Propagation Of Fibroblasts Research Articles

Optimizing the in vitro colony-forming assay for more efficient delineation of the interaction between lung epithelial stem cells and their niche.

The use of in vitro 3D organoid/colony forming assay (CFA); which mimics the in vivo environment have provided insight into the mechanisms by which lung stem cells maintain and repair the lung. In recent years, the use of CFA has markedly expanded. However, variations among laboratories in lung cell isolation methods, media used, type, origin, and processing methods of mesenchymal cells used as feeders for the epithelial colonies, and terms utilized to describe and quantify the growing colonies, have caused difficulty in reproducing results among different labs. In this study, we compared several previously described methods for lung cell isolation and culture media, to identify their influence on retrieved cells and growing colonies. We also characterized the effect of freeze/thaw, and propagation of fibroblasts on their ability to support epithelial colonies. Importantly, we suggested markers to identify fibroblast subtypes that offer the best support to alveolar stem cell proliferation. Then, we used our optimized assay to confirm the in vitro identity of recently described epithelial progenitors. We also tested the effect of hyperoxia on lung stem cells, and examined the expression of the receptors for the SARS-COV-2 virus's entry into epithelial cells, on our organoids. In summary, our findings facilitate CFA standardization, help understand how niche cell variations influence growing colonies, and confirm some of the recently described lung stem cells.

Cytomegalovirus Strain TB40/E Restrictions and Adaptations to Growth in ARPE-19 Epithelial Cells.

Despite displaying broad tropism in vivo, human cytomegalovirus (CMV) contained in bodily fluids replicates inefficiently in most cultured cell types except fibroblasts. As propagation in fibroblasts leads to the accumulation of genomic changes, a number of strains were generated by serial passaging on endothelial cells. One of these, TB40/E, was shown to contain a mixture of genetically distinct virus variants, and to retain tropism for fibroblasts, endothelial and epithelial cells. Cloning of an endotheliotropic subpopulation produced the TB40-BAC4 variant, extensively used in CMV tropism studies. Because TB40-BAC4 represents only one of the different variants comprising TB40/E, we generated a series of epithelial-cell adapted stocks derived from a TB40/E mixed stock, rather than from TB40-BAC4. Within two passages on ARPE-19 cells, virus populations were produced with the ability to enter and initiate replication with similar efficiencies in both epithelial cells and fibroblasts. Although the ability to release progeny also increased, cell-free virus yields from ARPE-19 cells remained consistently two to three-logs lower than from fibroblasts, hinting at the existence of a post-entry and post-genome synthesis block in epithelial cells. Multinucleated syncytia also rapidly appeared exclusively in ARPE-19 cell cultures, where their numbers and dimensions increased with virus passage. Irrespective of the number of infected nuclei comprising each syncytium, however, only one cytoplasmic virion assembly compartment was consistently observed, leading us to speculate that improvements in entry efficiency associated with ARPE-19 cell adaptation lead to the development of syncytia, which may negatively affect progeny release by limiting the amount of resources available to maturing virions.

Lack of XBP-1 impedes murine cytomegalovirus gene expression.

The unfolded protein response (UPR) is an endoplasmic reticulum (ER)-to-nucleus signaling cascade induced in response to ER stress. The UPR aims at restoring homeostasis, but can also induce apoptosis if stress persists. Infection by human and murine cytomegaloviruses (CMVs) provokes ER stress and induces the UPR. However, both CMVs manipulate the UPR to promote its prosurvival activity and delay apoptosis. The underlying mechanisms remain largely unknown. Recently, we demonstrated that MCMV and HCMV encode a late protein to target IRE1 for degradation. However, the importance of its downstream effector, X Box binding protein 1 (XBP-1), has not been directly studied. Here we show that deletion of XBP-1 prior to or early after infection confers a transient delay in viral propagation in fibroblasts that can be overcome by increasing the viral dose. A similar phenotype was demonstrated in peritoneal macrophages. In vivo, acute infection by MCMV is reduced in the absence of XBP-1. Our data indicate that removal of XBP-1 confers a kinetic delay in early stages of MCMV infection and suggest that the late targeting of IRE1 is aimed at inhibiting activities other than the splicing of XBP-1 mRNA.

