MiniMACS System Research Articles

Is Acteoside Effects on Colon Cancer Stem Cells Via Inflamation or Apoptosis?

AIM: Colon cancer is one of the main health problems worldwide. Cancer stem cells (CSCs) are referred to as tumor-initiating cells involved in tumor heterogeneity and dormancy. CSCs can cause drug resistance, metastasis, and recurrence of primary and metastatic cancers. The interactions and survival trends of colon cancer stem cells with other cells may be an alternative route for effective treatment. In our study, we aimed to evaluate the effects of asteoside on stem cell properties, apoptotic and inflammatory processes in primary (HCT-116) and metastatic (Colo-741) colon CSCs. METHERIALS AND METHODS: CSCs were obtained from both types of colon cancer cell lines with the MINIMACS system using the anti-CD133 reagent. Metastatic Colo-741 and non-metastatic HCT-116 CD133+ and CD133- cells were cultured with or without Acteoside for 48 hours. Expressions of Caspase-3, Bcl, Bax, and Fas-L for apoptosis, and IL-1β, TNF-α, IL-6, IL8 and IL-10 for inflammation were analyzed by indirect immunocytochemistry technique by performing H-Score. Changes in cell morphology were examined under an inverted microscope. RESULTS AND CONCLUSION: It was observed that Oct-4 expression decreased after acteocyte administration in both metastatic and non-metastatic colon cancer cells. In addition, Colo-741 increased the intensity of the Bax/Bcl ratio in both CD133+ and CD133- cells and increased caspase 3 expression. While acteoside did not immunohistochemically affect inflammation in metastatic COLO-741 cells, it contributed to the apoptotic process by changing the Bax/Bcl ratio. When the morphology of the cells was evaluated, it was observed that the number of apoptotic cells increased in COLO-741 CD133+ and CD133- cells.

Boswellia sacra essential oil manages colon cancer stem cells proliferation and apoptosis: a new perspective for cure

ABSTRACT Boswellia sacra Flueck essential oil is obtained from frankincense obtained from Boswellia sacra trees. The aim of this study was to investigate the in vitro anti-cancer activity of Boswellia sacra (BS) essential oil in CD133+ and CD133- Colo-320 cells. Essential oil was analyzed by GC-MS. CD133+ cells, cancer stem cells, were obtained from the Colo-320 by the MiniMACS system. Cytotoxicity were evaluated with MTT assay. Anti-cancer activity of BS essential oil was investigated by immunocytochemistry. 41 compounds were identified in the essential oil. 1:2 dilution BS essential oil was found to be active against all the cells for 24 h incubation. β-catenin immunoreactivity was significantly decreased in both BS essential oil-treated CD133+ and CD133- Colo-320 cells. Caspase-3 immunoreactivity significantly increased in BS essential oil-treated CD133- Colo-320 cells. We conclude that BS essential oil decreased β-catenin signaling molecules which play a crucial role in cancer cell proliferation. BS essential oil promoted apoptosis in CD133- Colo-320 cells.

The Effect of Resveratrol and Quercetin on Epithelial-Mesenchymal Transition in Pancreatic Cancer Stem Cell

Resveratrol and quercetin are phytochemicals that are found in a variety of plants. The aim of this study was to investigate the effect of resveratrol and quercetin on epithelial-mesenchymal transition (EMT) of CD133+ and CD133− pancreatic cancer cells. Cancer stem cells (CD133+ cells) were obtained from the PANC-1 cells by the MiniMACS system. CD133+ and CD133− PANC-1 cells were treated with different concentrations (5, 10, 25, 50, and 100 µM) of resveratrol and quercetin. Cell growth and cytotoxicity were evaluated by MTT assay. Anticancer and anti-metastatic properties of resveratrol and quercetin were determined by immunocytochemistry using antibodies (ACTA-2, IL-1β, N-cadherin, TNF-α, and vimentin). The immunostaining intensity of CD133+ cells was stronger than CD133− cells. ACTA-2, IL-1β, and N-cadherin immunoreactivities were significantly decreased, whereas TNF-α and vimentin immunoreactivities significantly increased in quercetin-treated CD133+ cells. Moreover, N-cadherin and TNF-α immunoreactivities significantly decreased in resveratrol-treated CD133+ cells. The reduction in N-cadherin and ACTA-2 immunoreactivities was higher than the increase in vimentin immunoreactivity, quercetin could prevent EMT to a greater extent than resveratrol in pancreatic cancer stem cells because of the reduced expression of N-cadherin. Quercetin could be more effective in inhibiting metastasis compared to resveratrol.

