Development of Schistosoma mansoni worms in mice analyzed by bright field and confocal microscopy

The blood flukes of mammals (Digenea: Schistosomatidae) are among trematodes unique whose adult worms have separated sexes which are dissimilar in appearance. The developmental features, growth and organogenesis of Schistosoma mansoni were studied in Swiss Webster mice by a digital system for image analysis and confocal microscopy. Data so far obtained showed two phases with significative morphological changes at 3-4 weeks post-infection, and a gradual similar development onwards in the reproductive system and tegument. Our male-dependent phase demonstrated that mating occurs before sexual maturing. At week three, the majority of male worms (59%) had formed the gynaecophoric canal although testicular lobes and tegumental tubercles were absent. By this time, 33% females had an incipient ovary (without cellular differentiation). At week four, 77.2% males presented testicular lobes with few germinative cells while 26% had developing tegumental tubercles. The immature ovary was observed in 69% females. Suckers followed different pattern of growth between male and females. The size of oral and ventral suckers from six-week-old male worms grew abruptly (3.0 fold) more than that of three-week-old. In female worms, maximum growth was attained at week four, reducing in size thereafter. From sixth week onwards, all specimens showed the fully developed reproductive system. Probably, these features are morphological traits which schistosome has experienced from hermaphrodite to dioecy.     

Effect of N-Methyl-N-nitrosourea and N-Methyl-N-nitrosourethane upon the growth of tissue cultures ofNicotiana tabacum L.

N-Methyl-N-nitrosourea and N-methyl-N-nitrosourethane at concentrations of 0.1 mM to 1 mM inhibited the growth of tissue cultures ofNicotiana tabacum L. The inhibitory effect was proportional to the mutagen concentration applied. The primary expiants (pith slices) and a 3-year tissue culture strain exhibited a different sensitivity to the same mutagen concentrations. The variability in sensitivity of tissue culture inocula to mutagen effects was reduced by previous fractionation of the culture and by standardization of the age and size of inocula. The changes investigated in the ratio of relative growth rates between the controls and treated cultures give evidence of a fluctuating expression of mutagen effect in the course of the subculture interval and may demonstrate a recovery of the cultures from the mutagen effect.     

Immunohistochemical localizations of albumin and transferrin accumulated in Rhodamine fibrosarcoma tissue of rats for supporting growth of the tumor cells

Our recent studies indicated that Rhodamine fibrosarcoma (RdF) tissue of rats accumulated large amounts of albumin and transferrin and that these proteins were essential for growth of RdF cells in primary cultures in serum-free medium. Therefore, the localizations of albumin and transferrin accumulated in RdF tissue were examined by immunohistochemical stainings. Both anti-rat albumin IgG and anti-rat transferrin IgG stained the cell surface of the tumor cells strongly, but the intracellular area only weakly.     

Factors controlling the proliferative rate, final cell density, and life span of bovine vascular smooth muscle cells in culture

Low density vascular smooth muscle (VSM) cell cultures maintained on extracellular-matrix(ECM)-coated dishes and plated in the presence of either plasma or serum will proliferate actively when serum-containing medium is replaced by a synthetic medium supplemented with three factors: high density lipoprotein (HDL, 250 micrograms protein/ml); insulin (2.5 micrograms/ml) or somatomedin C (10 ng/ml); and fibroblast growth factor (FGF, 100 ng/ml) or epidermal growth factor (EGF, 50 ng/ml). The omission of any of these three factors from the synthetic medium results in a lower growth rate of the cultures, as well as in a lower final cell density once cultures reach confluence. When cells are plated in the total absence of serum, transferrin (10 micrograms/ml) is also required to induce optimal cell growth. The effects of the substrate and medium supplements on the life span of VSM cultures have also been analyzed. Cultures maintained on plastic and exposed to medium supplemented with 5% bovine serum underwent 15 generations. However, when maintained on ECM-coated dishes the serum-fed cultures had a life span of at least 88 generations. Likewise, when cultures were maintained in a synthetic medium supplemented with HDL and either FGF or EGF, an effect on the tissue culture life span by the substrate was observed. Cultures maintained on plastic underwent 24 generations, whereas those maintained on ECM-coated dishes could be passaged repeatedly for 58 generations. These experiments demonstrate the influence of the ECM-substrate only in promoting cell growth but also in increasing the longevity of the cultures.     

