Preservation of differentiation and clonogenic potential of human hematopoietic stem and progenitor cells during lyophilization and ambient storage.
Progenitor cell therapies show great promise, but their potential for clinical applications requires improved storage and transportation. Desiccated cells stored at ambient temperature would provide economic and practical advantages over approaches employing cell freezing and subzero temperature storage. The objectives of this study were to assess a method for loading the stabilizing sugar, trehalose, into hematopoietic stem and progenitor cells (HPC) and to evaluate the effects of subsequent freeze-drying and storage at ambient temperature on differentiation and clonogenic potential. HPC were isolated from human umbilical cord blood and loaded with trehalose using an endogenous cell surface receptor, termed P2Z. Solution containing trehalose-loaded HPC was placed into vials, which were transferred to a tray freeze-dryer and removed during each step of the freeze-drying process to assess differentiation and clonogenic potential. Control groups for these experiments were freshly isolated HPC. Control cells formed 1450+/-230 CFU-GM, 430+/-140 BFU-E, and 50+/-40 CFU-GEMM per 50 microL. Compared to the values for the control cells, there was no statistical difference observed for cells removed at the end of the freezing step or at the end of primary drying. There was a gradual decrease in the number of CFU-GM and BFU-E for cells removed at different temperatures during secondary drying; however, there were no significant differences in the number of CFU-GEMM. To determine storage stability of lyophilized HPC, cells were stored for 4 weeks at 25 degrees C in the dark. Cells reconstituted immediately after lyophilization produced 580+/-90 CFU-GM ( approximately 40%, relative to unprocessed controls p<0.0001), 170+/-70 BFU-E (approximately 40%, p<0.0001), and 41+/-22 CFU-GEMM (approximately 82%, p = 0.4171), and cells reconstituted after 28 days at room temperature produced 513+/-170 CFU-GM (approximately 35%, relative to unprocessed controls, p<0.0001), 112+/-68 BFU-E (approximately 26%, p<0.0001), and 36+/-17 CFU-GEMM ( approximately 82%, p = 0.2164) These studies are the first to document high level retention of CFU-GEMM following lyophilization and storage for 4 weeks at 25 degrees C. This type of flexible storage stability would potentially permit the ability to ship and store HPC without the need for refrigeration.
Buchanan
Sandhya S
SS
Center for Pharmaceutical Biotechnology and Department of Pharmaceutical Sciences, University of Colorado, Aurora, Colorado, United States of America. Sandhya.Buchanan@Merck.com
Pyatt
David W
DW
Carpenter
John F
JF
D002454:Cell Differentiation; D049109:Cell Proliferation; D005312:Fetal Blood / Q000166:cytology; D005612:Freeze Drying / Q000379:methods*; D055014:Granulocyte-Macrophage Progenitor Cells / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D023461:Myeloid Progenitor Cells / Q000378:metabolism; D013696:Temperature
Bone Marrow Transplant. 1998 Dec;22(11):1091-6
9877272
EMBO J. 1996 Aug 15;15(16):4174-81
8861946
Annu Rev Physiol. 1998;60:73-103
9558455
Biochem J. 2001 Oct 1;359(Pt 1):227-33
11563987
Appl Environ Microbiol. 1990 May;56(5):1386-91
2339891
Biochim Biophys Acta. 1987 Jan 20;923(1):109-15
2948571
Cryobiology. 2001 Mar;42(2):79-87
11448110
FEBS Lett. 2000 Dec 29;487(2):199-202
11150509
J Natl Cancer Inst. 1951 Aug;12(1):197-201
14874130
ILAR J. 2000;41(4):187-96
11123179
J Neurosci. 2001 Sep 15;21(18):7143-52
11549725
Cryobiology. 2001 Sep;43(2):168-81
11846471
Nat Biotechnol. 2000 Feb;18(2):168-71
10657122
J Pharm Sci. 2001 Jul;90(7):860-71
11458335
J Hematother Stem Cell Res. 2000 Apr;9(2):161-73
