Characterization of Human Anulus Fibrosus– and Nucleus Pulposus–Derived Cells During Monolayer Expansion and in Hydrogel Cultures

2014 ◽  
Vol 5 ◽  
pp. BTRI.S13604 ◽  
Author(s):  
Gundula Schulze-Tanzil ◽  
Marion Lemke ◽  
Carola Meier ◽  
Wolfgang Ertel ◽  
Benjamin Kohl ◽  
...  
Keyword(s):  
Nucleus Pulposus ◽  
Cell Types ◽  
Type I ◽  
Collagen Gels ◽  
Anulus Fibrosus ◽  
Tenascin C ◽  
In Situ Conditions ◽  
Extracellular Matrix Components

In vitro-expanded intervertebral disc (IVD) cells could be a source for disc repair. However, IVD cell characterization still remains challenging and is demanded to detect phenotypical shifts. Therefore, the aim of the present study was to determine IVD cell expression profile during two- and three-dimensional culturing in direct comparison to in situ conditions. Human IVD tissue was analyzed immunohistologically and anulus fibrosus (AF) and nucleus pulposus (NP) cells were isolated and characterized for cytoskeletal architecture and expression of typical markers (type I, II, and III collagens, aggrecan, decorin, cartilage oligomeric protein, the chondrogenic transcription factor sox9, the tendon markers scleraxis and tenascin C) during 6 monolayer passages using real-time detection polymerase chain reaction and/or immunolabellings. Cells were introduced in alginate and collagen hydrogels and cell morphology and viablility was determined after 7 days. In addition to typical extracellular matrix components, IVD tissue and isolated cells revealed scleraxis expression. In early passages of cell expansion, genes of sox9, scleraxis, and the small proteoglycan decorin were expressed higher, but type I and III collagen genes were expressed lower in NP cells compared with AF cells. However, in passage 6, actin stress fibers increased and the expression levels of sox9 were nearly similar in NP and AF cells. The immunolabeling indicated that the fibroblast marker tenascin C could only be detected in vitro in both cell types but not in situ. Decorin protein expression decreased in both cell types in vitro in passage 6. IVD cells survived in both hydrogel cultures, and some cells elongated in collagen gels.

scholarly journals In Vitro Studies of Mechanisms of Lung Injury in the Rodent

1991 ◽  
Vol 19 (4_part_1) ◽  
pp. 419-427 ◽  
Author(s):  
Leah A. Cohn ◽  
Kenneth B. Adler
Keyword(s):  
Epithelial Cells ◽  
Cell Types ◽  
Lung Cells ◽  
Serum Free Medium ◽  
Collagen Gels ◽  
Respiratory Epithelial Cells ◽  
Respiratory Epithelial ◽  
Air Liquid Interface

In order to better define the responses of lung cells to potentially pathogenic insults, primary cell cultures of dissociated respiratory epithelial cells have been established. These epithelial cells have been obtained from various areas of the respiratory tract ranging from the trachea to the alveolus and the cultures have been demonstrated to mimic the differentiated state of these cell types as observed in situ. Several procedures which enhance the differentiated state have been evaluated, which include maintenance on more physiologically-relevant substrata, such as collagen gels, use of defined serum-free medium and use of air/liquid interface systems. These approaches have allowed intracellular responses of respiratory epithelium to toxic insult to be better defined.

scholarly journals The biosynthesis of extracellular-matrix components by bovine retinal endothelial cells displaying distinctive morphological phenotypes

1986 ◽  
Vol 235 (2) ◽  
pp. 375-383 ◽  
Author(s):  
A E Canfield ◽  
A M Schor ◽  
S L Schor ◽  
M E Grant
Keyword(s):  
Endothelial Cells ◽  
Type I ◽  
Collagen Gels ◽  
Early Passage ◽  
Experimental Conditions ◽  
Retinal Endothelial Cells ◽  
Type Iv ◽  
Extracellular Matrix Components ◽  
Cells Cultured

Previous studies have indicated that the morphology and behaviour of bovine retinal microvessel endothelial cells are influenced by culture conditions in vitro. Data are presented here concerning the biosynthesis of matrix macromolecules by bovine retinal endothelial cells cultured under conditions in which the cells display either the ‘cobblestone’ or the ‘sprouting’ phenotype. Newly synthesized matrix proteins were identified by their characteristic electrophoretic mobilities, immunoprecipitation with specific antibodies, susceptibilities to enzymic digestions and chromatographic behaviour. Type IV procollagen was the major collagenous species synthesized by early-passage cells forming a ‘cobblestone’ monolayer. In contrast, cells displaying the ‘sprouting’ morphology switched to the predominant synthesis of interstitial fibrillar collagens (types I and III). Fibronectin was synthesized by retinal endothelial cells under all the experimental conditions studied. A non-collagenous glycoprotein of Mr approx. 47,000 was also a major biosynthetic product of these cells. The synthesis of thrombospondin was very much dependent on the nature of the substratum on which the cells were cultured. This glycoprotein was synthesized in large amounts by ‘cobblestone’ endothelial cells cultured on gelatin-coated dishes, whereas its synthesis was markedly decreased by culturing the cells on collagen gels, and the protein appeared to be absent when the cells were plated within collagen gels (‘sprouting’ cells). Late-passage retinal cells synthesized predominantly type I procollagen, variable amounts of type III procollagen and only traces of type IV procollagen, irrespective of whether the cells displayed a ‘cobblestone’ or ‘sprouting’ morphology.

