Recovery Of Mononuclear Cells Research Articles

A fully automated method for mononuclear bone marrow cell concentration

We describe our experience in processing 40 bone marrow aspirates harvested for autotransplantation from patients with several hematological diseases using the CS-3000 blood cell separator. The bone marrow of the first 30 patients was processed by a semiautomated method, and a fully automated procedure was used for the remaining 10 cases. Both procedures were developed in our laboratory and yielded a similar average mononuclear cell recovery of 87.78% and 86.98%, respectively, and similar nucleated cell recovery (27.39% and 27.11%). The cloning efficiency of hematopoietic progenitor cells, measured as the total CFU-GM colony recovery in the in vitro cultures, did not differ between processed and recovered mononuclear cells. On the other hand, all the patients with transplants showed complete hematologic recovery, and the time to engraftment was similar to that described for other procedures. The automated procedure resulted in an average red cell removal of 97.81%, similar to the semiautomated procedure (94.19%), though with a narrower range (96.31-98.6% vs. 80.34-98.34%). The time taken to process a similar amount of bone marrow cell suspension was very different for each method: 1 hour for the fully automated vs. 2 1/2 hours for the semiautomated method to process 1,000 ml. Furthermore, the semiautomated procedure required the addition of homologous or irradiated plasma in a laminar air flow chamber, while the automated method is performed in a closed sterile system. We conclude that our procedure using the CS-3000 processor is an efficient method for fully automated large-scale processing of human bone marrow cells.

169. Phase I and pharmacokinetic evaluation of all- trans retinoic acid in pediatric patients and pharmacokinetic evaluation in the nonhuman primate

An effective method for concentrating bone marrow is described. Concentration was achieved using an intermittent flow cell separator. Elimination of mature haemopoietic cells was enhanced by the addition of a density separation medium (Ficoll-metrizoate) which was then removed by washing. All procedures were undertaken using the cell separator, this allowed for standardization of procedure and less manipulation which is associated with enhanced mononuclear cell recovery and less risk of microbial contamination. Mature granulocytes were depleted by 86.8% and red cells by 97.7% whilst mononuclear cells showed a 49% recovery. Marrows processed in this way and subsequently purged and/or cyropreserved were shown to successfully engraft when reinfused.

Concentration of bone marrow mononuclear cells using a programmable blood cell separator

Bone marrow was collected from adult patients with various solid tumors who consented to participate in a study of myelo-ablative chemotherapy followed by autologous bone marrow rescue. Twenty marrow suspensions were processed by using standard Procedure 3 (PRO-3) for lymphocytapheresis without modification. A modified Procedure 1 (M-PRO-1) for plateletpheresis was employed for processing 34 marrow suspensions. For PRO-3, mononuclear cell (MNC) recovery was 68 +/- 22% of the starting marrow suspension (baseline), in a concentrate volume of 234 +/- 53 ml. MNC represented 59 +/- 27% of the total WBC count of the concentrate. The residual volume of RBC was 49 +/- 47 ml. For M-PRO-1, MNC recovery was 63 +/- 22% of the baseline in a concentrate volume of 200 +/- 8 ml. MNC comprised 94 +/- 7% of the total WBC count of the concentrate. RBC contamination was 7 +/- 3 ml. Hematopoietic recovery, defined as the post-transplant days when a sustained granulocyte count of 500/microL and a platelet count of 50,000/microL were achieved, was similar in the two groups and comparable to other reports utilizing other methods and equipment for bone marrow concentration. Personnel time was significantly reduced compared to other procedures for bone marrow concentration due to increased automation. Although there was no significant difference in MNC recovery between the two groups (P greater than 0.5), M-PRO-1 was clearly superior to PRO-3 because of the consistently high degree of purity of the MNC in the concentrate and minimal RBC contamination.(ABSTRACT TRUNCATED AT 250 WORDS)

Density gradient separation of peripheral blood stem cells: comparison of an automated cell processing device and manual methods