SPARC promotes the development of erythroid progenitors

Secreted protein acidic and rich in cysteine (SPARC) is a well-recognized regulator that affects cell proliferation, differentiation, and survival. Here, we investigated the impact of SPARC on erythropoiesis. Erythropoiesis in bone marrow (BM) from SPARC(-/-) mice was analyzed by flow cytometry and colony-forming assays. The mechanisms that affected erythropoiesis were investigated by BM transplantation experiments. CD34(+) cells from umbilical cord blood were isolated by MiniMACS, and the effect of SPARC on human erythropoiesis was assessed by colony-formation assay and erythroid differentiation culture. The hemoglobin level in peripheral blood (PB) was lower in SPARC(-/-) mice. Neither red blood cell count in PB nor the percentage of Ter119(+) erythrocytes in BM showed significant difference between SPARC(-/-) and WT mice. However, the ability of SPARC(-/-) bone marrow cells (BMCs) to form erythroid burst-forming units (BFU-E), as well as spleen colony-forming units (CFU-S8), was significantly decreased. The addition of exogenous SPARC could promote the formation of BFU-E from SPARC(-/-) BMCs in vitro. And the impaired ability of SPARC(-/-) BMCs to form BFU-E could be restored by transplanted into WT BM microenvironment, whereas the BFU-E formation of WT BMCs was impaired after transplanted into SPARC(-/-) recipients. Furthermore, exogenous SPARC promoted umbilical cord blood CD34(+) cells to form erythroid colonies and the hemoglobinization of erythroid, but did not affect the expression levels of glycophorin A and CD71. In conclusion, our results indicate that SPARC promotes the development of erythroid progenitors, but does not affect terminal erythroid differentiation.

CD34(+) stem cells from umbilical cord blood.

We describe the relation between umbilical cord clamping time and two different enrichment system of CD34+ stem cells from umbilical cord blood with the proliferative ability and bone marrow reconstitution of the stem cells obtained. After an obstetrician performed the cord blood collection, the purification of stem cells was performed either with a combination of monoclonal antibodies (negative selections) using the Stem Sep method, or with a positive cells selection based on their surface CD34 antigens using the Mini Macs system. An excellent recovery of haematopoietic progenitors [Burst Forming Unit Erythroids (BFUE); Colony Forming Unit Granulocytes and Macrophages (CFU-GM); and Colony Forming Unit Granulocytes, Erythroids, Monocytes and Macrophages (CFU-GME)], inversely related to the increase in clamping time, was performed with the Mini Macs system (54% of colonies, with 90% purity). With Stem Sep method, haematopoietic progenitor's recovery was 35% (with 80% purity). By applying early clamping of umbilical cord blood we obtained a greater number of CD34+ cells and their clonogenic activity was increased with enrichment. This is a useful technique considering that the number of CD34+ stem cells usually contained from a unit of placental blood is enough for the transplant to a child, but not for an adult. Thus, using these methods, we can get a larger number of CD34+ stem cells which reduces the risk of Graft versus Host Disease also in adult patients, producing survival rates similar to those obtained with transplantation of bone marrow from unrelated donors.

Deficient Th-1 Responses from TNFα-Matured Dendritic Cells.

