Persistence of murine myeloid leukaemic cells in long-term bone marrow cultures

Patterns of growth in long-term bone marrow culture were compared for a number of murine myeloid leukaemias. A leukaemic pattern of haematopoiesis was maintained in culture for a period of at least 15 weeks for one of the lines. However in the other five murine myeloid leukaemic lines studies, the leukaemic cells appeared not to survive in culture and normal haematopoiesis was established. Transplantation of cells from these established cultures at weeks 7 or 11 into syngeneic recipients revealed that leukaemic cells were present. Thus leukaemic cells seem to persist in long-term bone marrow cultures even in morphologically normal cultures.     

Characterization of engraftable hematopoietic stem cells in murine long-term bone marrow cultures

OBJECTIVE: Long-term bone marrow cultures (LTBMC) are a potential source of hematopoietic stem cells (HSC) for transplantation. Previous reports indicate that feeding LTBMCs induces hematopoietic progenitor cycling, and other studies link HSC cycle phase with engraftability. Our study was initiated to further characterize LTBMC engraftability and determine if a cycle phase-related engraftment defect affects HSC from LTBMCs. MATERIALS AND METHODS: Competitive repopulation of lethally irradiated BALB/c females was used to examine engraftability of LTBMCs under "fed" or "unfed" conditions at 3 to 5 weeks culture. Tritiated thymidine suicide was used to determine the cycle status of HPP-CFC and CFU-S from LTBMCs. RESULTS: Total cell number in LTBMCs decreases from input. Quantitatively, both fed and unfed 3-, 4-, or 5-week cultures compete strongly with fresh marrow for 2 and 8 weeks, but not 6 months, after transplantation. Short-term engraftable HSCs expand between 3 and 5 weeks of culture. Clonal assays indicate no peak in S-phase of CFU-S at 24 and 48 hours after feeding, and fluctuation in both content and cycle status of HPP-CFC after feeding. CONCLUSIONS: Our LTBMCs engraft in all conditions, and the level of engraftment capability does not correlate with cell-cycle phase of CFU-S or HPP-CFC, or with time from feeding. Although the total cell number decreases from input, the proportion of short- and intermediate-term engrafting HSC in whole LTBMCs approximates that of fresh marrow and expands from 3 to 5 weeks in culture, whereas long-term engraftable HSCs are decreased in culture.     

Regulation of megakaryopoiesis in long-term murine bone marrow cultures

Megakaryocytes and their precursor cells were sustained in mouse bone marrow suspension cultures for over 4-6 wk. Megakaryocyte precursor cells were detected by their capacity to form colonies of megakaryocytes in semisolid agar cultures. Colony formation was dependent on the presence of medium conditioned by a myelomonocytic leukemic cell line (WEHI-3CM). Megakaryocytes from the liquid and semisolid cultures were identified by cytoplasmic acetylcholine esterase and by ultrastructural analysis. The suspension medium from the bone marrow liquid cultures which sustained megakaryopoiesis was not directly acitive in stimulating megakaryocyte colony formation in the semisolid agar cultures, but potentiated the number of colonies detected when WEHI-3CM was present. Bone marrow-conditioned medium increased the sensitivity of megakaryocyte progenitor cells to the stimulus in WEHI-3CM. Addition of the activities present in the two sources produced a quantitative assay for the detection of mouse megakaryocyte progenitor cells. These studies showed: (1) that no inductive regulator of in vitro clones of megakaryocytes was present in the supernatants from the long-term marrow cultures and, (2) that at least two factors were necessary for the induction of megakaryocyte progenitors to proliferate and differentiate in semisolid cultures in vitro.     

Expression of human glucocerebrosidase in murine long-term bone marrow cultures after retroviral vector-mediated transfer

