Establishment of tight junctions between cells from different animal species and different sealing capacities

Summary Epithelial cells establish tight junctions (TJs) that offer an ample range of transepithelial electrical resistances (TER), in adjustment to physiological requirements. In the present work, we demonstrate that cells from different animal origins, co-cultured in monolayers, can make sealed TJs, suggesting that this structure has a basic universal structure. TJs cannot be established, however, if one of the partners does not normally express TJs, indicating that each neighbor has to contribute its moiety. Furthermore, we observe that clones of the same cell line, with widely different values of TER, do not differ, in the number and length of their junctional trands, suggesting that the difference is due to their ability to express ionic channels traversing their strands. The value of TER achieved in mixed monolayers of cells of the same or different lines is the one that may be expected by taking into account the proportion of each type in the mixture and adding in parallel the electrical resistance that they exhibit in pure monolayers. Therefore, epithelial TJs appear to behave as parallel resistances.     

Comparative studies of two types of “spontaneous” malignant alteration of ST/A mouse lung fibroblasts propagated in vitro

Summary Two types of apparently spontaneous malignant alterations of fibroblastlike ST/a mouse lung cells (ST-L cells) grown in vitro are described. One type is characterized by a high tumorigenic potential of the altered cells in nonconditioned syngeneic recipients, a fibro-blastlike morphology with cell surface showing very few microvilli by scanning electron-microscopy (SEM), and a growth pattern typical of nontransformed cells. These cells were described as R⁻ cells. The other type is characterized by a low tumorigenic potential in nonconditioned, immunocompetent syngeneic recipients, rounding up of the cells which by SEM showed numerous microvilli on the surface, and a growth pattern typical of transformed cells. These cells were described as round cells or R⁺ cells. In immunoincompetent mice, R⁺ cells readily produced sarcomas, which grew faster than those produced by R⁻ cells. Both types of ST-L cells expressed murine leukemia virus (MuLV) when tested in a peroxidase anti-p30 plaque test. The concentration of murine leukemia virus envelope glycoprotein (gp70) has previously (5) been shown to be threefold higher in R⁺ cells compared to R⁻ cells. Furthermore, round-cell transformation was accompanied by the development of crossreacting rejection antigens protective against a secondary challenge with Ehrlich ascites tumor and with syngeneic dimethylbenzanthracene induced ST/a mouse leukemia (STABAL). A similar protection was obtained by preimmunization with a cloned embryonic feral mouse cell line (SC-1) infected with ST-L virus as well as with virus-free SC-1 cells, suggesting the presence of rejection antigens both of viral (gp70) and nonviral origin. Sponsored by the Danish Cancer Society. Supported by the Kankerfonds van de Algemene Spaar-en Lijfrentekas. The SC-1 cell line was originally provided by contract E-73-2001-N01 within the Special Virus-Cancer Program NIH, USPHS, through the courtesy of Walter A. Nelson-Rees, Naval Biomedical Research Laboratory, Oakland, California, and Wallace P. Rowe, Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland. The financial support of the Daell Foundation, the Schepler Foundation, the Jorgen Holm Foundation and the Danish National Research Foundation is gratefully acknowledged.     

Cell surface modifications in the epithelium of rat ventral prostate during adaptation to in vitro conditions: An ultrastructural study

Summary Sequential changes in epithelial cells of collagenase-dissociated rat ventral prostate were studied by thin-section and freeze-fracture electron microscopy. Epithelial cells did not attach to the substrate for 48 h. Pelleted cells obtained 1, 24, and 48 h after dissociation were assigned to three categories depending on morphology and cellular associations. (a) Solitary epithelial cells degenerated as determined by extensive vacuolization in the cytoplasm and aggregation of intramembranous particles (IMP). (b) Epithelial clusters consisted of a homogeneous population of well-maintained, closely packed cells. Aggregation of IMP was minimal. Tight junctions that formed between cells at the periphery of the clusters appeared normal and provided an effective permeability barrier demonstrated by the exclusion of ruthenium red tracer. (c) Tissue fragments were comprised of varying combinations of epithelial, endothelial, and smooth muscle cells as well as fibroblasts and erythrocytes. Maintenance of tissue fragments was variable. Plasma membranes often displayed aggregated IMP and proliferated tight junctional strands. An effective permeability barrier was absent. After the 48 h “latent period”, epithelial cells in the clusters lost interdependence, disassociated from one another, and attached to the substrate. These isolated cells, which did not display aggregated IMP, retained the ability to form an effective permeability barrier upon reaching confluency. During the first 48 h, epithelial cells did not tolerate solitary existence, yet as participants in clusters they were well maintained. After this interval, they no longer required interactions with neighbors in order to survive. These results indicate that under our experimental conditions, an adaptation period is required by prostatic epithelial cells. The enhanced quality of maintenance associated with epithelial clusters suggests that control over the internal microenvironment, provided by a tight junctional barrier, may be important during the initial period of adaptation in vitro. This work was supported by funds from NIH Grants CA 26063, 29513, and CA 15776; National Cancer Institute; DHHS; and Charlton Fund, Tufts University School of Medicine (awarded to P. K.).     

