Wak Mycoplasma CryoSure-DEX40

STERILE A Sterilized by sterile-filtration
Steril durch Anwendung aseptischer
Verfahrenstechniken 2 °C
8 °C Store at 2°C – 8°C.
Bei 2°C – 8°C lagern.
Use until: see product labelling
Verwendbar bis: siehe Produktetikett
LOT Protect from strong light.
Vor Sonnenlicht geschützt aufbewahren.
Follow instructions for use.
Gebrauchsanweisung beachten.
Do not use if packaging is damaged.
Bei Beschädigung Verpackung nicht verwenden.
Do not re-sterilize.
Nicht erneut sterilisieren.
Lotnumber: see product labelling
Chargennummer: siehe Produktetikett
WAK-DEX40-25 (25 x 8 ml)

Instructions for use CryoSure-DEX40

Introduction:
CryoSure-DEX40 is a ready-to-use cryoprotective solution for the addition to a volume reduced buffy coat suspension from cord
blood according to the method of Rubinstein et al (1).
CryoSure-DEX40 is a solution consisting of 50 % v/v DMSO and 50 % v/v of a 10% aqueous solution of Dextrane 40.
DMSO (Dimethyl Sulfoxide) is a cryoprotectant which penetrates the cell wall and takes its cryoprotectant effect within the cell.
It reduces the osmotic stress on the cells during freezing and thawing (2, 3, 4, 5, 6) and antagonizes the osmotic shock (7). Also
DMSO protects the cells by reducing dehydration and shrinkage of the cells during the freezing process (5, 8). After thawing
DMSO has to be removed from the stem cell suspension by means of wash centrifugation.
According to the protocol of Rubinstein et al the DMSO-concentration in the volume-reduced ready-to-freeze endvolume is 10%
v/v.
Before freezing and after thawing DMSO is potentially cytotoxic. The cytotoxicity is dependant on the DMSO-concentration, the
time of exposure and the temperature of the stem cell suspension during the time of exposure to DMSO (9, 10, 11, 12, 13, 14,
15, 16).
Therefore before freezing respectively after thawing the stem cell suspension has to be kept cool at 2°C whilst CryoSure-DEX40
is added to the stem cell suspension, respectively before removal of CryoSure-DEX40 from the stem cell suspension after thawing.
Immediately after addition of DMSO the freezing process has to be started.
Likewise immediately after thawing the wash out process has to be started.
In case of adequate cooling of the stem cell suspension during DMSO-exposure in the unfrozen state, no relevant adverse
effects on the cells are observed at end volume concentrations of DMSO between 5 and 10 % (15, 16, 17, 18). Since DMSO is
a strong aprotic solvent, special care has to be taken to only use DMSO-compatible materials for withdrawal of the DMSO from
the vial and during transition of the DMSO to the target suspension and to minimize the contact time of DMSO with such materials. All processes related to the application and elimination of CryoSure-DEX40 have to be validated by the user.
EN
Addition of CryoSure-DEX40 to the stem cell suspension
Zugabe von CryoSure-DEX40 zur Stammzellsuspension
CryoSure-DEX40
WAK – Chemie

Withdrawal of cryoprotectant
Entnahme des Kryoprotektivums
Freezing bag
Einfrierbeutel
Immediately rate-freeze
Unverzüglich einfrieren
Store in freeze tank
Aufbewahren im Gefriertank
Cryoprotectant
Kryoprotektivum
2oC
15 min
1. 2. 3. 4.
EN Addition of CryoSure-DEX40 to the stem cell suspension
CryoSure-DEX40 is added to the volume-reduced cord blood suspension as the last step before initiating the freezing process.
2. Withdrawal of CryoSure-DEX40
from the vial and preparation of
the cryoprotective solution
Before addition to the stem cell
suspension CryoSure-DEX40 is to
be cooled to 2°C. In order to reach
the envisaged concentration of
CryoSure-DEX40 in the endvolume
the necessary amount is to be taken
volumetrically from the vial.
