Viability (CFU-C recovery) of human frozen bone marrow: Frozen period, aging, and malignant diseases specificity

Viability (CFU-C recovery) of human frozen bone marrow: Frozen period, aging, and malignant diseases specificity

524 ABSTRACTS, 25th ANNUAL hypotonic stress test indicated a slight impairment. The viability of platelets after both freezing techniques was signi...

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524

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hypotonic stress test indicated a slight impairment. The viability of platelets after both freezing techniques was significantly reduced with respect to fresh platelets, but 1.4 M glycerol ensured slightly better preservation than the mixture. 45. Cryopreservation of Human Platelets: A Comparison of Different Viability Assays. A. SPUTTEK, CH. K~RBER, W. R. MAYR,* AND G. RAU (Helmholtz-Inst. filr Biomedizinische Technik an der RWTH Aachen, *Abtl. fur Transfusionsmedizin der medizinischen Einrichtungen der RWTH Aachen, Aachen, West Germany). Effective cryopreservation of platelets for clinical use requires a suitable freezing procedure. Although the hemostatic effectiveness in vivo is the decisive criterion for frozen/thawed platelets, reliable in vitro assays may be helpful to estimate their viability. The development of new freezing procedures, the investigation of different cryoprotectants, and the comparison of already existing procedures can be performed using viability assays before in vivo studies become feasible. The influence of different methods to obtain platelet-rich plasma (PRP), to store it either unfrozen or in the frozen state, and addition or removal of cryoprotective compounds have been investigated. A variety of parameters were evaluated including hypotonic stress test (HST), release of adenosinetriphosphate (ATP), cell number recovery (CNR), cell volume distribution (CVD), resonance thrombography as an in vitro stimulation of the clotting process (RTG), and stimulated aggregation with different agents (SAG). After the determination of standard values for unfrozen thrombocytes (n = 13), the influence of two common blood bank stabilizers (CPD-Al and ACD formula A) was investigated (a = 3). In addition the viability parameters mentioned above were measured for 3-day-old platelets, which had been obtained from a cell separator and were stored in conventional PVCbags at room temperature (n = 10) or cryopreserved (n = 7). The quality of these thrombocytes was compared to those prepared by fractionated centrifugation and cryopreserved with hydroxyethyl starch (HES) using a well-investigated freezing procedure (n = 32). When the results from the different viability tests were taken into account, a pronounced decrease of activity could be detected when 3-day-old platelets stored unfrozen were investigated. Platelets from fractionated centrifugation could be cryopreserved with better results than those obtained from a cell separator. 46. Functional and Immunological Characteristics of Cryopreserved Myeloid Leukemic Cells. A. LA&SE, L. CAMPOS,Z. H. SHI, J. P. BOURGEOT, D. GUYOTAT, AND A. EHRSAM (Centre de Transfusion Sanguine, Lyon, France).

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Banking myeloid leukemic cells seems to be useful in order to increase knowledge about differentation of this cellular population. Among 102 stored leukemias defined by the FAB cytological classification, some groups support liquid culture during several weeks, and others do not. We present a study concerning surface antigens and their functional characteristics after cryopreservation. A preliminary study (1) showed that viability estimated by vital dye exclusion is maintained after freezing. Correlation curves indicated that antigenie expression is not modified. Three main groups have been defined in culture related to their kinetics. Nevertheless, we did not find any correlation in these groups before and after freezing. These different results are presented and discussed. The proliferation peaks are decreased in 50%. In 40% cases, freezing stress is recovered in the first 2 days. All leukemias are tested in B-glucoronidase content, a lysosomal marker. At this time we have not found any relation between the enzyme content of the leukemia and the recovery after freezing. REFERENCE

1. Campos, L., et al. Cryobiology 25, 18-22 (1988). 47. Viability (CFU-C Recovery) of Human Frozen Bone Marrow: Frozen Period, Aging, and Malignant Diseases Specificity. S. SUMIDA, F. MORISHIGE,T. TOYOMASU,R. MIURA, AND R. MATSUKI (Department of Cardiovascular Surgery, Ntl. Fukuoka Ctr. Hospital, Fukuoka, Japan). Bone marrow was collected from 216 patients with malignant advanced unresectable cancer by aspiration before megadose anticancer chemotherapy. In a series of 289 collection procedures the mean volume of heparinized marrow was 510 f 190 ml with a mean of 8.9 + 5.1 x 10’ nucleated cells. Ficoll-Hipaque density gradients were used for the separation of marrow cells. The packed marrow cells were cryoprotected in 10% Me,SO in TC-199 medium and frozen in liquid N, for 30 to 68 months. The thawed marrow was diluted with the same volume of TC-199 in 5 min and centrifuged at 4000g to remove the supematant. The marrow cells were resuspended in 50 ml of TC-199 and infused into the circulation of original patients. A small sample of those resuspended marrow cells was used to test the viability of CFU-C recovery. CFU-C assay was performed by the methylcellulose method, in which colony-stimulating factors produced in large quantities from the conditioned medium of a thyroid cancer cell line were used. The results were (1) CFU-C recovery decreased little during 68 months, (2) CFU-C was mostly normal even in those patients 80 years old, (3) CFU-Cs of advanced cancer of the alimentary canal were the worst, and the recovery decreased less than