INTRODUCTION: The ultimate success of dendritic cell (DC) vaccination for the active immunotherapy of neoplasia involves a wide variety of variables including the method of DC generation, loading, and maturation; the DC dose and route of administration. A maturation cocktail comprising IL-1b, TNF-a, IL-6, and PGE2 (ITIP) is commonly employed clinically, as is the use of the single inflammatory cytokine TNF-a. Here we compared these two methods with respect to DC maturation, phenotype, and function.METHODS: CD14+ monocytes were isolated from normal donor G-CSF-mobilized peripheral blood progenitor cells using the mini-MACS system (Miltenyi Biotec), and cultured for 6 days in AIM-V medium containing GM-CSF and IL-4 (Cellgenix). Immature DCs were loaded with both acute myelogenous leukemia (AML) tumor lysate and mRNA as previously described1 and then matured with the ITIP cocktail, TNF-α alone, or a combination of TNF-α and CD40 agonism (Becton-Dickinson). Mature DCs were analyzed phenotypically and then cultured with autologous T-cells.RESULTS: Data generated from three different normal donors indicated that DCs matured with TNF-α were deficient in important costimulatory surface molecules CD80, CD83, and CD40 as well as in IL-12 production (p=0.001 in representative experiment). Analysis of T-cell functionality by IFN-γ ELISpot also demonstrated greatly decreased Th-1 functionality among lymphocytes stimulated by TNFα-matured DCs in comparison to ITIP-matured DCs (p=0.004 in representative experiment). Additionally, lymphocytes stimulated by TNFα-matured DCs were not able to proliferate significantly whereas ITIP-matured DCs mediated robust lymphocyte expansions consisting of up to 70% CD8+ cells. The addition of a CD40 agonist antibody to TNF-α during the DC maturation process could partially, but not completely overcome these observed functional deficiencies.CONCLUSIONS: The data suggest that DCs loaded with primary tumor antigens and then matured with TNF-a might be deficient in the induction of type 1 T-helper responses. The absence of critical costimulatory markers and IL-12 secretion suggest that DCs matured with TNF-α might be suboptimal for the priming of naïve CD8 responses. These findings might help to improve the clinical efficacy of future DC vaccination studies.

Characteristics of Ex Vivo expansion of endothelial progenitor cells

The characteristics for the ex vivo expansion of the endothelial progenitor cells (EPCs) were explored. CD34+ cells were selected from umbilical cord blood mononuclear cells (MNC) by MiniMACS system, expanded under the same conditions as those for total MNC, coincubation of CD34+ and CD34- from the same donor for EPCs. In addition, the effects of vessel endothelial growth factor (VEGF) and passage on cell differentiation, expansion kinetics and apoptosis were examined. EPCs were determined and quantified by immunocytochemistry and flow cytometry. The results showed that both coculture of CD34+ and CD34- and total MNC led to a significant increase in the expansion of CD34+ cells as compared with CD34 enrichment (P < 0.05). There was a tendency toward decreased apoptosis in cultures when early passage was performed immediately after cord like structures appeared. VEGF had no significant effect on apoptosis (P > 0.05). These differentiated EPCs were positive for CD34+, von Willebrand factor (vWF), KDR, CD31 staining and phagocytized acetylated low-density lipoprotein (LDL). CD34+ cells accounted for (68.2 +/- 6.3)% of attaching (AT) cells at day 7 of culture. It was suggested the most efficient method to ex vivo expansion of EPCs was coculture of CD34+ and CD34- or total MNC. Early passage makes cell apoptosis rate decrease. VEGF had no significant effect on ex vivo expansion of EPCs.

Comparison of two immunomagnetic separation technologies to deplete T cells from human blood samples

The objective of this study was to compare the performance of two immunomagnetic separation technologies to deplete T cells from buffy coats of human blood. Specifically, two versions of the commercial MACS(R) Technology: MiniMACS and SuperMACS, and a prototype, flow-through system, the QMS, were evaluated. Peripheral blood mononuclear leukocytes (PBL) were isolated from buffy coats and an immunomagnetic separation of CD3(+) cells was conducted using company and optimized labeling protocols. To mimic peripheral blood containing bone marrow purged hematopoietic stem cells, HSC, CD34 expressing-cells (KG1a) were spiked into PBL prior to T-cell depletion once optimized depletion conditions were determined. Once the labeling protocol was optimized, the MiniMACS system performed well by producing a highly enriched CD3(+) fraction, and a respectable level of depletion of T cells and recovery of KG1a cells in the depleted fraction; an average log(10) depletion of T cells of 2.88 +/- 0.17 and an average recovery of the KG1a cells of 60.8 +/- 5.94% (n = 14). The performance of the SuperMACS system was very similar with an average log(10) depletion of T cells of 2.89 +/- 0.22 and an average recovery of KG1a of 63.1 +/- 8.55% (n = 10). In contrast, the QMS system produced an average log(10) depletion of T cells of 3.98 +/- 0.33 (n = 16) with a corresponding average recovery of 57.9 +/- 16.6% of the spiked CD34+ cells. The aforementioned QMS performance values were obtained using sorting speeds ranging from 2.5 x 10(4) to 1.7 x 10(5) cells per second. It is suggested that the lack of a 100% recovery of the unlabeled KG1a cells is the result of a previously reported "drafting" phenomena which pulls unlabeled cells in the direction of the magnetically labeled cells thereby resulting in loss of the unlabeled cells.