Growth of H-35 rat hepatoma cells in unsupplemented serum-free media: Effect of transferrin,insulin and cell density

Summary Serum-free tissue culture medium consisting of a 1∶1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 medium is herein shown to support growth of Reuber H-35 cells over several days in culture. Cells were initially plated in serum containing DMEM medium for 3 h. After cell attachment, serum is removed and replaced with a serum-free 1∶1 mixture of these two commercially available tissue culture media. The doubling time of cell growth in this unsupplemented serum-free medium was 46 h in lightly plated cultures over the first 5 d. The presence of transferrin (5 μg/ml) and insulin (3.3 nM) results in a cell doubling time of 17 h, which equaled the growth rate in medium containing 10% fetal bovine serum. In the absence of transferrin, growth rates in serum-free medium were correlated with the cell density of cultures. Conditioned medium from dense, serum-free cultures has growth-stimulating activity in recipient lightly plated cultures. This simple, serum-free culture medium will facilitate studies on the growth regulation of H-35 rat hepatoma cells. This work was funded by a feasibility grant from the American Diabetes Association, as well as by the National Institutes of Health grants CA 24604-09 and CA 16463-14.     

Cell cultures of fetal rat brain : Growth and marker enzyme development

Growth and enzyme development in cell cultures of fetal rat brain were influenced by type of growth medium, cell density, and age of fetal tissue source. Cells grew better in one medium (DMEM), but the other (F12G) enhanced development of choline acetyltransferase activity. One type of growth medium (DMEM) lost efficacy 2 weeks after preparation of complete medium. Cell division rate was density dependent, and choline acetyltransferase development was related to time in culture and cell concentration. Some results suggested division of choline acetyltransferase producing cells. Differences in age of tissue source resulted primarily in differences in growth: cultures of 21 day fetal cells developed more protein per 10⁶ cells inoculated than cultures of cells from younger animals; there was little difference in enzyme activity per culture. Conditions may be controlled such that fetal rat brain cells will grow and express differentiated functions in culture in a predictable manner.     

Schistosoma mansoni host-exposed surface antigens characterized by sera and recombinant antibodies from schistosomiasis-resistant rats

Antibodies from Schistosoma mansoni-infected rats, unlike mice, show a higher titer for schistosome apical tegumental antigens compared with non-apical membrane antigens. These antibodies bind to the surface of living lung-stage worms and to formaldehyde-fixed adult worms. We produced a single-chain antibody Fv domain (scFv) phage library displaying the antibody repertoire of rats highly immune to schistosome infection and we selected for scFvs that recognize the host-exposed surface of worms. Five unique rat scFvs (Teg1, Teg4, Teg5, Teg20 and Teg37) were obtained which recognize schistosome surface epitopes. Each of the scFvs recognizes the surface of living schistosomula and lung-stage schistosomules and/or the surface of formaldehyde-fixed adult worms. None of these scFvs reproducibly stained living adult worms. This suggests that a change occurs during the transition from lung schistosomules to 4-week adults such that at least some surface antigens, although remaining on the surface in living adult worms, can no longer be immunologically stained. Teg1 and Teg4 scFvs both recognize specific bands on Western blots. No bands were observed for the other three scFvs, suggesting that these scFvs may recognize non-protein or conformationally-dependent epitopes. Teg1 was unambiguously identified as recognizing the S. mansoni tetraspanin antigen, SmTSP-2, within the large extracellular domain. Teg4 recognizes a 35 kDa band tentatively identified as Sm29 by proteomic analysis. These scFvs can now be used to characterize schistosome epitopes at the host-parasite interface, to target worms in vivo, and to study the mechanisms by which these worms naturally evade immune damage to the tegument within permissive hosts.     

Identification of cells responsible for urokinase-type plasminogen activator synthesis and secretion in human diploid kidney cell cultures

Human urokinase-type plasminogen activator (uPA) is a serine protease that converts plasminogen to plasmin. It is produced and secreted by a variety of different human cells in vivo and in vitro. We have studied human diploid kidney cell (HKC) cultures prepared from neonatal kidney tissue and cultures of purified populations of HKC to determine which cells synthesize and secrete uPA into the culture medium. Antibodies against cell specific antigens and uPA were used to correlate specific kidney cell types with uPA synthesis. In addition, secretion of uPA activity into growth and uPA production media was determined for each cell type and cultures containing a mixture of cell types. The results of these studies demonstrated that glomerular visceral epithelial and kidney tubular epithelial cells synthesize and secrete uPA into the culture medium.     