Cell suspension
Zellsuspension
1. Calculation of the composition
of the cryoprotective solution
The amount of CryoSure-DEX40 to
be added to the stem cell suspension has to be chosen in a way so
that the envisaged endvolumeconcentration of DMSO is met. 8 ml
of CryoSure-DEX40 contain 4 ml of
DMSO (≙ 4,4 g DMSO) The specific
gravity of DMSO is 1,1 g/cm3. In
accordance with the protocol of
Rubinstein et al
5 ml of CryoSure-DEX40 are to be
added to 20 ml of volume-reduced
cord blood. Like this the added
2,5 ml of DMSO result in an
endvolume-concentration of DMSO
of 10% within the ready-to-freeze
suspension (1).
3. Addition of the cryoprotective
solution to the suspension of
hematopoietic stem cells
Before addition of CryoSure-DEX40
the stem cell suspension is placed
on an ice bed and cooled to 2°C.
Thereafter CryoSure-DEX40, which
has also been cooled to 2°C, is
added volumetrically at a constant
velocity within a period of 15 minutes
to the stem cell suspension until the
designated end volume is reached.
Preferably a calibrated syringe pump
is to be used for the addition of the
cryoprotective solution (1). The
decelerated addition of the DMSOcontaining CryoSure-DEX40
provides for the osmotic tolerance of
the hyperosmolaric DMSO and the
cells in the target suspension. During
the addition process the target
suspension is continuously and
consistently mixed in order to assure
a consistent dispension of the
conveyed DMSO within the target
suspension (1).
4. Begin of freezing process
Immediately after addition of the
complete designated amount of
CryoSure-DEX40 to the target
suspension the freezing process has
to be started. Until the beginning of
freezing the temperature of the
ready-to-freeze stem cell suspension
has to be kept at 2°C. For freezing
standard freezing procedures have
to be applied as specified in
literature. A freezing rate of
1°C/minute until the final freezestore temperature is reached has
been described as an applicable
freezing rate for hematopoietic stem
cells (16).
Calculation of required
quantity of cryoprotectant
Berechnung der Anteile
der kryoprotektiven Lösung
Wash out at + 2°C
Auswaschen bei +2°C
Centrifuge
Zentrifuge
Withdrawal of liquid supernatant
Abziehen des Überstandes
Repeat until cryoprotectant is eliminated from the cell suspension
Wiederholen bis das Kryoprotektivum entfernt ist
Cells
Zellen
Thawed cell suspension
Aufgetaute Zellsuspension
Withdrawal of cryoprotectant
Entnahme des Kryoprotektivums
Water bath 37°C
Wasserbad 37 °C
Bag enclosing frozen cell suspension with cryoprotectant
Gefrorene Zellsuspension mit Kryoprotektivum im Beutel
Thaw until all ice crystals have disappeared
Auftauen bis sich alle Eiskristalle aufgelöst haben
Wash out of cryoprotectant by centrifugation
Auswaschen des Kryoprotektivums durch Zentrifugation
Addition of wash solution
Zugabe der Waschlösung
Wash solution
Waschlösung
Cells
Zellen
Withdrawal of cryoprotectant from the stem cell suspension after thawing
Entfernung des Kryoprotektivums aus der Stammzellsuspension
Withdrawal of cryoprotectant from the stem cell suspension after thawing
Immediately after the completed thawing process the cryoprotective solution must be washed out of the stem
cell suspension. The washing process is executed in several washing steps consisting of centrifugation, withdrawal of liquid supernatant and resuspension of the cells with an appropriate wash solution. During the washing process until the quantitative elimination of the cryoprotectant from the stem cell suspension the suspension
has to be kept cool at 2°C. Consequently the wash out process has to be performed by means of a refrigerated
centrifuge. The validation of the elimination process ist the resonsibility of the user. For performing the wash out
process within a closed system several methods are available (19-25).
EN
Sources
1) Rubinstein, P.; Dobrila, L.; Roseneld, R.E.; Adamson, J.W.; Migliaccio, G.; Migliaccio, A.R.; Taylor, P.E.; Stevens, C.E .: “Processing and cryopreservation of
placental / umbilical cord blood for unrelated bone marrow reconstitution” . Proc. Natl. Acad. Sci. USA 92 , 10119-10122 (1995 )
2) Farrant 1969, Nature, 11755
3) Mazur 1970, Science 168, 939-949
4) Leibo 1977, Ciba Foundation Symposium No.52, Amsterdam S. 69-96
5) Merryman et al 1977 Cryobiology 14, 287-302).