A retroviral vector (N2-SV-GC) was constructed by inserting a normal human glucocerebrosidase (GC) cDNA under control of the SV40 early region promoter into the Moloney murine leukemia virus-derived N2 vector. N2-SV-GC produced human GC in murine 3T3 fibroblasts at levels in the range of the endogenous murine GC as determined by enzymatic assay and Western blot analysis. The N2-SV-GC retroviral vector was used for studies of gene transduction of murine hematopoietic progenitor cells (HPC). Infection of bone marrow cultured for 2 to 10 days in medium containing hematopoietic growth factors was significantly more efficient than infection of freshly isolated marrow cells (24% to 32% G418-resistant CFU-GM v 15%, respectively). The marrow infected by N2-SV-GC was maintained in long-term bone marrow culture (LTBMC) and had a stable level of G418-resistant HPC over 2 months of serial assays. The human GC gene of the vector was persistently expressed in the nonadherent cell fraction of the murine LTBMC as determined by Northern blotting, Western blotting, and immunohistochemical staining using a monoclonal antibody specific for human GC. N2-SV-GC also expressed the human GC gene in day 12 CFU-S. LTBMC represents a novel system for retroviral vector-mediated gene transduction of HPC and may accurately predict the activities of vectors in vivo.     

Extracellular matrix production by the adherent cells of long-term murine bone marrow cultures

We have studied the deposition of extracellular matrix proteins in the adherent stroma of long-term murine bone marrow cultures. Stable hematopoiesis was maintained for greater than 12 wk. At selected intervals, culture dishes were sacrificed by removing all nonadherent cells and air drying the dishes. The adherent stromal layer was analyzed for the presence of intracellular and extracellular collagen, fibronectin, and laminin using double immunofluorescent staining with specific antisera against these matrix components. In cultures examined during the first 2 wk, large numbers of stromal cells contained collagen, fibronectin, and laminin. Over the next 2 wk, an extensive extracellular network of fibronectin, laminin, and collagen was deposited on the dishes, which persisted throughout the life of the cultures. In contrast to a previous report, we detected substantial numbers of endothelial cells by means of immunofluorescent staining of stromal cells with antisera to type IV collagen, laminin, and factor VIII antigen. Although deposition of these extracellular matrix proteins coincides with onset of active hematopoietic cell production, the relative roles of the stromal cells and the extracellular matrix in supporting hematopoiesis in murine bone marrow cell cultures remain to be determined.     

Erythropoiesis in murine long-term marrow cultures following transfer of the erythropoietin cDNA into marrow stromal cells

Long-term bone marrow cultures (LTMC) have provided a useful in vitro system to study stem cell self-renewal and myeloid differentiation. However, standard murine LTMC are devoid of erythroid differentiation within 2 weeks of establishment. In an attempt to develop a model system to study erythropoiesis in vitro, we have used a recombinant retrovirus vector to transfer the erythropoietin cDNA into stromal cells making up the hematopoietic microenvironment of murine LTMC. Three weeks after infection, erythroid differentiation was evident macroscopically, with clumps of hemoglobinized red blood cells present in the infected cultures. Hemoglobinization was confirmed by benzidine staining of nonadherent cells, which showed that up to 70% of nucleated cells were benzidine positive. In combination with LTMC, the use of recombinant retrovirus vectors to transfer growth factor genes may provide useful models to study the interactions of hematopoietic stem cells, hematopoietic microenvironment, and growth factors in vitro.     

Impaired ability of bone marrow cells from immunodeficient mice to establish long-term cultures

Summary Haemopoiesis is often depressed in patients suffering from acquired immune deficiency syndrome (AIDS). Although several mechanisms have been postulated to be responsible for depressed haemopoiesis in AIDS patients, the aetiology of this disorder is still unknown. We hypothesized that failure of the stromal microenvironment may account for part of the haemopoietic defect observed in patients with AIDS. We therefore studied a murine model of AIDS (MAIDS) caused by infection with LP-BM5 virus to determine the ability of bone marrow cells from immunodeficient mice to establish long-term stromal cultures. In addition, normal and MAIDS mice received AZT (2 mg/ml) in their drinking water for up to 1 month to determine the effects of AZT treatment in vivo on the ability of bone marrow cells to support haemopoiesis in long-term cultures. Decreased numbers of non-adherent cells were observed in long-term bone marrow cultures (LTBMC) of MAIDS mice when compared to cultures derived from normal mice. Decreased numbers of non-adherent cells were observed in cultures of bone marrow cells from AZT-treated normal mice, when compared to untreated normal controls. Cells from AZT-treated MAIDS mice produced the smallest number of non-adherent cells. BFU-E and CFU-G/M were decreased in cultures of MAIDS mice when compared to those of normal mice. AZT-treatment further decreased the number of colony-forming cells in both MAIDS and normal cultures. Stromal cell function of MAIDS mice was also assessed by inoculating non-adherent cells from normal mice onto confluent irradiated MAIDS LTBMC. Stroma from MAIDS mice was unable to support haemopoietic function of normal bone marrow cells. Polymerase chain reaction (PCR) analysis of steady state levels of cytokine mRNAs of cells from confluent cultures revealed that levels of interleukin-6 mRNA were unchanged in MAIDS mice, as compared to normal controls, but the levels of GM-CSF were decreased in MAIDS mice. These data suggest that LP-BM5 MuLV infection alters the functioning of the haemopoietic stroma and that one mechanism of this depression in haemopoiesis may be via alterations of cytokine production.     