Cryopreservation of Toxoplasma gondii in infected murine tissues

Laboratory maintenance of the RH strain of Toxoplasma gondii is generally done by passage in mice, in vitro propagation in fibroblasts, or cryopreservation of peritoneal exudates from mice infected with T. gondii. To explore alternative techniques for preserving laboratory T. gondii tachyzoites, we propose a new method of freezing tissues from infected mice. The effect of storage of T. gondii tissue tachyzoites in two different cryoprotectant combinations and at two different temperatures was studied. The liver and spleen tissues, and peritoneal exudates from mice infected with RH-GFP strain of T. gondii, suspended in RPMI 1640 medium supplemented with 12 % glycerol plus 20 % calf serum, or 12 % dimethyl sulfoxide (DMSO) plus 20 % calf serum, were stored for 3 months at -20 °C in an ordinary refrigerator or at -80 °C in a deep freezer, respectively. The viability of tissue T. gondii tachyzoites was determined by animal inoculation method, which was assessed by monitoring survival and tissue parasitemia in recipient mice. Our data showed that toxoplasma tachyzoites in the above tissues remained viable after cryopreservation in 12 % DMSO plus 20 % calf serum at -80 °C, the infectivity of tachyzoites from the tissues and peritoneal fluids was demonstrated in inoculated murine tissues. Our data indicate that freezing infected murine tissues at -80 °C provides a simple and appropriate method for preservation of T. gondii tachyzoites in laboratory without the need for costly liquid nitrogen preservation procedures.

Isolation of Fibroblasts for Coating of Meshes for Reconstructive Surgery: Differences Between Mesh Types

An extensive colonization of surgical meshes with autologous fibroblasts may reduce complications. Therefore, we aimed to establish a technique that allows isolation and propagation of fibroblasts from vaginal biopsies. Using these cells we tested the applicability of several clinically applied meshes for fibroblast coating. Fibroblasts were isolated from vaginal tissue after digestion with collagenase. Characterization was performed by immunostaining for cytokeratin 5, 6 and 14, smooth muscle actin and vimentin. A semiquantitative technique was applied to determine the degree of mesh coating 5 h and 5 weeks after seeding of fibroblasts. Seven meshes of different mesh types have been tested. Cells with a fibroblast-like morphology have been isolated from vaginal tissue and could be propagated for at least 12 passages, resulting in a total number of 1.2 x 10(7) cells. Immunostaining showed that cells were positive for the mesenchymal cell marker vimentin and negative for smooth muscle actin, as well as the epithelial cell markers cytokeratin 5, 6 and 14, supporting their classification as fibroblasts. Clear differences in fibroblast colonization between the seven tested mesh types have been observed. Polypropylene mesh Obtape showed an acceptable covering with fibroblasts. The best coating was obtained for xenograft-based meshes, but under cell-culture conditions the mesh showed signs of decomposition. We have established a technique that allows isolation and propagation of vaginal fibroblasts. The result of vaginal fibroblast colonization of allograft-based meshes strongly depends on the mesh type, whereby the best coating could be achieved for a polypropylene mesh.

Human cytomegalovirus entry into epithelial and endothelial cells depends on genes UL128 to UL150 and occurs by endocytosis and low-pH fusion.

Human cytomegalovirus (HCMV) replication in epithelial and endothelial cells appears to be important in virus spread, disease, and persistence. It has been difficult to study infection of these cell types because HCMV laboratory strains (e.g., AD169 and Towne) have lost their ability to infect cultured epithelial and endothelial cells during extensive propagation in fibroblasts. Clinical strains of HCMV (e.g., TR and FIX) possess a cluster of genes (UL128 to UL150) that are largely mutated in laboratory strains, and recent studies have indicated that these genes facilitate replication in epithelial and endothelial cells. The mechanisms by which these genes promote infection of these two cell types are unclear. We derived an HCMV UL128-to-UL150 deletion mutant from strain TR, TRdelta4, and studied early events in HCMV infection of epithelial and endothelial cells, and the role of genes UL128 to UL150. Analysis of wild-type TR indicated that HCMV enters epithelial and endothelial cells by endocytosis followed by low-pH-dependent fusion, which is different from the pH-independent fusion with the plasma membrane observed with human fibroblasts. TRdelta4 displayed a number of defects in early infection processes. Adsorption and entry of TRdelta4 on epithelial cells were poor compared with those of TR, but these defects could be overcome with higher doses of virus and the use of polyethylene glycol (PEG) to promote fusion between virion and cellular membranes. High multiplicity and PEG treatment did not promote infection of endothelial cells by TRdelta4, yet virus particles were internalized. Together, these data indicate that genes UL128 to UL150 are required for HCMV adsorption and penetration of epithelial cells and to promote some early stage of virus replication, subsequent to virus entry, in endothelial cells.

Modification of human cytomegalovirus tropism through propagation in vitro is associated with changes in the viral genome.

