Freeze-drying of mononuclear cells and whole blood of human cord blood

Authors: Xiao H-H.; Hua T-C.; Li J.; Gu X-L.; Wang X.; Wu Z-J.; Meng L.; Gao Q-R.; Chen J.; Gong Z-P.

Source: Cryoletters, Volume 25, Number 2, 1 February 2004 , pp. 111-120(10)

Publisher: Cryoletters

Abstract:

The research on haematopoietic stem cells of human cord blood has become more important recently. People have concentrated on the preservation of cord blood stem cells. At present, cord blood can be preserved at ultra-low temperatures. In this study, we try to preserve cord blood and its constituents by freeze-drying. The experiments on both the mononuclear cell content and the whole blood of human cord blood were carried out respectively. The samples were frozen firstly by different cooling protocols in the presence of PVP, sucrose, and mannitol. Afterwards, they were vacuum-dried at a selected shelf temperature of -30ºC for the main drying stage, and then vacuum-dried at 15ºC for the second drying stage. The entire time of the freeze drying was 52 hours. Samples were stored at room temperature for 2 days prior to evaluation. Subsequently, the dried samples were suspended in an isotonic phosphate-buffered saline solution.

The recovery of the cells were tested by a haemacytometer, and the highest cell numerical count recovery of MNC was 75.0 ± 4.1% (P<0.01), obtained in the protocol of 40% VP + 20% Sucrose + 10% Mannitol. The viability of the nucleated cells measured by PI staining and the ratio of the number of CD34⁺ to the number of lymphocytes (by the FITC anti-human CD34⁺ conjugated antibody method) were measured using a flow cytometer (FCM). The protocol of 40% PVP + 20% Sucrose + 10% fetal bovine serum had the highest viability of 98.57±0.68%(P <0.01). The highest ratio of CD34⁺ to lymphocytes was 1.17%, and the highest recovery of CD34⁺ was 68.42 ± 39.5% (P<0.05). Comparing the results of the lyophilized MNC subfraction with that of the whole blood, the lyophilization of the isolated MNC was more successful than that of whole blood.

Keywords: FREEZE-DRYING; HUMAN CORD BLOOD; MNC; LYOPROTECTANT; CD34+ CELL

Document Type: Research article

Publication date: 2004-02-01

More about this publication?

• CryoLetters is a bimonthly international journal for low temperature sciences, including cryobiology, cryopreservation or vitrification of cells and tissues, chemical and physical aspects of freezing and drying, and studies involving ecology of cold environments, and cold adaptation

  The journal publishes original research reports, authoritative reviews, technical developments and commissioned book reviews of studies of the effects produced by low temperatures on a wide variety of scientific and technical processes, or those involving low temperature techniques in the investigation of physical, chemical, biological and ecological problems.

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• By this author: Xiao H-H. ;  Hua T-C. ;  Li J. ;  Gu X-L. ;  Wang X. ;  Wu Z-J. ;  Meng L. ;  Gao Q-R. ;  Chen J. ;  Gong Z-P.

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