Multipotentiality of CD34- Cells Extracted from Human Cord Blood

Purpose: We evaluated the differentiation potential of cells expanded from human cord blood into several differentiated cells, namely, osteoblasts, chondrocytes and adipocytes. Materials and Methods: cells, extracted from cord blood and isolated using a MiniMACS system, were cultured. Cells labeled with appropriate antibodies were analyzed by FACScan. The phenotypes of adipogenic and osteogenic differentiation were evaluated by Oil Red O, alkaline phosphatase, and von Kossa staining. Chondrogenic differentiation was evaluated by RT-PCR using primers for aggrecan, collagen types I, II and X. Results: cells showed a fibroblast-like morphology and expressed CD105, CD29, and CD44 antigens. These cells showed the deposition of a mineralized matrix and the expression of alkaline phosphatase in the osteogenic medium, and stained with Oil Red O in the adipogenic medium. In terms of chondrogenic differentiation, the expressions of aggrecan, and collagen types II and X showed a gradual increase, whereas the expression of type I collagen gradually decreased. Conclusion: Based on their differentiation potentials in at least three different tissue types, i.e., fat, bone, and cartilage, cord blood-derived cells can be visualized as attractive target cells for cellular or gene transfer therapeutic options.

Impaired stem cell function of CD34+ cells selected by two different immunomagnetic beads systems.

We have been investigating the hematopoietic stem cell (HSC) activity of peripheral blood-derived CD34(+) cells selected by two different laboratory immunomagnetic beads systems (MiniMACS and Isolex 50). In this study, the quality of purified CD34(+) cells was directly compared using clonal cell culture, a cobblestone area-forming cell (CAFC) assay, and an in vivo severe combined immunodeficiency (SCID)-repopulating cell (SRC) assay. It was found that CD34(+) cells selected by these two immunomagnetic methods showed a reduced yield of colony-forming cells and CAFCs compared with cells enriched by the StemSep device (a negative selection method). However, these CD34(+) cells still showed significant SRC activity, including multilineage lymphomyeloid reconstitution. The percentage of human CD45(+) cells in murine bone marrow after transplanting 5 x 10(5) CD34(+) cells selected by the Isolex 50 was significantly lower than after transplanting cells selected by the MiniMACS or the StemSep. Our findings clearly demonstrated that CD34(+) cells selected by the MiniMACS system had superior HSC functions, including SRC activity, compared with cells separated by the Isolex 50 system. More detailed functional analysis of immunomagnetically separated CD34(+) cells may provide useful knowledge for basic research on HSCs as well as for clinical HSC transplantation.

TWO CD34 + STEM CELLS FROM UMBILICAL CORD BLOOD ENRICHMENT METHODS

The authors describe the relation between clamping time and blood volume collected, and two enrichment systems of CD34 + stem cells from umbilical cord blood, to determine an excellent recovery with high proliferate ability and bone marrow reconstitution. After an obstetrician-based cord blood collection, the purification of stem cells was performed either with a combination of monoclonal antibodies (negative selections) using the Stem Sep method, or with a positive cells selection thanks to their surface CD34 antigens, using the Mini Macs system. An excellent recovery of hematopoietic progenitors, burst-forming unit erythroid, colony-forming unit granulocyte and macrophage, and colony-forming unit granulocyte, erythroid, monocyte, and macrophage, inversely related to the rising of clamping time, was performed with the Mini Macs system (54% of colonies, with a 90% purity), while with Stem Sep method, hematopoietic progenitor recovery was 35% (with an 80% purity). By applying early clamping of the umbilical cord blood a greater number of CD34 + cells was obtained and their clonogenic activity increased with enrichment. This is particularly useful, considering that the number of CD34 + stem cells contained in a unit of placental blood is enough for transplanting to a child, but not for an adult engraftment. Thus, using this method, a larger number of CD34 + stem cells can be obtained, which increases the possibility to reduce graft versus host disease also in adult patients, producing survival rates similar to the ones obtained with transplantation of bone marrow from unrelated donors.

Early clamping of umbilical cord blood and foetal CD34 enrichment.

