A separation chamber to sort cells,nuclei, and chromosomes at moderate g forces. II. Studies on velocity sedimentation and equilibrium density centrifugation of mammalian cells

A separation chamber having a surface of 50 cm² and a height of 2 cm is described for the rapid separation of cells and cell organelles at acceleration forces from 10 to 90g. To eliminate wall sedimentation artifacts, the chamber was positioned 20 cm from the rotor axis in a speed-controlled centrifuge. The chamber has flow deflectors for the undisturbed introduction of the sample layer and the gradient; an antivortex cross prevents swirling upon acceleration and deceleration. To illustrate the use of the separation chamber, examples of velocity sedimentation and of equilibrium density centrifugation are given: (i) human monocytes (70% were 90% pure) are separated from lymphocytes in 10 min at 20g; (ii) nonparenchymal rat liver cells are separated in 10 min at 16g in 97% pure endothelial cells and 99% pure Kupffer cells; (iii) equilibrium density centrifugation of human peripheral blood cells at about 90g permits the separation of erythrocytes, monocytes, lymphocytes, neutrophils, eosinophils, and basophils in one run. B cells are separated from T cells. The movement of swinging buckets is analyzed in mathematical terms and a simple method is offered to determine the position of cells in density gradients with the use of a small programmable calculator.     

Simian sarcoma virus transformation of normal rat kidney fibroblasts is associated with markedly increased basic fibroblast growth factor expression.

Transformation of normal rat kidney fibroblasts (NRK) by the simian sarcoma virus (SSV) occurs as a result of expression of p28v-sis, a homologue of platelet-derived growth factor-B chain. Chromatographic separation revealed that the bulk (85%) of the mitogenic activity in SSV-transformed NRK cells was not due to p28v-sis but rather two distinct endothelial cell growth factors that eluted off heparin-Sepharose between 1 and 2 M NaCl. Protein purification and Northern blot analysis revealed that one of these growth factors was the 18 kd form of bFGF, the expression of which was found to increase 15-fold with SSV-transformation of NRK cells. The pure 18 Kd bFGF had no effect on NRK cell growth but was a potent neurotrophic agent for fetal rat cortical neurones and a potent growth factor for fetal bovine heart endothelial cells, suggesting a paracrine but not autocrine role for this protein. The second endothelial cell growth factor activity in SSV-transformed NRK cells was due to an 18 Kd protein which could be distinguished immunologically, biochemically, and mitogenically from bFGF.     

Synthesis and secretion of retinol-binding protein by cultured rat Sertoli cells.

We report here that retinol-binding protein (RBP) is synthesized and secreted by rat Sertoli cells in culture. This was demonstrated in four ways. First, transthyretin (TTR) bound to Sepharose 4B retained a labeled protein from media collected from Sertoli cells provided with 35S-methionine, under the same conditions as authentic RBP is bound. The protein was co-eluted with authentic RBP by pure water. Second, this same radioactive protein co-eluted with pure RBP upon gel filtration. Third, when subjected to SDS-PAGE, the protein again migrated with pure RBP, as shown by radioautography. Finally, Sertoli cells were incubated with 35S-cysteine and the conditioned medium was put over a TTR-Sepharose column to isolate the radioactive protein, as characterized above. Cysteine residues were oxidized to cysteic acid residues, and the protein was submitted for sequencing through the first ten residues. Radioactivity was located only in the fourth residue, where a cysteine residue is found in rat RBP. This indicates that RBP is secreted by the Sertoli cell and may serve as the carrier of retinol to the developing germ cells, which are known to be dependent upon vitamin A.     

Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation

Mammalian spermatogenesis is a complex differentiation process that occurs in several stages in the seminiferous tubules of the testes. Currently, there is no reliable cell culture system allowing for spermatogenic differentiation in vitro, and most biological studies of spermatogenic cells require tissue harvest from animal models like the mouse and rat. Because the testis contains numerous cell types - both non-spermatogenic (Leydig, Sertoli, myeloid, and epithelial cells) and spermatogenic (spermatogonia, spermatocytes, round spermatids, condensing spermatids and spermatozoa) - studies of the biological mechanisms involved in spermatogenesis require the isolation and enrichment of these different cell types. The STA-PUT method allows for the separation of a heterogeneous population of cells - in this case, from the testes - through a linear BSA gradient. Individual cell types sediment with different sedimentation velocity according to cell size, and fractions enriched for different cell types can be collected and utilized in further analyses. While the STA-PUT method does not result in highly pure fractions of cell types, e.g. as can be obtained with certain cell sorting methods, it does provide a much higher yield of total cells in each fraction (~1 x 10⁸ cells/spermatogenic cell type from a starting population of 7-8 x 10⁸ cells). This high yield method requires only specialized glassware and can be performed in any cold room or large refrigerator, making it an ideal method for labs that have limited access to specialized equipment like a fluorescence activated cell sorter (FACS) or elutriator.     