Following extensive propagation in fibroblasts, human cytomegalovirus (HCMV) loses tropism for a number of otherwise natural host cells, in particular, endothelial cells. In this study, the hypothesis was tested that loss of endothelial tropism is associated with the appearance of genomic variants. Initial quantitative focus expansion assays on endothelial monolayers demonstrated that, while the laboratory strains AD169 and Towne failed to form detectable foci, 29 out of 30 recent clinical HCMV isolates had the potential to expand in endothelial cell culture. By long-term adaptation in fibroblast cultures, nonendotheliotropic strains could be selected from clinical HCMV isolates, while long-term endothelial-adapted strains of the same isolates retained both fibroblast tropism and endothelial tropism. Such differentially adapted isolate pairs always displayed genomic differences in restriction fragment length analyses. Coinfection of endothelial cells by two nonendotheliotropic HCMV strains yielded an endotheliotropic recombinant HCMV variant combining portions of the genomes of both parental viruses. When DNA purified from various isolates was transfected into fibroblasts, progeny virus retained the specific tropism of parental virus from which the DNA was isolated. These findings demonstrate that endothelial tropism is an inherent property of most clinical HCMV isolates and is determined by the viral genome. Although the specific determinants of HCMV cell tropism are still unknown, this study provides the first evidence for a genetic contribution.

Virus Growth in Tissue Culture Fibroblasts

Discussion and Summary The results of an investigation of the ability of a strain of influenza A virus and of herpes simplex virus to propagate in fibroblasts cultured from different tissues of the chick embryo have been presented. The data indicate that influenza virus, possessing rather narrow tissue trophisms, does not grow in fibroblasts derived from susceptible or nonsusceptible tissues of the chick embryo. Herpes virus, which has a broader affinity for different cell types, was readily cultivated and produced cellular cytopathogenicity in similar cultures. Influenza virus failed to multiply in fibroblasts from the lung, heart, skeletal muscle, or cornea tissue, although ability of this virus to grow in respiratory cells of the embryo itself has been reported (11). The same virus seemed to propagate in primary cultures of lung and cornea, presumably in susceptible cells carried over from the animal host. The growth of the virus in primary cultures of lung and cornea but not in skeletal muscle or heart agrees with results of Pearson and Enders (12). Although influenza virus appeared to grow in the primary explants of lung and cornea, the virus did not seem to produce cytopathogenic changes in the fibroblast outgrowth of these tissues Fibroblasts derived from lung tissue supported the growth of herpes simplex virus in plasma clot cultures; evidence for its propagation in fibroblasts from skeletal muscle was less striking. Cellophane cultures of both lung cells and heart fibroblasts produced unequivocal growth of the virus. These observations conform with the broad affinity of herpes virus for various cell types in the chick embryo described by Anderson (13) who found herpes inclusion bodies in cardiac muscle, in the interstitial cells of the lung, in cells of striated muscle and in other mesodermal elements. Similar findings for this strain of herpes virus and various cell types in newborn infants were described in an earlier paper from this laboratory (14). In contrast to influenza virus, not only does herpes virus multiply in fibroblasts, but with viral propagation destruction of the cells occurs until few normal cells remain and virus production ceases. Similar observations have been made by Bang and Gey (3), using Eastern equine encephalomyelitis virus in a cultured line of tumor cells. A direct cytopathogenic effect of herpes virus on the cells of plasma clot cultures was not evident, although in one experiment subcultures of cells from the infected cultures did not grow as well as uninfected control cultures. It seems, therefore, that cytopathogenic effects may be demonstrated more clearly in non-plasmatic cultures. This observation also has been made by Gey and Bang (2) who showed that Newcastle disease virus in plasma clot cultures of chick muscle fibroblasts may not be able to reach and destroy isolated strands of cells buried in the clot. The cytopathogenic effect of poliomyelitis viruses on the fibroblastic outgrowth of human embryonic tissues was demonstrated by Robbins and coworkers (15). Other workers (16, 17) have confirmed these findings and have demonstrated that strains of poliomyelitis viruses show differences in cellular pathogenicity for the flbroblast outgrowths. Since growth of the viruses in fibroblasts alone was not reported, the question remains whether the viruses were growing in the explanted tissues and exerting a toxic effect on the fibroblast outgrowth or whether degeneration of the fibroblasts resulted from the viruses growing within them. Either or both of two possibilities may account for the failure of influenza virus and the ability of herpes virus to multiply in fibroblast cultures. First, the fibroblasts may have been derived from connective tissue elements which were or were not susceptible to these viruses in the animal host. Second, the fibroblasts may have originated from susceptible cells in vivo but lost their ability to support the propagation of influenza virus after prolonged culture and transfers in vitro. The solution of this problem depends on whether fibroblasts derived from different tissues have varying inherent characteristics or whether they represent a common cell prototype in the animal host and subsequently develop individual characteristics in vitro (6).