6) Whittingham 1981, Gustav-Fischer Verlag, Stuttgart, S. 21-22
7) Lovelock 1953, Biochim. Biophys. Acta 10, 414-426
8) Merryman 1974, An. Rev. Of Biophys. And Bioengineering, Band 3 Paolo Alto, S.341-363
9) Goris A: ‘[Test of the toxicity of dimethyl sulfoxide (D.M.S.O.) on carrot tissue cultured in vitro]’, Ann Pharm Fr. 1966 Dec;24(12):781-4
10) Basch, H., and Gadebusch, H.H. In vitro antimicrobial activity of dimethyl sulfoxide. Appl. Microbiol. 16: 1953-1954 (1968).
11) Chang CY, Simon E: ‘The eect of dimethyl sulfoxide (DMSO) on cellular systems.’, Proc Soc Exp Biol Med. 1968 May;128(1):60-6
12) Da Violante G, Zerrouk N, Richard I, Provot G, Chaumeil JC, Arnaud P: ‚Evaluation of the cytotoxicity eect of dimethyl sulfoxide (DMSO) on Caco2/TC7
colon tumor cell cultures’, Bilo. Pharm. Bull. 25(12) 1600-1603 (2002)
13) Fahy GM: ‘The relevance of cryoprotectant ‘’toxicity’’ to cryobiology.’, Cryobiology 1986 Feb;23(1):1-13
14) Gurtuvenko AA, Anwar J: ‘Modulating the structure and properties of cell membranes: the molecular mechanism of action of dimethyl sulfoxide.’, J
Phys Chem B. 2007 Sep 6;111(35):10453-60
15) Yang H, Zhao H, Acker JP, Liu JZ, Akabutu J, McGann LE: ‘Eect of dimethyl sulfoxide on post-thaw viability assessment of CD45+ and CD34+ cells of
umbilical cord blood and mobilized peripheral blood.’, Cryobiology 2005 Oct;51(2):165-75
16) Hunt CJ, Armitage SE, Pegg DE: ‘Cryopreservation of umbilical cord blood: 2. Tolerance of CD34(+) cells to multimolar dimethyl sulphoxide and the
eect of cooling rate on recovery after freezing and thawing.’, Cryobiology. 2003 Feb;46(1):76-87
17) Rowley S.D., Anderson G.L.: ‘Eect of DMSO exposure without cryopreservation on hematopoietic progenitor cells’. Bone Marrow Transplant.11,
389-393 (1993)
18) Branch, D.R.; Calderwood, S.; Cecutti, M.A.; Herst, R.; Solh, H .: “Hematopoietic progenitor cells are resistant to dimethyl sulfoxide toxicity” . Transfusion
34, Nr.10 , 887-890 (1994 )
19) Calmels B, Houzé P, Hengesse JC, Ducrot T, Malenfant C, Chabannon C.: ‚ Preclinical evaluation of an automated closed uid management device:
Cytomate, for washing out DMSO from hematopoietic stem cell grafts after thawing.’, Bone Marrow Transplant. 2003 May;31(9):823-8.
20) Rodríguez L, Velasco B, García J, Martín-Henao GA.: ‘Evaluation of an automated cell processing device to reduce the dimethyl sulfoxide from hematopoietic grafts after thawing.’, Transfusion. 2005 Aug;45(8):1391-7.
21) Foïs E, Desmartin M, Benhamida S, Xavier F, Vanneaux V, Rea D, Fermand JP, Arnulf B, Mounier N, Ertault M, Lotz JP, Galicier L, Raoux E, Benbunan M,
Marolleau JP, Larghero J.: ‘Recovery, viability and clinical toxicity of thawed and washed haematopoietic progenitor cells: analysis of 952 autologous
peripheral blood stem cell transplantations.’, Bone Marrow Transplant. 2007 Nov;40(9):831-5. Epub 2007 Aug 27.
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postthaw and postwash samples.’, Transfusion. 2005 Dec;45(12):1909-16.
23) Lemarie C, Calmels B, Malenfant C, Arneodo V, Blaise D, Viret F, Bouabdallah R, Ladaique P, Viens P, Chabannon C.: ‘Clinical experience with the delivery
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improve the speed of engraftment of cord blood transplantation: follow-up of 46 adult patients with units shipped from a single cord blood bank.’,
Transfusion. 2003 Sep;43(9):1285-95.
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