Techno-economic modeling and assessment of cultivated meat: Impact of production bioreactor scale

Increases in global meat demands cannot be sustainably met with current methods of livestock farming, which has a substantial impact on greenhouse gas emissions, land use, water consumption, and farm animal welfare. Cultivated meat is a rapidly advancing technology that produces meat products by proliferating and differentiating animal stem cells in large bioreactors, avoiding conventional live-animal farming. While many companies are working in this area, there is a lack of existing infrastructure and experience at commercial scale, resulting in many technical bottlenecks such as scale-up of cell culture and media availability and costs. In this study, we evaluate theoretical cultivated beef production facilities with the goal of envisioning an industry with multiple facilities to produce in total 100,000,000 kg of cultured beef per year or ~0.14% of the annual global beef production. Using the computer-aided process design software, SuperPro Designer®, facilities are modeled to create a comprehensive analysis to highlight improvements that can lower the cost of such a production system and allow cultivated meat products to be competitive. Three facility scenarios are presented with different sized production reactors; ~42,000 L stirred tank bioreactor (STR) with a base case cost of goods sold (COGS) of $35/kg, ~211,000 L STR with a COGS of $25/kg, and ~262,000 L airlift reactor (ALR) with a COGS of $17/kg. This study outlines how advances in scaled up bioreactors, alternative bioreactor designs, and decreased media costs are necessary for commercialization of cultured meat products.     

Cell cycle reactivation in cultured pea embryo axes. Effect of abscisic acid

Summary We used cultured pea embryo axes to study the effect of exogenous abscisic acid (ABA) on the reactivation of cell proliferation at germination. ABA (100 μM) was administered in a complete medium or in a medium without KNO₃ (the main source of K⁺ in the complete medium). In the presence of ABA, only a few meristematic root tip nuclei progressed through the cell cycle during the first 24 h of germination. Compared to the control the percentage of G₀/G₁ cells remained high. In the absence of KNO₃, the inhibitory effect of ABA on the cell cycle increased and the transition from G₁ to S was almost completely inhibited after 24 h of germination. Moreover, the amount of a 47 kDa nuclear protein remained high in embryos treated with ABA whereas it decreased in the control.     

Effect of transforming growth factor-β3 on mono and multilayer chondrocytes

Articular cartilage is an avascular and flexible connective tissue found in joints. It produces a cushioning effect at the joints and provides low friction to protect the ends of the bones from wear and tear/damage. It has poor repair capacity and any injury can result pain and loss of mobility. Transforming growth factor-beta (TGF-β), a cytokine superfamily, regulates cell function, including differentiation and proliferation. Although the function of the TGF-βs in various cell types has been investigated, their function in cartilage repair is as yet not fully understood. The effect of TGF-β3 in biological regulation of primary chondrocyte was investigated in this work. TGF-β3 provided fibroblastic morphology to chondrocytes and therefore overall reduction in cell proliferation was observed. The length of the cells supplemented with TGF-β3 were larger than the cells without TGF-β3 treatment. This was caused by the fibroblast like cells (dedifferentiated chondrocytes) which occupied larger areas compared to cells without TGF-β3 addition. The healing process of the model wound closure assay of chondrocyte multilayer was slowed down by TGF-β3, and this cytokine negatively affected the strength of chondrocyte adhesion to the cell culture surface.     

Retention of cartilage oligomeric matrix protein (COMP) and cell death in redifferentiated pseudoachondroplasia chondrocytes

Cartilage oligomeric matrix protein (COMP) is a large extracellular glycoprotein that is found in the territorial matrix surrounding chondrocytes. Two skeletal dysplasias, pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EDM1) are caused by mutations in the calcium binding domains of COMP. In this study, we identified two PSACH mutations and assessed the effect of these mutations on redifferentiated chondrocyte structure and function. We confirmed, in vitro, that COMP is retained in enormous cisternae of the rough endoplasmic reticulum (rER) and relatively absent in the PSACH matrix. The rER accumulation may compromise chondrocyte function, leading to chondrocyte death. Moreover, while COMP appears to be deficient in the PSACH matrix, the matrix appeared to be normal but the over-all quantity was reduced. These results suggest that the abnormality in linear growth in PSACH may result from decreased chondrocyte numbers which would also affect the amount of matrix produced.