Peripheral blood stem cells were collected from normal donors by leukapheresis on a cell separator. The leukapheresis product contained 1.5 x 10(10) mononuclear cells (MNCs) and was divided into two aliquots that underwent either automated or manual density gradient separation with ficoll-hypaque and subsequent washing. In the automated process, recovery of MNCs was 85 percent, reduction in platelet content was 64 percent, and the final hematocrit (Hct) was less than 1 percent. The manual separation resulted in 76-percent MNC recovery, a 79-percent reduction in platelet content, and a final Hct of less than 1 percent. The purified MNCs were then placed in methylcellulose culture at a concentration of 4 x 10(5) MNCs per mL. Quadruplicate 1-mL aliquots were cultured, and colonies were counted and classified on Day 14. Comparison of automated and manual ficoll-hypaque separations demonstrated no differences in the total, erythroid, or granulocyte-macrophage colony numbers. The cell processor used is fast, reliable, uncomplicated, and provides a sterile product containing progenitor cells that are not adversely affected by the automated ficoll-hypaque separation.

Cryopreservation of equine mononuclear cells for immunological studies

A rapid and simple technique for the cryopreservation and recovery of equine mononuclear cells was developed. Buffy-coat leukocytes were frozen in autologous plasma containing 10% DMSO and mononuclear cells were recovered by gradient sedimentation using a standard Ficoll-Hypaque purification procedure. The total numbers of mononuclear cells recovered from cryopreserved samples were 94%–82% of those recovered from fresh blood samples. The functional capabilities of the mononuclear cells from cryopreserved buffy coat preparations were compared with those of mononuclear cells from fresh samples by measuring the ability of cells to proliferate in response to mitogens and specific antigens. Cell-surface antigen expression was measured using monoclonal antibodies in conjunction with flow cytometric techniques and alloantisera in a complement mediated cytotoxicity assay. Cryopreserved mononuclear cells were capable of proliferating normally when stimulated with several mitogens, pokeweed mitogen, phytohemagglutinin and concanavalin A, and a single specific antigen preparation, equine influenza-2 (Equi-2) proteins. The maximum levels of proliferation induced by varying the concentrations of mitogens or the Equi-2 proteins were the same for both the fresh and cryopreserved cells. However, the cryopreserved cells usually required one more day in culture to attain maximum proliferation levels. Flow cytometric analysis of the samples demonstrated that the relative proportions of different lymphocyte populations were not altered by the cryopreservation step. Similarly, MHS alloantigen expression was not altered. The simplicity of the technique coupled with the retained functional properties allows for the cryopreservation of large numbers of leukocytes and the ability to assay various immune functions at a later time.

The use of the Fenwal CS3000 cell separator to purify a mononuclear cell fraction from pooled buffy coats

Using a modified plateletpheresis program on the Fenwal CS3000, we have studied the recovery of mononuclear cells and clonogenic potential from pooled buffy coats. Recovery of mononuclear cells and CFU-GM was 90% and 67%, respectively. The final volume could easily be adjusted to 100 mL, and erythrocyte contamination was low (2.3%). This method should be readily applicable to bone marrow processing to achieve efficient and rapid separation for subsequent reinfusion, cryopreservation, and treatment with monoclonal antibodies.

A new technique for peripheral stem cell separation

Using a Haemonetics V50.1 cell separator we compared the new lymphocyte-surge-program for peripheral stem cell preparation with the conventional combined buffy coat and Ficoll techniques. Eleven surge and four conventional separations were performed in 15 healthy cytapheresis donors who gave informed consent to the procedure. Using the conventional methods, mean recovery of peripheral blood mononuclear cells (PBM) was 55% in a preparation time of 80 minutes for the buffy coat and 25 minutes for the Ficoll step. Eighty percent of the PBM loss occurred during the buffy coat step, and 20% during the Ficoll procedure. In a preparation time of 105 minutes, 2500 mL peripheral blood could be processed without flow or citrate problems. The donors' red cell losses were about 100 mL packed red cells. Granulocyte depletion was about 98%, red cell depletion higher than 99% (hematocrit 7 PBM) in both separation techniques. Therefore, both techniques seem to be adequate for T-cell depletion and preparation of bone marrow for cryopreservation. Since preparing bone marrow for purging techniques requires extreme stem cell purity, the surge technique may be less suitable for those purposes due to the awkward problem of considerably higher red cell contamination, despite a shorter preparation time.

