Summary
It was the purpose of this study to establish and evaluate a freezing and-thawing method for preservation of hemopoietic stem cells from the peripheral blood. Blood leukocytes collected by means of an IBM Blood-Cell-Separator were frozen in plastic bags using 10% DMSO and controlled cooling rates. Thawing was performed rapidly, and DMSO was diluted and removed prior to the in-vitro and in-vivo assays.
The mean recovery of mononuclear cells collected from 82 leukaphereses was 86%. To assess the recovery of cryopreserved hemopoietic stem cells, the soft agar culture method adapted for the dog was used. There was no significant difference in the CFUc recovery per 1 × 106 mononuclear cells or in per leukapheresis after different Cryopreservation times (1–6 and 7–27 months).
To evaluate the hemopoietic repopulation capability of cryopreserved blood stem cells, leukapheresis-derived leukocytes were transfused into 1200 R whole body x-irradiated dogs. The hemopoietic repopulation pattern at day 10 after transfusion of comparable numbers of fresh or frozen leukocytes was not significantly different, as measured in bone marrow smears and sections and by granulocyte concentration in the peripheral blood.
Zusammenfassung
Es war das Ziel der Untersuchungen, eine Einfriermethode für hämopoetische Blutstammzellen zu etablieren. Blutleukozyten wurden mittels eines IBM-Blood-Cell-Separator gewonnen und in Gegenwart von 10% DMSO in Plastik-Beuteln eingefroren, wobei der Kühlprozeß vorprogrammiert war. Der Auftauvorgang dauerte nur wenige Minuten; anschließend wurde das DMSO schrittweise verdünnt, bevor die aufgetaute Zellsuspension für in-vitro wie in-vivo-Versuche verwendet wurde.
Die durchschnittliche Ausbeute an mononukleären Zellen von 82 Leukapheresen betrug 86%. Zur Beurteilung der Ausbeute von eingefrorenen hämopoetischen Blutstammzellen wurde die “soft agar culture” Methode in einer Modifizierung für den Hund verwendet. Die CFUc-Ausbeute pro l × 106 mononukleäre Zellen und pro Leukapherese war nach verschieden langer Einfrierdauer (1 bis 6 Monate und 7 bis 27 Monate) nicht signifikant unterschiedlich.
Die hämopoetische Repopulationsfähigkeit eingefrorener Blutstammzellen wurde mittels Transfusion in ganzkörperbestrahlte Beagles untersucht. Dabei zeigte sich, daß das hämopoetische Repopulationsmuster am 10. Tag nach Transfusion vergleichbarer Zahlen an frischen oder eingefrorenen Leukozyten nicht signifikant unterschiedlich war, wie anhand von Knochenmarkausstrichen und histologischen Knochenmarkschnitten sowie anhand der Granulozytenkonzentration im peripheren Blut nachgewiesen werden konnte.
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Fliedner, T.M., Körbling, M., Calvo, W. et al. Cryopreservation of blood mononuclear leukocytes and stem cells suspended in a large fluid volume. Blut 35, 195–202 (1977). https://doi.org/10.1007/BF00999460
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DOI: https://doi.org/10.1007/BF00999460