Joseph Feder

Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis.

Vascular permeability factor (VPF) is an Mr 40-kD protein that has been purified from the conditioned medium of guinea pig line 10 tumor cells grown in vitro, and increases fluid permeability from blood vessels when injected intradermally. Addition of VPF to cultures of vascular endothelial cells in vitro unexpectedly stimulated cellular proliferation. VPF promoted the growth of new blood vessels when administered into healing rabbit bone grafts or rat corneas. The identity of the growth factor activity with VPF was established in four ways: (a) the molecular weight of the activity in preparative SDS-PAGE was the same as VPF (Mr approximately 40 kD); (b) multiple isoforms (pI greater than or equal to 8) for both VPF and the growth-promoting activity were observed; (c) a single, unique NH2-terminal amino acid sequence was obtained; (d) both growth factor and permeability-enhancing activities were immunoadsorbed using antipeptide IgG that recognized the amino terminus of VPF. Furthermore, 125I-VPF was shown to bind specifically and with high affinity to endothelial cells in vitro and could be chemically cross-linked to a high-molecular weight cell surface receptor, thus demonstrating a mechanism whereby VPF can interact directly with endothelial cells. Unlike other endothelial cell growth factors, VPF did not stimulate [3H]thymidine incorporation or promote growth of other cell types including mouse 3T3 fibroblasts or bovine smooth muscle cells. VPF, therefore, appears to be unique in its ability to specifically promote increased vascular permeability, endothelial cell growth, and angio-genesis.

Determination of the number of endothelial cells in culture using an acid phosphatase assay

A simple assay is described in which small numbers of endothelial cells in culture can be determined by measuring acid phosphatase activity. After removal of the growth medium from cells grown in 96-well culture plates, the cells are lysed in buffer containing the detergent Triton X-100 and the phosphatase substrate p-nitrophenyl phosphate. After 2 h at 37 degrees C, the reaction is stopped with sodium hydroxide, and color development is determined using a rapid multiwell plate reader. The assay detects 100 to 10,000 cells per well. The assay has been used to determine growth curves for endothelial cells in the presence and absence of endothelial cell growth factor from bovine hypothalamus and to monitor fractions during purification of the growth factor. Minor modifications in the assay allow it to be fully automated.

Chapter 3 - Large-Scale Cell Culture Technology

Publisher Summary Large-scale growth of vertebrate cells in vitro offers a method for the production of biomolecules not available from other sources. This has focused the attention of researchers on the development of mass culture systems that offer opportunities for large-scale growth of such cells for production of desired biomolecules. This chapter describes a variety of mass culture systems that can be operated antibiotic-free and can be selected to meet the specific requirements for each cell/product combination. It reviews the data on the vertebrate culture methods currently available and in use for the production of biologically important molecules with a particular focus on large-scale cell culture technology. The chapter presents a review of present large-scale cell culture technology and describes the development in the laboratory of significant advances in a new, multifaceted approach to this area. It would be useful both for the establishment of large-scale cell culture facilities dedicated to the production of biomolecules for clinical or commercial application and for the research laboratory.

Enhancement of angiogenesis by bFGF in mandibular bone graft healing in the rabbit

The effectiveness of basic fibroblast growth factor (bFGF) as an in vivo angiogenic factor was evaluated in autogeneic bone grafts to the rabbit mandible. Block cortical grafts harvested from the ilium were implanted into sites in the mandibular ramus or body. The basic fibroblast growth factor was continuously introduced over a period of 14 days through subcutaneous osmotic pumps. Increased vascularity, as assessed by vessel number and depth of penetration into the grafts, was noted at 10 days postoperatively in the bFGF stimulated side as compared to contralateral control grafts. At the fourteenth postoperative day, bFGF administration was discontinued and a decrease in angiogenesis was noted over the ensuing 2 weeks so that after 1 postoperative month, little difference could be detected between the stimulated and nonstimulated grafts. Assessment of osseous healing at the 1 month postoperative interval using triple fluorochrome labeling did not reveal evidence of accelerated osteogenesis on the previously stimulated side.

Human fibroblast-derived growth factor is a mitogen and chemoattractant for endothelial cells

Human fibroblasts were found to produce a potent mitogen and chemoattractant for fetal bovine aortic endothelial cells. Homogenates from AG1523 and AG1518 foreskin, CCD18Lu lung, and CCD18Co colon fibroblasts produced half-maximal stimulation of endothelial cell growth at concentrations of 1-7 micrograms/ml. The factor was purified from large-scale cultures of the CCD18Co fibroblasts using cation exchange chromatography and heparin-Sepharose chromatography. Such preparations were mitogenic for endothelial cells in vitro at concentrations of about 5-10 ng/ml, and promoted chemotaxis at 0.1-1 ng/ml. Heparinase treatment of the cells prevented the chemotactic response. These properties suggest that the factor may be related to fibroblast growth factor.