Preparation and characterization of human gingival cells.

A procedure was developed for the preparation of single cell suspensions from gingival and periodontal tissues using collagenase digestion followed by gentle mechanical disruption. The procedure was evaluated on rat gingival tissues. One‐hour incubation was found to produce the greatest number of cells with the highest viability, and the largest recovery of mononuclear cells. Collagenase did not appear to effect recovery, viability or B cell surface markers on human peripheral blood cells. Human gingival and periodontal tissues were obtained from 35 patients: 20 with adult periodontitis; 8 with juvenile periodontitis; 7 with normal gingivae or chronic gingivitis. Scaling, followed by probing measurements and then surgery of the affected sites was routinely performed. In single cell suspensions from tissue obtained from surgery, significantly fewer mononuclear cells were recovered from the tissues of normal/chronic gingivitis patients than cells/mg of tissue recovered from adult periodontitis or juvenile periodontitis groups. The maximum contribution of blood mononuclear cells to the gingival cells averaged 0.5+0.2%. This method is useful for recovering gingival (periodontal) cells for phenotypic and functional studies.

Erythrocyte depletion of ABO-incompatible bone marrow.

Bone marrow transplantation results in the infusion of 150 to 360 ml of erythrocytes. While this poses no problem to a recipient of marrow from an ABO-compatible donor, it clearly represents a serious risk of intravascular hemolysis for the recipient of an ABO-incompatible marrow. Thus, there is a need for removing incompatible erythrocytes from the marrow preparation. We removed erythrocytes from the marrow preparations by automated centrifugation. The erythrocyte-depleted marrow (EDM) contained a mean of 5 ml erythrocytes, representing an average reduction of 98%. The mean mononuclear cell recovery was 88%, resulting in a mean infusion of 0.6 X 10(8) cells/Kg of recipient's body weight in a final average volume of 155 ml. EDM was infused into 22 ABO-incompatible marrow recipients (21 patients with hematologic malignancies and one patient with aplastic anemia) without clinical evidence of hemolysis. The isohemagglutinin titers of recipients ranged from 4 to 4096 and were not lowered prior to infusion. Engraftment (i.e., recovery of peripheral leukocyte and platelet counts) and incidence of graft versus host disease were similar to those observed in recipients of ABO-compatible marrow transplantation. Erythrocyte engraftment was significantly delayed in only one patient who had a high isohemagglutinin titer. The post-transplantation red cell requirement was increased in EDM recipients: 9 units compared to 6 units in ABO-compatible bone marrow transplanted patients with neither hemolysis nor interference with successful engraftment.

Méthode de concentration des cellules-souches médullaires utilisant le laveur IBM 2991: Préalable essentiel avant traitement in vitro de la moelle osseuse par des moyens pharmacologiques ou immunologiques

The technique using the IBM 2991 blood cell processor is an effective technique for the concentration of mononuclear cells from large volumes of bone marrow. The marrow cells are layered on to Ficoll Metrizoate using the IBM processing set. The mononuclear cells and CFU-GM recoveries are in close relationship with the hematocrit of the cell suspension processed. Twenty two bone marrows have been collected and purified according to this protocol. The mononuclear cell recovery is an average of 78,3% (range: 44-92%) and the CFU-GM recovery is in average of 67,5% (range: 40-89%). At the end of the procedure the cell viability is satisfying (97,1% +/- 1,7 are trypan blue negatives). When it is necessary to remove from the bone marrow collected either malignant cells prior autologous bone marrow graft or T lymphocytes in an attempt to prevent GVHD in allogeneic BMT, the purity of marrow cell suspension become a fundamental parameter.