Purification and characterization of hormone-regulated isoforms of the regulatory subunit of type II cAMP-dependent protein kinase from rat ovaries

The regulatory subunit (R-II) of cAMP-dependent protein kinase type II is induced in rat ovarian granulosa cells by the synergistic actions of estradiol and follicle-stimulating hormone. The R-II from rat ovaries was compared with R-II from rat heart, rat brain, bovine heart, and bovine brain using immunological methods, 8-N3[32P]cAMP photoaffinity labeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three isoforms of R-II were identified in rat ovarian cell extract (R-II54 Mr = 54,000, R-II52 Mr = 52,000, R-II51 Mr = 51,000), two isoforms of R-II in rat brain cell extract (Mr = 54,000, Mr = 52,000), and one isoform of R-II in rat heart cell extract (Mr = 54,000). Rat ovarian R-II54, heart R-II, and brain R-II (Mr = 54,000) were recognized by antiserum against rat heart R-II, whereas rat ovarian R-II52/R-II51 and rat brain R-II (Mr = 52,000) were not. In contrast, an antiserum raised against bovine heart R-II recognized all three isoforms of ovarian R-II as well as the lower molecular weight form of rat brain R-II. Ovarian types R-II52 and R-II51 but not R-II54 were increased selectively in granulosa cells by estradiol and follicle-stimulating hormone. In addition: 1) ovarian R-II52/51 subunits were purified to homogeneity and shown to recombine with C subunit from bovine heart to form a cAMP-dependent protein kinase; 2) pure R-II52/51 were not interconvertible to a higher molecular weight form by C subunit-dependent phosphorylation; 3) pure rat heart R-II (Mr = 54,000) and ovarian R-II52/51 exhibited distinct differences based on one- and two-dimensional peptide mapping; and 4) by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis pure R-II52/51 were resolved as three (rather than two) isoelectric variants which were clearly different from pure rat heart R-II54. Thus, the hormone-regulated form of R-II in rat ovarian granulosa cells appears to represent a gene product distinct from R-II54 in rat heart.     

Transforming growth factor-beta. A very potent inhibitor of myoblast differentiation, identical to the differentiation inhibitor secreted by Buffalo rat liver cells

Transforming growth factor-beta (TGF-beta) is now known to have a number of actions in addition to the induction of phenotypic transformation in fibroblastic cells. In this paper, we characterize its inhibition of differentiation in rat myoblasts of Yaffe's L6 strain and demonstrate its identity or very close similarity to the differentiation inhibitor (DI) secreted by Buffalo rat liver cells cultured in serum-free medium. At concentrations as low as 60 pg/ml, TGF-beta gave detectable inhibition of differentiation measured as myoblast fusion and creatine kinase elevation; maximal inhibition was observed at and above 0.5 ng/ml (20 pM). The inhibition persisted as long as fresh TGF-beta was added at 48-h intervals, but it was reversed upon removal of the factor. By itself or in the presence of mitogens, TGF-beta had no mitogenic activity in the L6 cells. Concentration dependencies of human TGF-beta and the rat DI were closely parallel in three assays: inhibition of myoblast differentiation, stimulation of normal rat kidney cell growth in soft agar, and competition for displacement of labeled TGF-beta from binding sites on A549 human lung carcinoma cells. We conclude that most if not all of the DI activity found in medium conditioned by Buffalo rat liver cells can be attributed to the presence of TGF-beta or a very similar molecule. These observations also offer a potentially useful approach to study the control of myogenesis; the process(es) can be blocked in cloned L6 myoblasts by incubation with very small quantities of a pure protein in fully defined serum-free medium.     