Large-scale rotating filter perfusion system for high-density growth of mammalian suspension cultures

SummaryA system has been developed for growth and maintenance of mammalian cells in suspension culture at high density. In principle, the maintenance of constant levels of required nutrients coupled with the removal of toxic cell byproducts can support much higher suspension cell densities than may be obtained in conventional spinners. The system consisted of 4- or 40-liter reaction vessels equipped with a vertically supported rotating cylindrical filter. Agitation was provided by the magnetically driven, rotating filter. Fresh medium was supplied at a rate of 10 to 20 ml/h per 109 cells and the expended medium free of cells was withdrawn through the rotating filter. Both pH and dissolved O2 and CO2 were monitored and regulated. Walker 256 carcinosarcoma cells have been grown in these reactors to densities 10-to 30-fold greater than that obtained in Bellco spinners. In addition to high cell densities, the yield of cells per liter of medium used was 2- to 3-fold that obtained in the conventional systems. Both 4-and 40-liter versions of this reactor have been operated without the use of antibiotics. The 40-liter reactor also has been modified for chemostat operation. In a single run, for example, the Walker cell density was maintained between 6 and 10×106 cells/ml with a total yield of 8.7×1011 cells from 360 liters of medium.

Cell aggregate suspension culture for large-scale production of biomolecules

SummaryA method is described for the rapid reversible conversion of a number of continuous cell lines from anchorage-dependent growth to growth as aggregates of cells in suspension culture. Employing this technique, an inoculum of three 75-cm2 flasks of BALB/c SV3T3 cells was grown to 60 liters of aggregate suspension in 14 days. This yielded 120 ml of packed cells or 9.1×1010 cells. Similar results were obtained for other cell lines. Biomolecules such as migration-inhibition factor (MIF) and plasminogen activator were produced from these cultures.

Bacillus cereus neutral protease

Abstract 1. 1.Bacillus cereus protease has been purified from culture filtrates by acetone precipitation, treatment with active charcoal, (NH4)2SO4 fractionation and chromatography over hydroxylapatite and DEAE-cellulose. 2. 2.The enzyme has been shown to be a zinc-containing neutral protease. 3. 3.The specificity towards a series of furylacryloyl dipeptides was similar to that of the neutral proteases from Bacillus subtilis and Bacillus megaterium and thermolysin. 4. 4.The B. cereus enzyme partially crossreacted with anti-thermolysin serum with loss of activity but did not crossreact with antisera to either the B. megaterium nor B. subtilis neutral proteases.

An assay measuring the stimulation of several types of bovine endothelial cells by growth factor(s) derived from cultured human tumor cells

SummaryEndothelial cell growth factor(s) from several previously untested human tumor cell lines (i.e., SK-HEP-1, MG63, A375, TE671-C1, RD) were detected using a low cell inoculum growth assay. The final cell density in the 2-cm2 wells was determined by a highly sensitive DNA content measurement performed directly in the tissue culture plates. The sensitivity of the assay to human tumor cell growth factors depended critically on the low cell inocula, 2,000 to 5,000 cells/well. Most of the bovine endothelial cells used were cloned from primary cultures; all the cell lines obtained from various fetal and nonfetal sources responded to the growth factor(s) (up to a 16× stimulation) as well as to endothelial cell growth supplement. Dose response curves showing the cell specific response of bovine endothelial cells were obtained. The growth stimulatory activity and the in vivo chick embryo chorioallantoic membrane assay responses correlated sufficiently to imply that the assay is detecting tumor angiogenesis factor or some closely related activity. This in vitro assay should prove useful in the identification and purification of tumor-derived factors and in the elucidation of the role of these factors in the events comprising angiogenesis.

Cleavage site specificity of vertebrate collagenases.

A series of synthetic peptide substrates for vertebrate collagenase having the structure Ac-Pro-Leu-Gly-X-Leu-Gly-OC2H5, where X is Leu, Ile, Val, Phe and Ala, have been prepared. Collagenolytic enzymes from various sources cleave these substrates with differing relative rate patterns. This series of peptides should be valuable for characterization of collagenases.