We describe the relation between clamping time and blood volume collected, and two enrichment systems of CD34+ stem cells from umbilical cord blood, in order to determine the criteria for an excellent recovery with high proliferative ability and bone marrow reconstitution. After an obstetrician-based cord blood collection, the purification of stem cells was performed either with a combination of monoclonal antibodies, negative selections, using the Stem Sep method, or with a positive cells selection owing to their surface CD34 antigens, using the Mini Macs system. An excellent recovery of haematopoietic progenitors--burst forming unit erythroid; colony-forming unit granulocyte and macrophage; and colony-forming unit granulocyte, erythroid, monocyte, and macrophage--inversely related to an increase in clamping time, was achieved using the Mini Macs system (54% of colonies with a 90% purity), while the recovery of haematopoietic progenitors was 35% (with a 80% purity) using the Stem Sep method. By clamping umbilical cord blood at an early stage, we obtained a greater number of CD34+ cells, and their clonogenic activity increased with enrichment. This is particularly useful considering that the number of CD34+ stem cells contained in a unit of placental blood is sufficient for transplanting into a child but not for an adult engraftment. Thus, using these methods, we obtain a larger number of CD34+ stem cells. This increases the possibility of reducing graft versus host disease in adult patients and produces survival rates similar to the ones we observed in transplantation of bone marrow from unrelated donors.

Preparation of PEG-grafted immunomagnetoliposomes entrapping citrate stabilized magnetite particles and their application in CD34+ cell sorting

Immunomagnetic systems have been used for positive selection of a cell fraction from a mixture using appropriate surface markers with satisfactory results, as haematopoietic CD34+ cells. This work reports on the development of poly(ethylene glycol)-grafted (PEG) immunoliposomes loaded with citrate-magnetite stabilized particles as the separation vehicles for immunomagnetic separations. The magnetic ferrofluid was encapsulated into PEG-liposomes by the DRV methodology. The magnetoliposomes had a liposomal size of approximately 450 nm and a Fe/lipid molar ratio of 1.52+/-0.26, and were retained in the magnetic field created by the MiniMACS system. Anti-CD34 immunomagnetoliposomes with 100 mAb/vesicle were prepared by coupling the My10 mAb and bound specifically for CD34+ KG-1a cells in culture and in mixtures with CD34-cells (CHO or Jurkat). The magnetic cell sorting was carried out in cell mixtures KG-1a/CHO or KG-1a/Jurkat with different initial% of CD34+ Kg-1a cells. For 10(6) positive cells and 100 microM of immunomagnetoliposomes, the capture efficiency was > 85% and independent of the starting percentage of CD34+ cells. The decrease of the final purity, when the starting percentage of CD34+ cells decreases and, dependent of the CD34- cell line used, point to the degree of non-specific cell binding of My10-immunomagnetoliposomes as being crucial, among of the methodological aspects as the number of starting CD34+ cells. The CD34+ cells isolated retained the viability with an estimated recovery of 45-50%.

Clonogenic ability and apoptosis resistance are higher for CD34+ than for CD34− blasts in aml

Many cellular and functional characteristics of acute myeloid leukemia (AML) blasts have been correlated with clinical parameters; these include spontaneous apoptosis and response to chemotherapy (Smith et al., Br J Haematol, 1998, 102:1042; Wuchter et al., Leukemia, 1999, 13:1943). Since there is accumulating evidence that the leukemogenic cell in AML resides in the CD34+ cell population, CD34+ and CD34− AML cells were separated using the MiniMACS system and evaluated for a) clonogenic ability in the CFU-L assay and b) apoptosis resistance in a serum-free culture system. Initial percentage of CD34+ ranged between 4 and 88%. After correction for impurities in both fractions, the clonogenic ability of CD34− blasts in 10/17 samples was 0, while CD34+ blasts had a median of 547 CFU-L per 106 cells (range 71–3673). In 6/17 cases, both fractions had clonogenic ability, of which the CD34+ fraction had the highest in 4 cases; in 1/17 cases both fractions had no clonogenic ability. Spontaneous apoptosis was measured in 14 cases during 6 hours using Annexin-V/7-AAD and Syto16/PSC833/7-AAD, a sensitive method to detect early apoptosis (Schuurhuis et. al., Blood, 1998, 92:suppl 1:121a). Compared to the CD34+, the CD34− fraction displayed a significantly higher median value of spontaneous (early and late) apoptosis (mean % CD34−/ mean % CD34+ at t = 0 h: 32/19, p = 0.003; at t = 2 h: 36/22, p = 0.005; at t = 6 h: 42/27, p = 0.021). These results show that in a CD34+ AML, the CD34+ compartment differs from the CD34− compartment concerning at least spontaneous apoptosis and clonogenic ability. This should be considered when correlating any blast cell property with clinical outcome.