AKR murine leukemia viruses and long-term bone marrow cultures from AKR and SJL mice

We have evaluated the effects of AKR mouse retroviruses on a system of long-term bone marrow cultures which allows prolonged replication of hemopoietic stem cells (CFUs) and granulocyte-monocyte progenitors with production of mature granulocytes. Ecotropic-nononcogenic virus is expressed in all cultures and does not adversely effect stem cell replication. We found, however, that cultures established from mice treated in vivo with lymphomagenic AKR viruses (Gross murine leukemia virus and AKR SL3 murine leukemia virus) showed a more rapid decline in CFUs and CFUc than cultures from "normal" AKR mice. Only the former cultures were found to produce lymphomagenic viruses. Furthermore, it was shown that addition of lymphomagenic virus to three-week marrow cultures established from AKR and SJL mice also caused a prompt decline in progenitor cell production and granulopoiesis when compared to medium-treated control cultures. The fate of all cultures was a loss of CFUc and granulocytes with a continuous production of macrophages. The change to macrophage production occurred earlier in the cultures treated with lymphomagenic virus or in those derived from lymphomagenic virus-treated animals. A study of these bone marrow-drived macrophage cultures, as well as cultures of similar morphology but of thymic origin, showed that they could be maintained as continuous lines. Cells from three of the cultures from lymphomagenic virus-treated animals plroduced locally growing sarcoma when inoculated into mice. The macrophages from the declined marrow cultures differed in certain properties from those of the established lines.     

Generation of murine dendritic cells from flt3-ligand-supplemented bone marrow cultures

Murine dendritic cells (DCs) can be classified into at least 2 subsets, "myeloid-related" (CD11b(bright), CD8alpha(-)) and "lymphoid-related" (CD11b(dull), CD8alpha(+)), but the absolute relationship between the 2 remains unclear. Methods of generating DCs from bone marrow (BM) precursors in vitro typically employ granulocyte-macrophage colony-stimulating factor (GM-CSF) as the principal growth factor, and the resultant DCs exhibit a myeloidlike phenotype. Here we describe a flt3-ligand (FL)-dependent BM culture system that generated DCs with more diverse phenotypic characteristics. Murine BM cells cultured at high density in recombinant human FL for 9 days developed into small lymphoid-sized cells, most of which expressed CD11c, CD86, and major histocompatibility complex (MHC) class II. The CD11c(+) population could be divided into 2 populations on the basis of the level of expression of CD11b, which may represent the putative myeloid- and lymphoid-related subsets. The FL in vitro-derived DCs, when treated with interferon-alpha or lipopolysaccharide during the final 24 hours of culture, expressed an activated phenotype that included up-regulation of MHC class II, CD1d, CD8alpha, CD80, CD86, and CD40. The FL-derived DCs also exhibited potent antigen-processing and antigen-presenting capacity. Neutralizing anti-interleukin-6 (IL-6) antibody, but not anti-GM-CSF, significantly reduced the number of DCs generated in vitro with FL, suggesting that IL-6 has a role in the development of DCs from BM precursors. Stem cell factor, which exhibits some of the same bioactivities as FL, was unable to replace FL to promote DC development in vitro. This culture system will facilitate detailed analysis of murine DC development.     