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0.1% in comparison with that of 0.2-0.6 of others: ovarian, brain, pharyngo-laryngial cancer, etc., (4) 100% of human liquid nitrogen frozen bone marrow showed colony growth in methylcellulose medium under the stimulation of CSF from a squamous cell carcinoma of the thryroid gland (2). Our conclusions are any human bone marrows frozen in liquid nitrogen show CFU-C recovery; however, the colony growth is weak. This is an important finding when we treat those patients with high-dose anticancer chemotherapy combined with frozen autologous bone marrow transplantation. REFERENCES 1.

Sumida, S., et al. High-dose chemotherapy with frozen autologous bone marrow transplantation in patients with poor-prognosis tumors. Japan J. Clin. Oncol. (Suppl. 1) 149, 553-562 (1984).

2. Okabe, T., et al. Establishment and characterization of a human colony-stimulating factorproducing cell line from the squamous cell carcinoma of the thyroid gland. J. Natl. Cancer Inst. 69, 1235-1243 (1982). 48. Effects of Murine Multi-CSF (IL-3) and Cryopreservation on Bone Marrow and Hematopoietic Stem Cells in Murine. J. YANG, B. L. Ltu,

J. M. TANG, AND Y. Qu TANG (Department of Histology and Embryology, Beijing Medical University, Beijing, China). Multipotential colony-stimulating factor, also known as interleukin-3 (IL-3), is a regulatory glycoprotein able to stimulate self-generation of bone marrow (BM) by stem cells and various progenitor cells. Both IL-3 and cryopreservation of BM and stem cells are very important for those patients who suffer from hematopoietic deficiency and need BM transplantation. In our experiments, we have studied the effects of both cryopreservation and IL-3 on BM and stem cells of LACA mice by means of viability, CFU, cytochemistry, and ultrastructure. The cells of BM were suspended in RPM1 1640containing 15% FBS and 10% DMSO as protective agent. Cryopreservation was performed with the aid of a program Cell Freezer (Planer R-204, England) and Freezer Apparatus (YDS-Series, Chendu Liquid Nitrogen Vessel Factory, China). The cells were thawed in a 40°C water bath. The procedure gives a viability rate of 86%. The frozen cells showed no change in comparison with controls in the histochemistry of peroxidase, AcP, ANAE, glycogen, and DNA. Our results from CFU-S, CFU-E, and GMCFU techniques indicated that cryopreserved stem cells had retained the abilities of active proliferation and differentiation as well as self-replication compared with their normal counterparts. The cryopreserved hematopoietic stem cells were incubated with IL-3 for

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2a8 hr. Then assays of progenitor cell and CFU-S were performed. The spleens from mice injected with cell suspension treated with IL-3 showed a 70% increase in CFU-S. Two- to fivefold increases were observed in GM-CFU and CFU-E. There were many more hematopoietic islets and erythroblastic islets in spleens of the IL-3 group than that in the control under LM and EM. The significant tendency of ditferentiation was observed in the cell suspension treated with IL-3. The results indicate that IL-3 and cryopreservation should be useful in stimulating hematopoietic repopulation and function in vivo. SESSION

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49. Effects of Suljhydryl Group Reagents and Freezing on Human Erythrocyte Membranes. GH.

BENGA (Department of Cell Biology, Faculty of Medicine, Cluj-Napoca, Romania). A comparison was made of the appearance of freeze-etched membranes of control erythrocytes and erythrocytes following incubation with the sulfhydryl reagents. It was found that on many of the control and N-ethylmaleimide (NEM)-treated cells, small (W-100 nm) elevated patches and etch faces are devoid of any intramembrane particles (IMPS). In some cases the patches span the fracture/etch face boundary. The patch elevations were never observed in the mambranes of p-chloro-mercuribenzene sulfonate (PCMBS)-treated cells. In those fracture faces of control and NEM-treated cells which showed the presence of the patch elevations, no effect on the density or particle diameter of the IMPS could be detected. It must be stated however that the relative area of the patch elevations is so slow that it is not possible to draw any conclusions regarding changes in particle density in the membrane arising from the creation or insertion of the nonparticle regions. The patch elevations were also present in freeze-fracture samples of control cells incubated at a more acid pH value. The observations raise two interesting questions. The elevated IMP-free patches on freeze-etched erythrocyte membranes represent membrane-associated single, or multilamellar, vesicles. Vesicle formation by “slow” freezing seemed to be promoted by prolonged exposure of the cells to the concentrated extracellular solution resulting from extracellular ice crystal formation. It is known that erythrocyte vesiculation can be induced by a variety of conditions. However, such vesicles are always characterized by the absence of cytoskeleton proteins. From the present results we suggest that mercurials such as PCMBS inhibit the vesiculation process by their effect on the cytoskeleton network. 50. Differential

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