Control of lipoprotein lipase secretion in mouse macrophages

The regulation of secretion of lipoprotein lipase (LPL) was studied in in vitro-derived mouse bone marrow macrophages (BMM), peritoneal exudate and resident macrophages and in the macrophage-like tumor cell line J774.1. BMM in cultures initiated with low concentrations of bone marrow cells (LC-BMC cultures) secrete more LPL per cell than BMM in cultures initiated with high concentrations of bone marrow cells (HC-BMC cultures). The suppressed state of LPL secretion in HC-BMC cultures could be alleviated by the addition of a colony-stimulating factor source (L-cell-conditioned medium; L-CM) onto the culture medium or exchanging the medium of HC-BMC cultures with medium from LC-BMC cultures for short periods (4 h). Addition of L-CM increased LPL secretion also in LC-BMC cultures. Addition of L-CM to fresh culture medium had little or no effect, suggesting that, in addition to requirement for L-CM, optimal expression depended also on factors released by the growing cells, probably providing optimal growth conditions. L-CM enhanced LPL secretion by thioglycollate-elicited peritoneal macrophages and had no effect on LPL secretion by resident peritoneal macrophages. Secretion of LPL from adherent J774.1 cells showed a biphasic effect. Secretion increased with cell density up to the point when growth inhibition was observed. In dense cultures in which cell proliferation was almost arrested, LPL secretion was remarkably suppressed (80-90%). Change of medium of dense cultures to fresh medium or medium conditioned by sparse cultures (for the last 4 h of culture) led to enhancement of LPL secretion to levels similar to those optimally expressed by sparse cultures. L-CM did not enhance LPL secretion from J774.1 cells. Dense cultures of both BMM and J774.1 cells did not contain a stable inhibitor of LPL secretion and medium from sparse cultures did not contain an inducer of LPL secretion. The data suggest that proliferating macrophages secrete large amounts of LPL, whereas in nonproliferating, quiescent cells, this activity is much reduced. L-CM enhances LPL secretion in quiescent BMM and peritoneal exudate cells to levels expressed by proliferating cells. Since this effect is already expressed after a 4 h incubation period, it is not dependent on cell cycling but could be one of the early responses to this macrophage mitogen. In J774.1 cells, a change of medium is a sufficient signal for enhancement of LPL secretion in quiescent cells.     

Continuous activation of primitive hematopoietic cells in long-term human marrow cultures containing irradiated tumor cells.

Human hematopoietic cells can be maintained in vitro for many weeks in the absence of exogenously provided hematopoietic growth factors if an adequate stromal cell containing adherent layer is present. We have now extended the use of this type of long-term culture (LTC) system to create a model of perturbed hematopoiesis in which human tumor cells that constitutively produce a variety of factors are co-cultured together with normal human marrow cells. In the present study, we used the human bladder carcinoma cell line (5637) because these cells were known to produce not only a variety of factors active directly on hematopoietic cells but also factors that can stimulate hematopoietic growth factor production by human marrow stromal cells. Analysis of mRNA extracted from the adherent layer and measurement of growth factor bioactivity in the medium of established LTC of human marrow containing irradiated 5637 cells, showed increased levels of interleukin-1 and -6, as well as granulocyte and granulocyte-macrophage colony-stimulating factor production by comparison to control cultures. As in normal cultures, high proliferative potential clonogenic hematopoietic cells were found almost exclusively in the adherent layer of these co-cultures, but these primitive cells were maintained in a state of continuous turnover, in contrast to control cultures where the same cell types showed the expected oscillation between a quiescent and a proliferating state following each weekly change of the medium. A similar perturbation of primitive progenitor cycling was achieved by adding medium conditioned by 5637 cells twice a week to otherwise normal LTC. The presence of irradiated 5637 cells in the LTC or the addition of 5637 conditioned medium also resulted in modest (2- to 3-fold) but sustained increases in the total hematopoietic progenitor population, as well as in the final output of terminally differentiated granulocytes and macrophages. These findings indicate that primitive hematopoietic cells in LTC can be kept in a state of continuous activation for many weeks by appropriate endogenous or exogenous hematopoietic growth factor provision and that this does not necessarily lead either to their rapid exhaustion or to a large amplification in output of mature progeny.     