Role of colony-stimulating factor in myelopoiesis in murine long-term bone marrow cultures

Weekly medium change or midweek feeding of long-term bone marrow cultures (LTMBCs) results in a significant increase in total myeloid cell production. Proliferative myeloid cells peak 48 hours after feeding, and nonproliferative myeloid cells reach maximum levels at 72 hours. This increase in myelopoiesis is invariably preceded by a significant elevation in biologically and immunologically measurable colony-stimulating factor (CSF) in the supernatants of LTBMC. The level peaks 24 hours after medium change, then gradually returns to basal values. The decrease in CSF relates to its consumption by generating myeloid precursors because no fluctuation in the levels occur in cultures without active myelopoiesis. No significant inhibitors or promoters of CSF were detected. When highly purified L cell CSF, CSF in lung-conditioned medium, or CSF concentrated from LTBMC supernatant is added to cultures, an identical increase in myelopoiesis occurs. Anti- CSF antiserum, added to culture at the time of medium change, totally neutralizes supernatant CSF levels but does not affect myelopoiesis. These findings suggest a potential regulatory role for CSF in myelopoiesis in LTBMC. CSF appears to function within the microenvironment through a mechanism involving cell:cell interactions or by causing the production of other substances that stimulate myelopoiesis. Because exogenous CSF stimulates myelopoiesis, it is likely that it too can react either directly or through microenvironmental cells to stimulate primitive myeloid cells to divide.     

Homing and engraftment of bone marrow cells derived from different donors in a murine model of sensitization

Sensitized recipients are at a high risk of graft rejection in hematopoietic stem cell transplantation. To explore the trace of donor cells, we tried to explore homing and engraftment of bone marrow cells (BMCs) derived from different donors in a murine model of sensitization. Sensitized BALB/c mice were used as transplanted recipients, which received BMCs derived from C57BL/6 or BALB/c donors after lethal irradiation. The homing study showed that the donor cells decreased along with time in recipients of the C57BL/6 donor group, but the donor cells increased along with time in recipients of the BALB/c donor group. For the engraftment assay, all the sensitized recipients transplanted with BMCs derived from C57BL/6 donors died after lethal irradiation. In contrast, all the recipients transplanted with BMCs derived from BALB/c donors got long-term survival. Our results suggest that it is crucial to have human leukocyte antigen identical donors for sensitized recipients during hematopoietic stem cell transplantation.     

Partial engraftment of donor bone marrow cells associated with long-term remission of haemophagocytic lymphohistiocytosis

Summary. We used polymerase chain reaction amplification of minisatellite sequences or of a Y chromosome-specific sequence and Southern blotting to analyse long-term engraftment (12–82 months) after bone marrow transplantation (BMT) for familial haemophagocytic lymphohistiocytosis (FHL). Six children aged from 1 to 18 months were transplanted with bone marrow from an HLA-identical sibling in five cases and from an HLA-nonidentical related donor (one mismatched HLA antigen) in one. The conditioning regiment included VP 16–213 (900 mg/m²), busulfan(16 mg/kg), cyclophosphamide (200 mg/kg) and, in one case. aracytine (2 g/m²).
Four patients are alive without therapy more than 3 years after BMT; the other two relapsed 1 year after BMT. DNA was extracted from separated polymorphonuclear cells and mononuclear cells, as well as from separated E + and E — cells in one case and CD16 + (natural killer) and CD16 — cells in two cases. Engraftment was partial in the four long-term survivors. Recipient cells were largely predominant in three of them as well as in one of the patients who relapsed (the donor also developed FHL 18 months after BMT). E +, E-, CD16 + and CD16— cells presented the same pattern of chimaerism. Engraftment failed to occur in the patient who received an HLA-nonidentical bone marrow. These results indicate that partial engraftment is compatible with longterm remission of FHL and that the presence of a small proportion of cells of donor origin can prevent FHL-related lymphocyte and macrophage activation.     

Stromal growth factor production in irradiated lectin exposed long-term murine bone marrow cultures

Hematopoietic regulatory factors produced by adherent (stromal) cells in long-term murine bone marrow cultures have been investigated. Using an in situ double layer agar overlay system, we demonstrated that exposure of the stromal cells to 1,100-rad irradiation increased their activities in stimulating colony formation of FDC-P1, an interleukin 3 (IL 3)-responsive cell line. The colony-stimulating activities (CSAs) of the irradiated stroma also stimulated normal marrow cells to form granulocyte-macrophage, megakaryocyte, and mixed lineage colonies. Addition of the lectin pokeweed mitogen to the irradiated stroma increased the level of CSAs. The FDC-P1 CSA of the irradiated stroma was inhibited by antibodies directed against murine granulocyte- macrophage colony stimulating factor (GM-CSF) but not by those against murine IL 3. Stromal-derived CSA for marrow cells was also partially blocked by anti-GM-CSF antibodies, probably reflecting the presence of other CSAs such as CSF-1. This latter growth factor has been found to be present in conditioned media from Dexter stroma, but levels are not increased after irradiation or lectin exposure. Partially purified GM- CSF, like IL 3, stimulated FDC-P1 proliferation and granulocyte, macrophage, and megakaryocyte colony formation. These results indicate that the major terminal differentiating hormone elicited by irradiation or lectin exposure of murine marrow stromal cells is GM-CSF. This growth factor, along with CSF-1, can account for the differentiated progeny produced in this system: macrophages, granulocytes, and megakaryocytes.     