Osteoblastic regulation of osteoclast survival: effect of calcitriol

Throughout life, bone is remodelled in a dynamic process which results in a balance between bone formation by osteoblasts and bone resorption by osteoclasts. It is now clearly established that osteoblasts/stromal cells are crucial for differentiation of osteoclasts, through a mechanism involving cell-to-cell contact. However, the possible involvement of osteoblasts and stromal cells in the survival of osteoclasts has not yet been clearly demonstrated. In this study, we assessed the influence of cellular microenvironment, especially osteoblasts, on the osteoclast survival. Our results have shown significant differences in osteoclastic survival between unfractionated bone cells and pure osteoclasts. Furthermore, we have shown that addition of 1.25(OH)2D3 to unfractionated bone cells resulted in a dose-dependent increase in osteoclast survival. Finally, we have shown that a conditioned medium obtained from rat osteoblastic cells cultured with calcitriol was able to increase significantly survival of pure osteoclasts. Taken together, these results strongly suggest that osteoblastic cells present in the bone microenvironment might play a role in the osteoclastic survival by producing soluble factor which modulate osteoclast apoptosis.     

The separation of different cell classes from lymphoid organs. X. Preparative electrophoretic separation of lymphocyte sub-populations from mouse spleen and thoracic duct lymph

Conditions have been established for the separation of viable mouse lymphoid cells by continuous free-buffer film preparative electrophoresis. The detailed electrophoretic distribution profiles of T and B lymphocytes from mouse spleen and thoracic duct have been determined. Cell surface θ-antigen was used as a marker for T cells, and high surface-density of immunoglobulin as a marker for B cells. Spleen cells from athymic “nude” mice were also studied. In the unselected normal spleen cell populations B lymphocytes are heterogeneous, about 60% being of low mobility with the remainder distributing broadly, and extending into the highest mobility fractions. T lymphocytes are predominantly of high mobility. Lymphoid cells lacking markers of either the B or T lineage are of intermediate mobility. There is only partial separation of T and B cells because of the extensive overlap between the populations. The high mobility B cells, which separate along with T cells, include a substantial proportion of large cells, and include cells with high surface density of immunoglobulin. The majority of these large B cells can be selectively eliminated by their adherence on passage through a glass-bead column. By pre-selecting the 50% non-adherent lymphocytes from spleen as the starting material, a very sharp and more extensive separation of B and T cells can be achieved, with 100% pure B cells and 90% pure T cells in many fractions. However these samples are not representative of the total T and B cell populations of spleen. In thoracic duct lymph high mobility B-cells are absent, there is little overlap between T and B cell mobility. 100% pure T and B cells can be isolated.     

Identification of the Schwann cell as a peripheral nervous system cell possessing a differentiation antigen expressed by a human lung tumor.

Recent studies of the plasma membrane antigens of a human lung tumor (oat cell carcinoma) indicated that the tumor expressed at least two normal differentiation antigens undetectable in normal respiratory epithelium. One antigen was characteristic of certain endodermally derived epithelial cells of the digestive system; the other antigen was characteristic of certain neural crest-derived cells in the peripheral nervous system. The present studies were undertaken to identify the reactive cell type in the peripheral nervous system. Since similar cells in the rat peripheral nervous system expressed a cross-reactive form of this antigen, and since pure cultures of different rat nerve cell type were available, the following approach was possible. Cultures of pure neurons, pure Schwann cells, pure fibroblasts, neurons and Schwann cells, and neurons, Schwann cells, and fibroblasts were assayed for this antigen with rabbit anti-oat cell carcinoma plasma membrane antiserum absorbed with normal lung and liver. The indirect immunofluorescence method on both whole, viable cell and fixed cell substrates was used. Only Schwann cells expressed the antigen; Schwann cells in the presence of neurons expressed the antigen much more strongly than did pure Schwann cells. It was concluded that the oat cell carcinoma of the lung expressed a differentiation antigen present on Schwann cells.     