Initial observations on the effect of medium composition on the differentiation of murine embryonic stem cells to alveolar type II cells

The pluripotency and high proliferative index of embryonic stem (ES) cells make them a good potential source of cells for tissue engineering purposes. We have shown that ES cells can be induced to differentiate in vitro into pulmonary epithelial cells (type II pneumocytes) using a serum-free medium designed for the maintenance of mature distal lung epithelial cells in culture (SAGM). However, the resulting cell cultures were heterogeneous. Our aim in this study was to attempt to increase pneumocyte yield and differentiation state by determining which medium components enhance the differentiation of pneumocytes and modifying the medium accordingly. Quantitative RT-PCR was used to measure changes in the expression of a type II pneumocyte-specific gene, surfactant protein C (SPC), in response to alterations in the cell culture medium. Results suggested that most individual SAGM growth factors were inhibitory for type II pneumocyte differentiation, with the largest increases in SPC expression (approximately threefold) being observed upon removal of retinoic acid and triiodothryonine. However, large standard deviations occurred between replicates, illustrating the highly variable nature of ES cell differentiation. Nevertheless, these observations represent an initial step towards achieving directed differentiation of pneumocytes from stem cells that could lead to their purification for tissue engineering purposes.     

THE RELATIONSHIP BETWEEN CELL-SUBSTRATE ADHESIVENESS AND CELL GROWTH: A STUDY ON CHONDROCYTES CULTURED IN VITRO WITH CONDITIONED MEDIUM

Sparsely cultured chondrocytes from the sternae of 14-day chick embryos grew vigorously with a liquid medium using Falcon tissue culture dishes, when the conditioned medium (CM) prepared from the high-density cultures of neural retinae was used as a growth medium. In CM most cells adhered to the culture substrate, while in the fresh medium, in which cell growth was very slow, many viable cells remained unattached to the substrate. Enhancement of cell growth by CM was much less marked in cultures of chondrocytes suspended in agarose. The main effect of CM in enhancing cell growth is thus considered to be related to the increase of cell adhesiveness to the substrate. The main active factor in CM is non-dialyzable and heat-stable.     

Establishment and characterization of SV40 large T antigen-immortalized cell lines derived from fetal bovine brain tissues after prolonged cryopreservation

Bovine brain cell lines with specific characteristics are useful in vitro experimental systems for molecular and cellular investigation of the interactions between bovine specific neuropathogenic agents and the host. Here, we established two novel cell lines from cultures of cryopreserved fetal bovine brain tissue by the transfection of SV40 large T antigen. Both cell lines showed cobblestone morphology in DMEM/F12 medium supplemented with 10% fetal bovine serum, epidermal growth factor and basic fibroblast growth factor. They were immunostained with endothelial marker, Von Willebrand Factor. Endothelial properties, such as capillary-like tube formation on matrigel and the incorporation of DiI-AcLDL were confirmed with these cells. Removal of growth factors increased the number of cells expressing alpha-smooth muscle actin, suggesting the potential of these cell lines to differentiate into smooth muscle cells. This study suggests an efficient protocol to immortalize brain endothelial cell lines from fetal bovine brain tissue culture.     

Comparison of Trichoplusia ni BTI-Tn-5B1-4 (high five) and Spodoptera frugiperda Sf-9 insect cell line metabolism in suspension cultures

Nutrient utilization and byproduct accumulation were monitored in Spodoptera frugiperda Sf-9 and Trichoplusia ni BTI-Tn-5B1-4 (High Fivetrade mark) cell lines during growth and following viral infection in suspension cultures in order to develop a better understanding of cell metabolism and to acquire information relevant to large scale fed-batch bioreactors. The utilization of glucose, dissolved oxygen, and amino acids were monitored in Sf-9 cell cultures grown in Sf-900 II serum-free medium (SFM) and in High Fivetrade mark cell cultures grown in both Sf-900 II and Express Five SFM. Using the optimal medium for each cell line, i.e., Sf-900 II SFM for Sf-9 cells and Express Five SFM for High Fivetrade mark cells, the cell growth rate, maximum cell density, specific glucose and glutamine utilization rates, and specific alanine production rate were comparable during cell growth. In addition, the expression level of recombinant human tissue plasminogen activator was comparable in the two cell lines on a per cell basis. It was found, however, that lactate and ammonia accumulated in High Fivetrade mark cell cultures, but not in Sf-9 cell cultures. In addition, High Fivetrade mark cells utilized asparagine more rapidly than glutamine, whereas Sf-9 cells consumed only minimal asparagine, and the oxygen utilization rate was significantly higher in High Fivetrade mark cell cultures. It was also found that the medium had a significant effect on High Fivetrade mark cell metabolism, e.g., the specific glucose utilization rate and the specific lactate and alanine production rates were significantly higher in Sf-900 II SFM than in Express Five SFM. In addition, the maximum cell density and specific asparagine utilization rate were significantly higher in Express Five SFM. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55:909-920, 1997.     