Resistance of the stromal cell in murine long-term bone marrow cultures to damage by ionizing radiation

The ability of bone marrow stromal cells to survive and function after exposure to ionizing radiation remains controversial. Therefore, we used the murine long-term bone marrow culture system to analyze the effects of single doses of ionizing radiation (9-500 Gy) on the function of a preexisting, nearly confluent stroma that was supportive of hematopoiesis. Hematopoiesis ceased promptly in all the irradiated cultures and did not recover unless fresh marrow cells were inoculated. Radiation doses less than or equal to 100 Gy caused no obvious morphologic change in the cells. Total RNA, total protein, and collagen synthesis declined by 35%-60% within two days after even 9 Gy; but radiation doses up to 100 Gy caused minimal or no additional decline. Although RNA synthesis recovered nearly to normal within three weeks after radiation doses less than 100 Gy, total protein and collagen synthesis remained suppressed. Normal adherent layers irradiated with 9-50 Gy supported long-term hematopoiesis by fresh Sl/Sld marrow cells, although Sl/Sld marrow did not demonstrate sustained hematopoiesis when cultured in plain culture dishes or over normal stroma irradiated with 200 Gy. Thus, bone marrow stromal cells in long-term cultures did not show evidence of substantial cell death over at least the six-week period studied after irradiation with as much as 100 Gy, and they maintained hematopoietic supportive functions when irradiated with up to at least 50 Gy.     

Chemotherapy prior to autologous bone marrow transplantation impairs long-term engraftment in mice

OBJECTIVE: Autologous bone marrow transplantation in cancer patients is often preceded by multiple cycles of chemotherapy. In this study, we assessed in a mouse model whether stem cells were affected by prior chemotherapy. METHODS: Donor mice were treated with three consecutive injections of 150 mg/kg 5-fluorouracil (5-FU). Peripheral blood counts were allowed to recover before the subsequent dose of 5-FU was given. Mice recovered from three doses of 5-FU and showed normal steady-state hematopoiesis. Bone marrow cells from these mice were mixed with congenic competitor cells and transplanted into lethally irradiated recipients. RESULTS: Although in vivo homing of cells from these mice was not impaired, donor leukocyte contribution steadily decreased posttransplantation. In contrast to in vivo homing, both in vitro migration toward stromal-derived factor (SDF)-1 and the average CXC chemokine receptor-4 (CXCR4) expression were lower in 5-FU-treated cells. Moderate reductions in L-selectin and CD11a expression were observed on stem cells of 5-FU-treated mice. CD43, CD44, CD49d, and CD49e were normally expressed and could thus not explain the reduced engraftment of these cells. CONCLUSION: We therefore conclude that 5-FU either directly damages stem cells or that the replicative stress induced by 5-FU causes a decline in stem cell reconstitution potential resulting in lower chimerism levels posttransplantation, that declines in time.     

High-efficiency gene transfer to human hematopoietic cells maintained in long-term marrow culture

We used a helper-free recombinant retrovirus carrying the neomycin resistance (neor) gene to investigate methods for improving gene transfer efficiencies to clonogenic hematopoietic progenitor cells of human origin and to assess the possibility of gene transfer to the more primitive cells from which clonogenic cells are derived after several weeks in long-term human marrow cultures. The proportion of neor CFU-GM in methylcellulose assays of infected fresh marrow was increased by six- to eightfold (mean 37.4%) by the addition of extra GM colony-stimulating factor and interleukin-1 beta or medium conditioned by a human marrow "stromal" cell line to medium conditioned by agar-stimulated human leukocytes both during the infection and the colony growth period. Similar increases were also noted in the proportion of neor BFU-E, although the efficiencies overall were somewhat lower (up to 25.7%, mean 16.3%). Initiation of long-term cultures with marrow exposed to virus under the same growth factor-supplemented conditions but without any immediate selection step resulted in sustained production of a high proportion of neor CFU-GM and BFU-E for 6 weeks in both the nonadherent and adherent fractions. Molecular analysis was used to confirm the presence of the neo gene after culture. These results demonstrate that stable, high-efficiency gene transfer can be accomplished to the most primitive class of human hematopoietic cells currently detectable that may also have in vivo reconstituting potential. Further use of this approach should provide new insights into human hematopoietic stem cell regulation and allow continued development and assessment of gene therapy procedures.     