A novel method of cell separation based on dual parameter immunomagnetic cell selection

We describe a novel method of cell purification involving two stage immunomagnetic selection which permits isolation of cells based on a second cell surface marker without the need for removal of beads used in the first selection step. This approach takes advantage of the size differences between commercially available immunomagnetic beads and/or particles and their differing properties in terms of attraction to magnetic fields of various strengths. The first stage of separation involves positive selection of cells using the Miltenyi MiniMacs system, utilising 50 nm MicroBeads and a MiniMacs magnet. Cells obtained from this procedure—still rosetted with 50 nm MicroBeads—can then be subjected to further positive or negative selection using either streptavidin M280 or anti-rat M450 Dynabeads, without the need for prior bead removal, since the strength of the magnetic field of the Dynal separator is sufficient to attract the larger Dynabeads but not the MicroBeads. Here, we show that this system can be used to isolate a number of cell types including very rare target cell populations such as haemopoietic stem cells, using two different surface markers without perturbing subsequent functional capacity.

Quantitative immunophenotypic characterization, cryopreservation, and enrichment of second- and third-trimester human fetal cord blood hematopoietic stem cells (progenitor cells)

Objective: The aims of this study were (1) to assess the hematopoietic stem cell (progenitor cell) contents of umbilical cord blood samples from second-trimester and early-third-trimester fetuses versus term fetuses and (2) to determine the feasibility of cryopreservation and enrichment of cord blood from fetuses of different gestational ages. Study Design: Cord blood between 13 and 42 weeks’ gestation (n = 31) was sampled after delivery or fetal expulsion. Fluorescence-activated cell sorting was used to measure CD34 + and CD34 + CD38 – cell numbers. Samples were cryopreserved with 10% dimethylsulfoxide, and CD34 + enrichment was performed by magnetically activated cell sorting with the MiniMACS system (Miltenyi Biotech, Bergisch Gladbach, Germany). Kruskal-Wallis analysis of variance and the Mann-Whitney U test were used for analysis of data. Results: CD34 + and CD34 + CD38 – cell contents were significantly higher in second- and early third-trimester fetuses than in term fetuses (CD34 + 2.57% ± 0.38%, 1.48% ± 0.31%, and 0.7% ± 0.13%, respectively, P = .0067; CD34 + CD38 – 0.72% ± 0.26%, 0.18% ± 0.05%, and 0.06% ± 0.02%, respectively, P = .0132). Mononuclear cell recovery, viability, and CD34 + cell purity after cryopreservation and enrichment were similar among different gestational ages. Conclusion: Cord blood stem cell content decreases significantly from the second trimester to term. Cryopreservation and enrichment of these cells from earlier gestational ages is feasible. This might be especially useful for allogeneic stem cell transplantation and for in utero gene therapy. (Am J Obstet Gynecol 1998;179:1228-33.)

Combined negative and positive selection of mobilized CD34+ blood cells

We tested four negative and two positive selection methods for separation of CD34+ cells from mobilized blood cells, and analysed fold-enrichment, purity and recovery of CD34+ cells after selection procedures. The elimination of mature CD34- cells was achieved by adhesion to nylon-wool fibre (5.9 +/- 1.0 mean fold-enrichment and 65.2 +/- 2.3 mean recovery of CD34+ cells). Standard or modified Ficoll-Hypaque and Percoll density gradients, as well as phagocytosis with magnetic beads, were less effective in eliminating CD34- cells, both purity and fold-enrichment of CD34+ cells being lower than those obtained with separation by nylon-wool. Both positive selection methods tested. Ceprate and MiniMacs System, generated highly purified CD34+ cell populations ranging from 80% to 90%. The recovery of CD34+ cells was optimal with MiniMacs (77.9 +/- 3.6) and low with Ceprate (28.8 +/- 2.8). Based on these results, in two large-scale experiments we combined nylon-wool fibre and MiniMacs System in a two-step separation procedure obtaining a 36.9 +/- 2.6 mean fold-enrichment and a 50.5 +/- 0.3 mean recovery of CD34+ cells. In this way we achieved optimal enrichment and recovery of CD34+ cells, with a substantial saving of cost compared to either selection method alone.