Growth and differentiation of alveolar bone cells in tissue-engineered constructs and monolayer cultures

The ability to enhance bone regeneration by implanting autologous osteoblasts in combination with an appropriate scaffold would be of great clinical interest. The aim of our study was to compare the growth and differentiation of alveolar bone cells in tissue-engineered constructs and in monolayer cultures, as the basis for developing procedures for routine preparation of bone-like tissue constructs. Alveolar bone tissue was obtained from four human donors and explant cultures of the cells were established. Expanded cells were seeded on macroporous hydroxyapatite granules, and cultured in medium supplemented with osteogenic differentiation factors for up to 3 weeks. Control monolayer cultures were established in parallel, and cultured in media with or without osteogenic supplements. Cell proliferation, alkaline phosphatase (AP) activity and gene expression of AP, osteopontin and osteocalcin were determined under different culture conditions at weekly intervals. Cells in tissue constructs exhibited growth patterns similar to those in control monolayer cultures: enhanced proliferation was noted during the first 2 weeks of cultivation, followed by a decrease in cell numbers. AP activity at 3 weeks was higher in all cultures in osteogenic medium than in control medium. Gene expression levels were stable in monolayer cultures in both types of media whereas, in tissue constructs, they exhibited patterns of osteogenic differentiation. Light and scanning electron microscopy examination of the cell-seeded constructs showed uniform cell distribution, as well as cell attachment and growth into the interior region of the hydroxyapatite granules. Our results show that bone-like constructs with viable cells exhibiting differentiated phenotype can be prepared by cultivation of alveolar-bone cells on the tested hydroxyapatite granules.     

Schistosome immunomodulators

Schistosomes are long lived, intravascular parasitic platyhelminths that infect >200 million people globally. The molecular mechanisms used by these blood flukes to dampen host immune responses are described in this review. Adult worms express a collection of host-interactive tegumental ectoenzymes that can cleave host signaling molecules such as the “alarmin” ATP (cleaved by SmATPDase1), the platelet activator ADP (SmATPDase1, SmNPP5), and can convert AMP into the anti-inflammatory mediator adenosine (SmAP). SmAP can additionally cleave the lipid immunomodulator sphingosine-1-phosphate and the proinflammatory anionic polymer, polyP. In addition, the worms release a barrage of proteins (e.g., SmCB1, SjHSP70, cyclophilin A) that can impinge on immune cell function. Parasite eggs also release their own immunoregulatory proteins (e.g., IPSE/α1, omega1, SmCKBP) as do invasive cercariae (e.g., Sm16, Sj16). Some schistosome glycans (e.g., LNFPIII, LNnT) and lipids (e.g., Lyso-PS, LPC), produced by several life stages, likewise affect immune cell responses. The parasites not only produce eicosanoids (e.g., PGE2, PGD2—that can be anti-inflammatory) but can also induce host cells to release these metabolites. Finally, the worms release extracellular vesicles (EVs) containing microRNAs, and these too have been shown to skew host cell metabolism. Thus, schistosomes employ an array of biomolecules—protein, lipid, glycan, nucleic acid, and more, to bend host biochemistry to their liking. Many of the listed molecules have been individually shown capable of inducing aspects of the polarized Th2 response seen following infection (with the generation of regulatory T cells (Tregs), regulatory B cells (Bregs) and anti-inflammatory, alternatively activated (M2) macrophages). Precisely how host cells integrate the impact of these myriad parasite products following natural infection is not known. Several of the schistosome immunomodulators described here are in development as novel therapeutics against autoimmune, inflammatory, and other, nonparasitic, diseases.     

Cholinergic differentiation in neurogenic basal forebrain cultures.