Origin of stroma cells in long-term bone marrow cultures from patients with acute myeloid leukemia

 Bone marrow stroma cells from patients with acute myeloid leukemia (AML) display a variety of functional abnormalities. In order to determine whether this is related to an imbalance in the proportion of different stroma cell types or to integration of leukemic progeny into the regulatory cell network, stroma layers were established in mycophenolic acid-treated long-term marrow cultures from 16 patients with AML and 42 controls and analyzed by means of simultaneous membrane immunofluorescence and interphase cytogenetics. Macrophages were identified by CD14 expression, fibroblasts by staining with the AS02 antibody, and malignant cells by leukemia-specific numerical chromosome aberrations, including monosomy 7 and trisomy 8. Compared with normal controls, there was a slight decrease in the proportion of stroma fibroblasts (52±27% versus 77±5%) in 10-week-old cultures from patients with AML. Two of five AML patients with trisomy 8 and both patients with monosomy 7 had evidence of leukemic stroma cells. Most malignant cells were CD14+ macrophages (3.8–98.1% of all CD14+ cells), but some were AS02+ (2.8–5.2%). AML stroma layers showed a reduced capacity to support the growth of normal hematopoietic cells in standard two-stage long-term cultures, but this was unrelated to the presence or absence of leukemic stroma elements. In conclusion, AML populations vary with respect to their ability to produce a malignant microenvironment. Functional defects in the hematopoietic microenvironment, however, are not limited to AML patients with cytogenetically abnormal stroma cells, but extend to cases without evidence of malignant stroma cells. Received: December 9, 1998 / Accepted: February 2, 1999     

Retrovirally marked CD34-enriched peripheral blood and bone marrow cells contribute to long-term engraftment after autologous transplantation

We report here on a preliminary human autologous transplantation study of retroviral gene transfer to bone marrow (BM) and peripheral blood (PB)-derived CD34-enriched cells. Eleven patients with multiple myeloma or breast cancer had cyclophosphamide and filgrastim-mobilized PB cells CD34-enriched and transduced with a retroviral marking vector containing the neomycin resistance gene, and CD34-enriched BM cells transduced with a second marking vector also containing a neomycin resistance gene. After high-dose conditioning therapy, both transduced cell populations were reinfused and patients were followed over time for the presence of the marker gene and any adverse effects related to the gene-transfer procedure. All 10 evaluable patients had the marker gene detected at the time of engraftment, and 3 of 9 patients had persistence of the marker gene for greater than 18 months posttransplantation. The marker gene was detected in multiple lineages, including granulocytes, T cells, and B cells. The source of the marking was both the transduced PB graft and the BM graft, with a suggestion of better long-term marking originating from the PB graft. The steady- state levels of marking were low, with only 1:1000 to 1:10,000 cells positive. There was no toxicity noted, and patients did not develop detectable replication-competent helper virus at any time posttransplantation. These results suggest that mobilized PB cells may be preferable to BM for gene therapy applications and that progeny of mobilized peripheral blood cells can contribute long-term to engraftment of multiple lineages.     

Kinetics of myeloid progenitor cells in human micro long-term bone marrow cultures

Hemopoiesis was analyzed in a miniaturized long-term culture of human bone marrow cells by quantifying the production of granulocyte-macrophage progenitor cells. As in the conventional long-term culture system, hemopoiesis was dependent on the presence of a marrow-derived adherent layer. Adipocytes proved to be essential for long-term proliferation. Optimal growth conditions were maintained by incubation in McCoy's medium supplemented with hydrocortisone, fetal calf serum, and horse serum. When calculated back to the volume of conventional cultures, the numbers and kinetics of nucleated cells and granulocyte-macrophage colony-forming cells were comparable in both culture systems. The microsystem is therefore suitable for performing multiple analyses on small samples of cells.