.
) 130-(
.
.

DMSO
) ( Polyols .
.

.
Verification
.

.


Phenosafranine blue

Trypan

chloride

Triphenyltetrazolium

Fluorescein diacetate .
76



.



Agrobacterium tumefacience
.
) ( Pollen
DNA .

.

4
70 20
.

2
4 .

77


70 -.

.

.
.
Anhydrobiotic Engineering
) (Anhydrobiosis

).(Anhydrobiotes


Pseudomonas putida

.
Cyanobacteria
.
Escherichia coli
.
Trehalose synthetase , Sucrose synthetase
.
78


) (Anti-freezing peptides

.
Recovery
%5


.



40-30
1 60 -30 .
. .




) ( Metabolic load

79


.

.
.


) ( ATP
.

:
-1

.
-2
.
- 3
-4 .


.
80








.


.
.


.

.-

.
.-

81

 14 10- 1011 /.

10108 10 \ . /610 )
(.
.-

: .

- .

-1
-2

-: .

-:
) ( .
.

.
) (
.
.

82


1. Billi , D., Wright ,D .J ., Helm, R.F., Prickett , T.,Potts, M.&
Crowe,J.H.2000. Engineering desiccation tolerance in Escherichia
coli .Appl. Environ .Microbiol. 66 : 1680-1684.
2. Brown ,A.D.1976.Microbial water stress. Bact. Rev.40:803-847.
3. Buitink, J., Dries, I.J., Hoekstra, F.A., Alberda, M.& Hemminga,
M.A.2000. High critical temperature above Tg may contribute to
the stability of biological systems. Biophys.J.79:1119-1128.
4. Castro, A.G., Bredholt, H., Strm, A.& 4 Tunnacliffe, A.2000.
Anhydrobiotic engineering of Gram- negative bacteria .Appl.
Environ. Microbiol.66:4142-4144.
5. Colaco, C., Sen, S., Thangavelu, M., Pinder,S. & Roser, B.1992.
Extraordinary stability of enzymes dried in terhalose simplified
molecular biology. Biotechnol.10:1007-1111.
6. Crowe, J.H.& Crowe, L.M. 1984. Preservation of membranes in
anhydrobiotic engineering organisms : The role of trehalose.
Science,223: 701-703.
7. Crowe,J,H.,Carpenter,J.F&Crowe,L.M.1998.The role of verification
in anhydrobiosis. Ann.Rev.Physiol.60:73-103.
8. Dinnbier,U., Limpinsel,E., Schmid,R.& Bakker, E.P.1988.Transient
accumulation of potassium glutamate and the replacement by
trehalose during adaptation of growing cells, of Escherichia coli K12 to elevated sodium chloride concentrations,
Arch
Microbiol.150:348-357.
9. Efiuvwewere, B.J.O.Gorris,L.G.M., Smid,E.J.& Kets, E.P.W.1999.
Mannitol enhanced survival of Lactococcus lactis subjected to
drying .Appl. Microbiol. Biotechnol.51:100-104.
10. Horlacher, R& Boss,W.1997. Characterization of the Escherichia
coli trehalose system . J. Biol .Chem. 272. 13026-13032 .
11. Hunter Cevera, J.& Belt, A. 1996. Maintaining Cultures for
Biotechnology and Industry. Academic Press: San Diego& New
York.
83

12. Kets,E.P.W.&Bont,J.A.M.1994. Protective effect of betaine on

survival of Lactobacillus plantarum subjected to drying . FEMS
Microbiol. Lett.116:251-256.
13. Kets,E.P.W.&Bont,J.A.M .1997. Effect of carnitine on
Lactobacillus plantarum subjected to osmotic stress. FEMS
Microbiol .Lett. 146:205-209.
14. Kets, E.P.W. , Teunissen , J. M.&Bont ,J.A.M.1996 Effect of
compatible solutes on survival of lactic acid bacteria. Recent Res.
Devel. in Microbrology 1, 189-199.
15. Kets,E.P.W., Galinski, E.A.& Bont, ,J.A.M.1994. Carnitine :a
novel compatible solute in Lactobacillus plantarum . Arch
Microbiol 162: 243-248.
16. Kets, E.P.W., Galinski, E.A., Wit, M., Bont, J.AM.& Heipieper,
H.J.1996. Mannitol, a novel bacterial compatible solute in
Pseudomonas putida S12.J. Bact. 178, 6665-6670.
17. Kets, E.P.W, Groot, M.N, Galinski, E.A.& Bont, J.A.M.
1997. Choline and acetylcholine : novel cationic osmolytes in
Lactobacillus plantarum. Appl. Microbiol. Biotechnol.48:94-98.
18. Kets, E.P.W., Teunissen, J.M. & Bont, J. A. M. 1996. Effect of
Compatible solutes on survival of lactic acid bacteria subjected to
drying. Appl. Environ. Microbial. 62: 259-261.
19. Koster, K.L., Lei,Y.P, Anderson, M., Martin, S.&Bryant, G. 2000.
Effects of vitrified and non vitrified sugars on phosphatidylcholine
fluid- to gel phase transitions. Biophys.J.78:1932-1946.
20. Leslie, S.B., Lighthart, B., Crowe, J.H.& Crowe, L.M.1995.
Trehalose and sucrose protect both membranes and proteins in
intact bacteria during drying .Appl.Environ.Microbiol.61:35923597.
21. Manzanera, M., Castro, A.D.,Tndervik, A., Rayner-Brandes, M.,
Strm , A.R. & Tunnacliffe, A.2002. Hydroxyectoine is superior to
trehalose for anhydrobiotic engineering of Pseudomonas putida
KT2440.Appl.Environ. Microbiol.68:4328-4333.
22. Matsuo,T.2001.Trehalose protects corneal epithelial cells from
death by drying .Br.J.Ophthalmol.85:610-612.
84

23. Potts, M.1994 . Desiccation tolerance of prokaryotes. Microbiol &

Mol. .Biol .Rev. 58:755-805.
24. Prescott,L.M. , Harley, J.P.& Klein ,D.A. 1999. Microbiology.4th
ed .wcb McGraw- Hill: Boston Burr Ridge &New York.
25. Rawn,J.D.1989. Biochemistry. Neil Patterson Publishers. Carolina
Biological Supply Comp: Burlingtom, North Carolina.
26. Shaffer, B.T. Lighthart, B.1993. Protection of freeze-dried
Escherichia coli by trehalose upon exposure to environmental
conditions. Cryobiology.30:519-523.
27. Singleton, P. 1997. Bacteria in Biology, Biotechnology and
Medicine. John Wiley & Sons : Chichester& New York.
28. Sneath, P.H. 1962. Longevity of microorganisms. Nature, 195:643646.
29. Tsvetkova, N.M., Plillips, B.L., Growe, L.M., Crowe, J.H. &
Risbud, S.H. 1998 . Effect of sugars on head group mobility in
freez-dried dipalmitoyl phosphatidy choline bilayers. Biophys .J.
75:2947-2955.
30. Walker, G.M. 1999. Yeast Phsiology and Biotechnology. John
Wiley & Sons: Chichester& New York.
31. Wood, J.M.1999.Osmosensing by bacteria :Signals and membrane
based sensors. Microbiol .&Molecular Biol.Review.63:230-262.
32. Yancey , P.H., Clark, M.E., Hand, S.C., Bowlus, R.D.&
Somero,G.N.1982.Living with water stress: Evolution of osmolyte
systems . Science, 217:1214-1222.

85

571.84
723   
" #  
Cell Cultures Preservation
.    ('&
) ('& 2005
,- 24
-1
)1
.
2005 / 197