10813529
Diabetes. 1997 Mar;46(3):519-23
9032112
J Cell Sci. 1999 Feb;112 ( Pt 3):297-305
9885283
Cryobiology. 1995 Aug;32(4):366-78
7544712
Fertil Steril. 2002 Jan;77(1):152-8
11779606
Annu Rev Immunol. 2003;21:759-806
12615892
Blood. 1990 Feb 1;75(3):781-6
2297578
Nat Biotechnol. 2000 Feb;18(2):163-7
10657121
Cryobiology. 1988 Jun;25(3):244-55
3396389
Neuroreport. 1996 Nov 4;7(15-17):2533-7
8981418
Trends Neurosci. 1996 Aug;19(8):312-8
8843599
Blood. 2004 Jul 1;104(1):81-8
15016651
Cryobiology. 2003 Apr;46(2):121-34
12686202
Cryobiology. 1999 Sep;39(2):103-29
10529304
Arch Biochem Biophys. 1986 Nov 1;250(2):505-12
2946263
Mem Inst Oswaldo Cruz. 2000 May-Jun;95(3):415-28
10800201
Pharm Res. 1994 Jan;11(1):54-9
8140056
Am J Physiol. 1998 Dec;275(6):F962-71
9843914
J Cell Physiol Suppl. 1957 Dec;50(Suppl 1):103-8
13513681
Blood. 1997 Dec 1;90(11):4313-20
9373242
Biochem J. 1987 Feb 15;242(1):1-10
2954537
Cancer Biother Radiopharm. 1999 Feb;14(1):11-22
10850282
Am J Physiol. 1984 Sep;247(3 Pt 1):C125-42
6383068
J Clin Oncol. 1994 Oct;12(10):2176-86
7523609
Proc Natl Acad Sci U S A. 1989 Jan;86(2):520-3
16594011
Biochem J. 1986 Jan 15;233(2):309-19
3006665
Transfusion. 1991 Jul-Aug;31(6):521-6
1853447
Biochemistry. 1989 May 2;28(9):3916-22
2526652
Leukemia. 1998 Apr;12(4):563-70
9557615
Arch Biochem Biophys. 1993 Jun;303(2):456-64
8512328
Cryobiology. 1988 Aug;25(4):331-7
3409707
Nat Biotechnol. 1997 Mar;15(3):278-82
9062930
Transfusion. 2002 Dec;42(12):1573-80
12473137
Naunyn Schmiedebergs Arch Pharmacol. 2002 Feb;365(2):168-71
11819036
Transfusion. 2001 May;41(5):667-73
11346704
J Biol Chem. 2003 Nov 28;278(48):47554-61
12968021
J Biol Chem. 2003 Mar 7;278(10):8853-60
12496266
Cryobiology. 1999 Aug;39(1):80-7
10458903
J Clin Oncol. 2005 Sep 20;23(27):6699-711
16170178
J Biol Chem. 2003 Jan 17;278(3):1549-60
12424250
Biochim Biophys Acta. 1988 Jun 9;947(2):367-84
3285894
Immunol Today. 1995 Nov;16(11):524-8
7495489
Cryobiology. 2001 Sep;43(2):182-7
11846472
Biophys J. 1988 Sep;54(3):509-18
2462928
Blood. 2000 Aug 1;96(3):996-1005
10910915
Biochim Biophys Acta. 1992 Feb 19;1134(1):31-45
1311958
Biochim Biophys Acta. 1975 Oct 6;406(2):187-96
1191647
PLoS One. 2009;4(4):e5240
19381290
J Invest Dermatol. 2003 Nov;121(5):1145-9
14708618
J Hematother Stem Cell Res. 2001 Dec;10(6):725-38
11798499
J Biol Chem. 1987 Jun 25;262(18):8884-8
3597398
Arch Biochem Biophys. 1999 May 15;365(2):289-98
10328824
Stem Cells Dev. 2004 Jun;13(3):295-305
15186725
Cryobiology. 1978 Aug;15(4):382-90
81120
Bone Marrow Transplant. 1999 Mar;23(6):533-7
10217182
Br J Pharmacol. 1998 Nov;125(6):1194-201
9863647
Adv Food Nutr Res. 1995;38:103-269
15918292
Circ Res. 1998 Jul 27;83(2):196-203
9686759
Biochim Biophys Acta. 1996 Jan 12;1278(1):125-30
8611600
Neuropharmacology. 1997 Sep;36(9):1277-83
9364482
J Cell Physiol. 1989 Aug;140(2):323-34
2663885
Am J Physiol Cell Physiol. 2001 Apr;280(4):C943-53
11245611
Am J Physiol Regul Integr Comp Physiol. 2005 Feb;288(2):R501-10
15458972
Methods Cell Biol. 1989;31:45-61
2506408
J Biol Chem. 2004 Oct 22;279(43):44749-55
15304508
Blood. 2001 Feb 1;97(3):587-600
11157473
Blood. 1989 Feb;73(2):375-80
2537111
Cryobiology. 2005 Dec;51(3):290-305
16185682
Hematology. 2003 Oct;8(5):313-8
14530173
Arch Biochem Biophys. 1985 Oct;242(1):240-7
4051504
Science. 1996 May 3;272(5262):735-8
8614837
Cryobiology. 1987 Oct;24(5):455-64
2958239
Plant Physiol. 1991 Jun;96(2):660-3
16668237
Cryobiology. 2001 May;42(3):207-17
11578120
N Engl J Med. 1957 Sep 12;257(11):491-6
13464965
J Immunol. 2001 Jun 1;166(11):6754-63
11359833
Exp Hematol. 2001 Mar;29(3):309-14
11274758
J Immunol. 1997 Jun 1;158(11):5095-105
9164924
Ann N Y Acad Sci. 1999 Apr 30;868:716-29
10414359