To study early events in the central nervous system (CNS) cholinergic development, cells from rat basal forebrain tissue were placed in culture at an age when neurogenesis in vivo is still active [embryonic day (E) 15]. The rapid mortality of these cells in defined medium, with 50% mortality after 5-10 h, was blocked completely by soluble proteins from the olfactory bulb (a basal forebrain target), extending earlier observations (Lambert, Megerian, Garden, and Klein, 1988). Treated cultures were capable of incorporating thymidine into DNA, and most cells incorporating 3H-thymidine (greater than 90%) also stained positive for neurofilament, confirming neuronal proliferation in the supplemented cultures. A small percentage of 3H-thymidine labelled cells were glial fibrillary acidic protein (GFAP) positive, but growth factors that support astroglial proliferation [epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and insulin-like growth factor (IGF-1)] were not sufficient for neuronal support. After 5 culture days with supplemented medium, almost 50% of the cells showed choline acetyltransferase (ChAT) immunofluorescence. The cholinergic neurons typically formed clusters separate from noncholinergic cells. These mature cultures did not develop if young cultures were treated with aphidicolin to block DNA synthesis. The data show that cultures of very young rat basal forebrain cells can be neurogenic, giving rise to abundant cholinergic neurons, and that early cell proliferation is essential for long-term culture survival.     

Glycosaminoglycan accumulation by normal and malignant human mammary epithelial cells in primary culture

The effects of glycosaminoglycans (GAGs) on cell growth and differentiation appear to vary with cell type and stage of development. This study describes the types and distribution of GAGs accumulated by normal and malignant human mammary epithelial cells in primary culture during their exponential and stationary phases of growth. Cultures incubated with [3H]glucosamine or [35S]sulfate were separated into medium, extracellular matrix (ECM), and cell fractions. Labelled GAGs were identified by chemical and enzymatic degradations and cellulose acetate electrophoresis. Cultures of normal cells in the exponential phase of growth released the most labelled GAGs into the medium fraction, the majority of which was hyaluronic acid (HA). The increase in labelled GAG accumulation, the increase in sulfated GAGs localized in the ECM fraction correlated with the reduced proliferative activity and increased cell density of cells in stationary cultures. In contrast, cultures of mammary tumour cells had the same labelled GAG profile, regardless of their growth status. Although there was variation among tumours, in general, the majority of the labelled GAGs were secreted into the medium fraction and the predominant GAG was HA. The results are comparable with those obtained from studies on mammary tissue in vivo. Primary cultures of human mammary epithelial cells should be useful for determining how modulations of GAGs affect growth and differentiation of these cells.     

Failure of in vitro-cultured schistosomes to produce eggs: how does the parasite meet its needs for host-derived cytokines such as TGF-β?

When adult schistosome worm pairs are transferred from experimental hosts to in vitro culture they cease producing viable eggs within a few days. Female worms in unisexual infections fail to mature, and when mature adult females are separated from male partners they regress sexually. Worms cultured from the larval stage are also permanently reproductively defective. The cytokine transforming growth factor beta derived from the mammalian host is considered important in stimulating schistosome female worm maturation and maintenance of fecundity. The means by which schistosomes acquire TGF-β have not been elucidated, but direct uptake in vivo seems unlikely as the concentration of free, biologically active cytokine in host blood is very low. Here we review the complexities of schistosome development and male–female interactions, and we speculate about two possibilities on how worms obtain the TGF-β they are assumed to need: (i) worms may have mechanisms to free active cytokine from the latency-inducing complex of proteins in which it is associated, and/or (ii) they may obtain the cytokine from alpha 2-macroglobulin, a blood-borne protease inhibitor to which TGF-β can bind. These ideas are experimentally testable.     

Growth limitation in hybridoma cell cultures: The role of inhibitory or toxic metabolites

Hybridoma cells usually grow to fairly low cell densities in batch cultures (1–3×10⁶ cells/ml). The reason for this is either that essential nutritional components of the medium are consumed, or that the cells produce some kind of inhibitory or toxic metabolite. This investigation presents evidence for the latter. Spent medium from cultures of hybridoma cells did not support growth of cells at lower cell densities (1–3×10⁵ cells/ml). The ability to support cell growth could not be restored by adding additional serum, energy sources (glucose, pyruvate) or L-glutamine. Furthermore, the consumption of amino acids could not account for this growth inhibition. On the contrary, the spent medium contained a substance that inhibited cell growth. This substance or metabolite was found in a fraction eluted from a gel filtration column when spent medium was applied to the column. This substance was found in the spent medium from all hybridoma and myeloma cell lines that were tested. The molecular weight of the substance was about 5 kD. Spent medium from two hybridoma cell lines also contained a substance that was eluted in the same fraction as albumin (67 kD). It is likely that this (or these) substance(s) is responsible for the growth limitation in hybridoma cell cultures.Abbreviations PBS phosphate buffered saline