A single partitioning step in aqueous polymer two-phase systems reduces hypotonized rat erythrocyte heterogeneity

Rat carrier erythrocytes prepared by hypotonic dialysis (80 mOsm/kg) are a heterogeneous cell population that can be fractionated into two-well-defined cell subpopulations by a single partition step, in charge-sensitive dextran-poly(ethylene glycol) aqueous two-phase systems. One subpopulation (65% of total cells) has a decreased cell surface charge and is partitioned at the interface in a single step and then fractionated by counter-current distribution as a low-G subpopulation. The other subpopulation (35% of total cells) has charge surface properties more like those of the untreated control rat erythrocytes. These last cells are partitioned in the top phase in a single step and then fractionated by counter-current distribution as a high-G subpopulation. Partitioning is more effective in reducing cell heterogeneity in hypotonized rat erythrocyte populations than is density separation in Ficoll-paque which only separates a small less dense cell subpopulation (5% of total cells), with the most fragile cells, from a larger and more dense cell subpopulation (95% of total cells), with a mixture of fragile and normal cells. This simple cell separation procedure quickly reduces carrier erythrocyte heterogeneity in a single partitioning step so it can be used to prepare cells for in vivo studies.     

Enzymatic production and in?situ separation of natural β-ionone from β-carotene

A biotechnological process concept for generation and in?situ separation of natural β-ionone from β-carotene is presented. The process employs carotenoid cleavage dioxygenases (CCDs), a plant-derived iron-containing nonheme enzyme family requiring only dissolved oxygen as cosubstrate and no additional cofactors. Organophilic pervaporation was found to be very well suited for continuous in?situ separation of β-ionone. Its application led to a highly pure product despite the complexity of the reaction solution containing cell homogenates. Among three different pervaporation membrane types tested, a polyoctylmethylsiloxane active layer on a porous polyetherimide support led to the best results. A laboratory-scale demonstration plant was set up, and a highly pure aqueous–ethanolic solution of β-ionone was produced from β-carotene. The described process permits generation of high-value flavor and fragrance compounds bearing the desired label “natural” according to US and European food and safety regulations and demonstrates the potential of CCD enzymes for selective oxidative cleavage of carotenoids.     

Isolation of CD4+ T cells from mouse lymph nodes using Miltenyi MACS purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are > or = 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!     

Application of preferential crystallization to resolve racemic compounds in a hybrid process

The application of preferential crystallization is at present limited to conglomerate forming systems, which cover only a minor part of chiral substances. In this paper, a hybrid process is proposed that extends the applicability of the preferential crystallization principle to the more common racemic compound forming systems. It comprises a preliminary (e.g., chromatographic) enantiomeric enrichment step and preferential crystallization to finally produce the desired pure enantiomer(s). The applicability of preferential crystallization to racemic compounds is demonstrated on the example of mandelic acid as a model system. Direct monitoring of the separation progress is performed using combined online polarimetry and online density measurements. A cyclic crystallization process, which provides alternating the pure mandelic acid enantiomer and the racemic compound, is feasible and allows the resolution of rac-mandelic acid as part of the proposed hybrid approach.     

Optimal operating mode for enantioseparation of SB-553261 racemate based on simulated moving bed technology

The performance of the simulated moving bed (SMB) technology and its modification, the Varicol process, was optimized using an experimentally verified model for the enantioseparation of SB-553261 racemate. Single and multiobjective optimizations have been carried out for both existing as well as design stage and their efficiencies were compared. The optimization problem involves a relatively large number of decision variables, both continuous variables such as flow rates, switching time and length of the columns, as well as discrete variables like number and distribution of columns. A state-of-the-art new optimization technique based on a genetic algorithm (nondominated sorting genetic algorithm with jumping genes) was utilized which allows handling of these complex optimization problems. The optimization results showed that significant improvement could be made to the chiral drug separation process using both the SMB and the Varicol process. It was found that the performance of a Varicol process is superior to that of a SMB process in terms of treating more feed using less desorbent or increasing productivity while at the same time achieving better product quality. Optimum results were explained using equilibrium theory by locating them in the pure separation region.     

Stages of normal tracheo-bronchial development in rat embryos: resolution of a controversy

The embryonic events surrounding tracheo-esophageal separation remain controversial. The present study was undertaken to clarify early tracheo-bronchial development in the rat embryo at a critical period of organogenesis. Twenty-seven timed-mated Sprague-Dawley rats were divided into nine groups of three rats. Their embryos were harvested on gestational days 11-15 at intervals of 8 h, processed and sectioned transversely. The sections were stained with hematoxylin and eosin and examined serially. The foregut is a single tube on gestational day 11. During the following 16 h, there is localized and rapid growth of the respiratory epithelium and a laterocaudal expansion to form the bronchial buds and a protuberance on the ventral wall of the foregut (future tracheal carina). From gestational days 12-12 + 8, cellular debris and apoptotic epithelial cells are specifically located in the tracheo-esophageal groove, resulting in collapse and fusion of the lateral walls of the foregut, effectively separating the trachea and esophagus. Afterwards, the epithelial proliferation dominates the process of tracheo-esophageal separation until it reaches the caudal end of the laryngeal epithelial lamina on gestational day 15. The present study shows that separation of the trachea from the esophagus involves three consecutive stages: (i) epithelial proliferation resulting in the formation of bronchial buds and the tracheal carina; (ii) epithelial apoptosis leading to separation of the trachea and esophagus; and (iii) epithelial proliferation to complete the separation process.     