Isolation and preliminary characterization of human intestinal macrophages

Methods for the isolation and culture of macrophages from normal human intestine are described. After disaggregation using sequential treatments of dithiothreitol, ethylenediaminetetraacetate, collagenase, and deoxyribonuclease, Percoll density gradients (1.064 SG) produced single cell suspensions from which macrophages were readily purified by adherence to plastic. Macrophages were characterized by morphology, phagocytosis, cytoplasmic staining for nonspecific esterase, presence of membrane Fc receptors, Ia-like antigens, and lysozyme synthesis and secretion. In 10 separate experiments, recovery of viable mononuclear cells was 2.9 ± 0.6 × 106 cells/g of mucosa. Thirty percent of these cells were phagocytic. After adherence to plastic, the macrophage recovery was 1.05 ± 0.2 × 106 cells/g mucosa and 90% ± 0.4% of the adherent cells in the monolayer were phagocytic. Fifty-five percent of the adherent cells showed Fc receptors for immunoglobulin G, while 94% expressed the Ia-like antigen on their membrane. The successful isolation and culture of human intestinal macrophages in large numbers will allow detailed study of their role in the mucosal immune response in health and disease.

A technique for the separation and cryopreservation of myeloid stem cells from human bone marrow

We have developed a technique for the cryopreservation of large volumes of human bone marrow, which reduces cell losses due to clumping and release of lysosomal enzymes from mature granulocytes. Mononuclear cells were separated from whole bone marrow by a large-scale Ficoll-Hypaque procedure. The agar colony assay for myeloid stem cells (CFU-C) was used to assess each step of the isolation and cryopreservation procedure. Conditions of varied cell and cryoprotectant concentrations and freezing and thawing rates were compared to obtain optimal recovery of mononuclear cells and CFU-C. This technique has been used to store bone marrow from 45 patients with hematologic and non-hematologic neoplasms. Up to 750 ml of marrow was obtained from each patient and separated by step-gradient centrifugation, and the cell fraction containing myeloid stem cells was cryopreserved. The mean recoveries following separation, cryopreservation, and thawing for 18 marrow storages from patients with hematological neoplasms were 8.8 ± 2.9% for mononuclear cells and 47.8 ± 20.8% for CFU-C. In comparison, values for 27 marrows from patients with non-hematological neoplasms were 14.5 ± 5.5% for cells and 57.7 ± 13.7% for CFU-C.

Strain-dependent cytotoxic effects of endotoxin for mouse peritoneal macrophages.

The cytotoxic effects of bacterial lipopolysaccharides (LPS) on mouse leukocytes have been examined in vivo and in vitro. Intraperitoneal injection of LPS into C57BL/6 mice greatly reduced the recovery of mononuclear cells; LPS was cytotoxic for macrophages, but had a mitogenic effect on lymphocytes. Similar effects of LPS on peritoneal leukocytes were observed in vitro. When monolayers of adherent peritoneal cells were studied in vitro, cytotoxicity was also observed, suggesting that the effect of LPS on macrophages is direct and does not require participation by lymphocytes. Entirely different results were obtained when peritoneal macrophages from LPS-resistant C3H/HeJ mice were studied. LPS failed to activate lymphocytes and was not cytotoxic for macrophages in vitro or in vivo. The effect of LPS on polymorphonuclear leukocytes appeared to be the same in all mouse stains studied. Lipid A was shown to be the most biologically active portion of the LPS molecule. Whereas polysaccharide-deficient endotoxins extracted from rough mutants of Salmonella typhimurium were cytotoxic for macrophages in vitro, polysaccharides that lacked esterified fatty acids did not exhibit this activity. Since LPS may mediate its effects through affinity for mammalian cell membranes, the cellular unresponsiveness of C3H/H3J mice to LPS may reflect an inability of cells from LPS-resistant strains to interact with LPS at the membrane level.

Cryopreservation of blood mononuclear leukocytes and stem cells suspended in a large fluid volume. A preclinical model for a blood stem cell bank.

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 X 10(6) 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.