Mineralization of marrow-stromal osteoblasts MBA-15 on three-dimensional carriers.

The present study describes a new three-dimensional (3-D) culture system that enables the maintenance and phenotypic expression of bone marrow stromal osteoblasts. This culture substratum is advantageous in that it provides suitable conditions for attachment, growth, and differentiation of cells forming 3-D layers. The MBA-15 cell line was grown in unlimited quantities on 3-D Fibro-Cel carriers. These cells mineralized when exposed to ascorbic acid and beta-glycerophosphate (beta GP). Under these mineralization conditions, mRNA expressions of procollagen alpha 2(I) and [3H]-proline-labeled protein were increased. The expression of mRNA for osteonectin and to a lesser extent, for osteopontin was increased, whereas alkaline phosphatase and biglycan remained unaffected under similar conditions. Exposure of mineralizing cultures to dexamethasone reduced mRNA of procollagen alpha 2 (I) and osteonectin to control level. Scanning electron microscopy revealed that cells were grown along the fabric's fibers and produced collagen fibrils. Under appropriate conditions, extensive mineralization had taken place. The mineralization process involves the formation of calcospherites, and correlates with an increase in calcium content. The Fibro-Cel carriers enable formation of 3-D architecture and mineralized tissue in vitro.

Dynamic changes in cytokine secretion by stromal cells during prolonged maintenance under protein-free conditions.

Stromal cells of bone marrow origin produce a variety of known cytokines and some factors exhibiting apparently new biological activities. Several of these were identified by the study of cell to cell interactions and were not found in detectable amounts in media conditioned by the cells. We describe here a culture system that enables the release of stromal cytokines into medium free of any added proteins and supplemented with peptides from casein hydrolysate (0.1%). The absence of serum proteins allows extensive concentration and monitoring of activities that are otherwise undetectable. Stromal cells of the MBA-2.1 clonal cell line were seeded in a stationary bed reactor packed with a carrier of non-woven fabric matrix. After a proliferation phase with serum containing medium, the cells were maintained for over 10 months in protein-free medium. Throughout this extended incubation in the absence of serum or serum replacing proteins, stromal cells retained their viability and continuously released transforming growth factor-beta (TGF-beta), macrophage-colony stimulating factor (M-CSF) and restrictin-P, a cytotoxic factor that specifically arrested the growth of plasmacytoma cells. In addition, interleukin-6 (IL-6) was first undetectable, and later in culture its titer reached a maximum of 180,000 international units (IU)/ml. Concomitantly, the production of restrictin-P diminished and reached its lowest levels at the end of 10 months. The results may imply a possible causal relationship between the expression of IL-6 and restrictin-P, since no similarly significant changes were observed in the titers of M-CSF and TGF-beta. This novel bioreactor system may be adaptable for efficient production of different cytokines under absolute serum-free conditions.

Use of a stationary bed reactor and serum-free medium for the production of recombinant proteins in insect cells.

Insect cells (Spodoptera frugiperda) have been cultured in a stationary bed reactor, packed with a fibrous polyester carrier. When the bioreactor was perfused with serum-supplemented medium, a cell density of 6 x 10(6) cells ml-1 packed carrier was reached. Scanning electron microscopy investigations have shown that the insect cells grew along the three-dimensionally oriented fibers of the Fibra-cel carrier. After infection of the logarithmically growing cells with a recombinant baculovirus (Autographa californica) containing the gene coding for beta-galactosidase, the medium in the bioreactor was changed to serum-free medium. At day 13 postinfection (p.i.), a beta-galactosidase level of 320 microgram ml-1 and, at day 17 p.i., a virus titer of 2.1 x 10(8) TCID50 units ml-1 (day 17 p.i.) were reached. In another bioreactor, operated in a similar way but with serum-containing medium, a beta-galactosidase concentration of 360 microgram ml-1 and a virus titer of 2.3 x 10(8) TCID50 units ml-1 were obtained. These results indicate the potential use of this production system for the production of recombinant protein and baculovirus in insect cells.

Restrictin-P: the first member of a putative family of novel inhibitors.

MBA-2.1 cells produce an activity, designated restrictin-P, which is specifically inhibitory to the growth of plasmacytomas and mature B cell lymphomas. We examined whether the activity of this stromally derived glycoprotein could be attributed to a well-characterized growth factor. Restrictin-P-producing cells were therefore screened for the expression of transcripts of a variety of growth suppressors. With the exception of TGF-beta 1, none was produced in detectable amounts by these cells. Furthermore, recombinant forms of the inhibitory molecules tested did not exert a biological effect similar to that of restrictin-P. Restrictin-P was shown to elicit a G0/G1 arrest in the cell cycle of its target cells, as soon as 24 h after their exposure to the inhibitor. This effect could not be mimicked by TGF-beta 1. We suggest that restrictin-P is part of a novel family of inhibitors which are required for the maintenance of cell-type specificities in the hematopoietic microenvironment.