In vivo and in vitro uptake and degradation of acetylated low density lipoprotein by rat liver endothelial, Kupffer, and parenchymal cells

Isolation and separation of rat liver cells into endothelial, Kupffer, and parenchymal cell fractions were performed at different times after injection of human 125I-acetyl low density lipoproteins (LDL). In order to minimize degradation and redistribution of the injected lipoprotein during cell isolation, a low temperature (8 degrees C) procedure was applied. Ten min after injection, isolated endothelial cells contained 5 times more acetyl-LDL apoprotein per mg of cell protein than the Kupffer cells and 31 times more than the hepatocytes. A similar relative importance of the different cell types in the uptake of acetyl-LDL was observed 30 min after injection. For studies on the in vitro interaction of endothelial and Kupffer cells with acetyl-LDL, the cells were isolated with a collagenase perfusion at 37 degrees C. Pure endothelial (greater than 95%) and purified Kupffer cells (greater than 70%) were obtained by a two-step elutriation method. It is demonstrated that the rat liver endothelial cell possesses a high affinity receptor specific for the acetyl-LDL because a 35-fold excess of unlabeled acetyl-LDL inhibits association of the labeled compound for 70%, whereas unlabeled native human LDL is ineffective. Binding to the acetyl-LDL receptor is coupled to rapid uptake and degradation of the apolipoprotein. Addition of the lysosomotropic agents chloroquine (50 microM) or NH4Cl (10 mM) resulted in more than 90% inhibition of the high affinity degradation, indicating that this occurs in the lysosomes. With the purified Kupffer cell fraction, the cell association and degradation of acetyl-LDL was at least 4 times less per mg of cell protein than with the pure endothelial cells. Although cells isolated with the cold pronase technique are also still able to bind and degrade acetyl-LDL, it appeared that 40-60% of the receptors are destroyed or inactivated during the isolation procedure. It is concluded that the rat liver endothelial cell is the main cell type responsible for acetyl-LDL uptake.     

A continuous multicolumn countercurrent solvent gradient purification (MCSGP) process

Biomolecules are often purified via solvent gradient batch chromatography. Typically suitable smooth linear solvent gradients are applied to obtain the separation between the desired component and hundreds of impurities. The desired product is usually intermediate between weakly and strongly adsorbing impurities, and therefore a central cut is required to get the desired pure product. The stationary phases used for preparative and industrial separations have a low efficiency due to strong axial dispersion and strong mass transfer resistances. Therefore a satisfactory purification often cannot be achieved in a single chromatographic step. For large scale productions and for very valuable molecules, countercurrent operation such as the well known SMB process, is needed in order to increase separation efficiency, yield and productivity. In this work a novel multicolumn solvent gradient purification process (MCSGP-process) is introduced, which combines two chromatographic separation techniques, which are solvent gradient batch and continuous countercurrent SMB. The process consists of several chromatographic columns, which are switched in position opposite to the flow direction. Most of the columns are equipped with a gradient pump to adjust the modifier concentration at the column inlet. Some columns are interconnected, so that non pure product streams are internally, countercurrently recycled. Other columns are short circuited and operate in batch mode. As a working example the purification of an industrial stream containing 46% of the hormone Calcitonin is considered. It is found that for the required purity the MCSGP unit achieves a yield close to 100% compared to a maximum value of a single column batch chromatography of 66%.     

Separation of intact rat hepatocytes and rat liver nuclei into ploidy classes by velocity sedimentation at unit gravity

A system is described which permits the separation of isolated hepatocytes and isolated rat liver nuclei belonging to different ploidy classes by velocity sedimentation at unit gravity.The problem of obtaining single cells suspensions is discussed and preparations were obtained that contained 96% single hepatocytes.By improving the sedimentation method, it took 2.5 h to separate rat liver nuclei on sucrose gradients into diploid and tetraploid ploidy classes. Recoveries were generally over 95%. The diploid band was 99% pure. DNA and protein content of the ploidy classes were measured. After partial hepatectomy and [³H]thymidine injection it was found that the label moved largely into the tetraploid compartment.Isolated hepatocytes were fractionated in 1 h on Ficoll gradients. Erythrocytes were separated from small nucleated cells and the population of hepatocytes was clearly separated from these two cell populations. Diploid hepatocytes were 80% and tetraploid hepatocytes were 99% pure. Viability was about 80% after fractionation.The gene dosage of NADPH cytochrome c reductase, succinate dehydrogenase and lactate dehydrogenase was estimated in diploid and tetraploid hepatocytes. Gene dosage was equal in diploid and tetraploid hepatocytes for succinate dehydrogenase and NADPH cytochrome c reductase. It is suggested, after correcting for non-viable tetraploid hepatocytes, that the gene dosage of lactate dehydrogenase was significantly lower in diploid than in tetraploid hepatocytes.     

Chronic iron overload inhibits protein secretion by adult rat hepatocytes maintained in long-term primary culture

Short-term pure cultures and long-term cocultures of adult rat hepatocytes with rat liver epithelial cells, presumably derived from primitive biliary cells, were used to define in vitro models of iron overloaded hepatocytes in order to understand the molecular mechanism responsible for liver damage occurring in patients with hemochromatosis. In vitro iron overload was obtained by daily addition of ferric nitrilotriacetate to the culture medium. A concentration of 20 microM ferric salt induced hepatocyte iron overload with minimal cytotoxicity as evaluated by cell viability, morphological changes of treated cells and cytosolic enzyme leakage into the culture medium. The effects of iron overload on protein biosynthesis and secretion were studied in both short-term pure cultures and long-term cocultures of hepatocytes. The amounts of intracellular and newly synthesized proteins were never modified by the iron treatment. Furthermore, neither the relative amounts of transferrin and albumin mRNAs nor their translational products were altered by iron overload. Moreover, no change in the transferrin isomeric forms were observed in treated cells. In contrast, a prolonged exposure of cocultured hepatocytes to 20 microM ferric salt led to a significant decrease in the amount of proteins secreted in the medium. This decrease included the two major secreted proteins, namely albumin and transferrin, and probably all other secreted proteins. These results demonstrate that iron loading alters neither the total nor the liver specific protein synthesis activity of cultured hepatocytes. They suggest that chronic overload may impede the protein secretion process.     

Influence of macrophages and macrophage-modified collagen I on the adhesion and proliferation of vascular smooth muscle cells in culture

The adhesion, proliferation and morphology of rat vascular smooth muscle cells (VSMC) in cocultures with macrophages or in cultures on type I collagen modified by activated macrophages were evaluated. In the first set of experiments, rat alveolar macrophages were added to 24-hour-old VSMC cultures. Between days 2 and 5 after VSMC seeding, the population densities and doubling times of cells were similar in both VSMC-macrophage and pure VSMC cultures. However, from day 5, the cocultures proliferated about two times more rapidly and on day 7, they reached higher cell population density by 40%. The pure macrophage cultures did not proliferate. In the second set of experiments, rat alveolar macrophages were activated by non-toxic TiO2 dust to produce reactive oxygen species and incubated for 120 min with collagen I. The collagen was then adsorbed on plastic culture dishes and seeded with VSMC. The collagen exposed for 10 min only, the unmodified collagen and pure culture dishes were used as control growth supports. On all four tested substrates, the number of initially adhered cells was similar, but on the collagen modified for 120 min, the cells were less spread. Moreover, on day 2 to 3 after seeding, some cells on this collagen became vacuolated and detached spontaneously from the growth support. The remaining VSMC, however, rapidly proliferated, so that on day 9, the cell population density on 120-min-modified collagen was similar as on both control collagens and significantly higher compared to that on uncoated dishes. Our results suggest that 1. The delayed growth-stimulating effect of macrophages on VSMC-macrophage mixed population is probably due to autocrine production of mitogens by both cell types rather than due to an acute effect of short-living oxygen radicals released from macrophages immediately after adding to VSMC cultures. 2. The effect of collagen I exposed to activated macrophages for 120 min is slightly cytotoxic, which could, however, stimulate a release of mitogens from damaged as well as surviving VSMC.