Abstract 418: Co-Culture of Endothelial Cells, Smooth Muscle Cells and Monocytes in Collagen Scaffold: A Novel i n vitro Model of Atherosclerosis

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Martin Liu ◽  
Angelos Karagiannis ◽  
Matthew Sis ◽  
Srivatsan Kidambi ◽  
Yiannis Chatzizisis
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Type I Collagen ◽  
Smooth Muscle Actin ◽  
Muscle Cells ◽  
Type I ◽  
Collagen Gels ◽  
Human Coronary Artery

Objectives: To develop and validate a 3D in-vitro model of atherosclerosis that enables direct interaction between various cell types and/or extracellular matrix. Methods and Results: Type I collagen (0.75 mg/mL) was mixed with human artery smooth muscle cells (SMCs; 6x10 5 cells/mL), medium, and water. Human coronary artery endothelial cells (HCAECs; 10 5 /cm 2 ) were plated on top of the collagen gels and activated with oxidized low density lipoprotein cholesterol (LDL-C). Monocytes (THP-1 cells; 10 5 /cm 2 ) were then added on top of the HCAECs. Immunofluorescence showed the expression of VE-cadherin by HCAECs (A, B) and α-smooth muscle actin by SMCs (A). Green-labelled LDL-C particles were accumulated in the subendothelial space, as well as in the cytoplasm of HCAECs and SMCs (C). Activated monocytes were attached to HCAECs and found in the subendothelial area (G-I). Both HCAECs and SMCs released IL-1β, IL-6, IL-8, PDGF-BB, TGF-ß1, and VEGF. Scanning and transmission electron microscopy showed the HCAECs monolayer forming gap junctions and the SMCs (D-F) and transmigrating monocytes within the collagen matrix (G-I). Conclusions: In this work, we presented a novel, easily reproducible and functional in-vitro experimental model of atherosclerosis that has the potential to enable in-vitro sophisticated molecular and drug development studies.

scholarly journals Revisiting the role of IRF3 in inflammation and immunity by conditional and specifically targeted gene ablation in mice

2018 ◽  
Vol 115 (20) ◽  
pp. 5253-5258 ◽  
Author(s):  
Hideyuki Yanai ◽  
Shiho Chiba ◽  
Sho Hangai ◽  
Kohei Kometani ◽  
Asuka Inoue ◽  
...  
Keyword(s):  
Immune Cell ◽  
Cell Types ◽  
Type I ◽  
Type I Ifn ◽  
Cell Type ◽  
Gene Ablation ◽  
Cell Type Specific

IFN regulatory factor 3 (IRF3) is a transcription regulator of cellular responses in many cell types that is known to be essential for innate immunity. To confirm IRF3’s broad role in immunity and to more fully discern its role in various cellular subsets, we engineered Irf3-floxed mice to allow for the cell type-specific ablation of Irf3. Analysis of these mice confirmed the general requirement of IRF3 for the evocation of type I IFN responses in vitro and in vivo. Furthermore, immune cell ontogeny and frequencies of immune cell types were unaffected when Irf3 was selectively inactivated in either T cells or B cells in the mice. Interestingly, in a model of lipopolysaccharide-induced septic shock, selective Irf3 deficiency in myeloid cells led to reduced levels of type I IFN in the sera and increased survival of these mice, indicating the myeloid-specific, pathogenic role of the Toll-like receptor 4–IRF3 type I IFN axis in this model of sepsis. Thus, Irf3-floxed mice can serve as useful tool for further exploring the cell type-specific functions of this transcription factor.

Temporal changes in the expression of the insulin-like growth factor II gene associated with tissue maturation in the human fetus

Development ◽  
1989 ◽  
Vol 106 (3) ◽  
pp. 543-554 ◽  
Author(s):  
A.L. Brice ◽  
J.E. Cheetham ◽  
V.N. Bolton ◽  
N.C. Hill ◽  
P.N. Schofield
Keyword(s):  
Growth Factor ◽  
Cell Types ◽  
Specific Cell ◽  
Insulin Like Growth Factor ◽  
Vitro Fertilization ◽  
Age Related ◽  
Factor Ii ◽  
Metanephric Blastema

The insulin-like growth factors are broadly distributed in the human conceptus and are thought to play a role in the growth and differentiation of tissues during development. Using in situ hybridization we have shown that a wide variety of specific cell types within tissues express the gene for insulin-like growth factor II at times of development from 18 days to 14 weeks of gestation. Examination of blastocysts produced by in vitro fertilization showed no expression, thus bracketing the time of first accumulation of IGF-II mRNA to between 5 and 18 days postfertilization. The pattern of IGF-II expression shows specific age-related differences in different tissues. In the kidney, for example, expression is found in the cells of the metanephric blastema which is dramatically reduced as the blastema differentiates. The reverse is also seen, and we have noted an increase in expression of IGF-II in the cytotrophoblast layer of the placenta with gestational age. The sites of expression do not correlate with areas of either high mitotic activity or specific types of differentiation, but the observed pattern of expression in the kidney, adrenal glands and liver suggests an explanation for the abnormally high IGF-II mRNA expression in developmental tumours such as Wilms' tumour.

scholarly journals In vitro production of human dermal equivalent

2010 ◽  
Vol 63 (7-8) ◽  
pp. 459-464 ◽  
Author(s):  
Zoran Milosavljevic ◽  
Biljana Ljujic
Keyword(s):  
Vitamin C ◽  
Collagen Type ◽  
Collagen Type I ◽  
Type I ◽  
Collagen Gels ◽  
In Vitro Production ◽  
Dermal Equivalent ◽  
Enzymatic Dissociation ◽  
Vitro Production

Introduction. Human dermal tissue is composed of loose and dense connective tissue. Main cell populations are fibroblasts and the dominant fibers are built from collagen type I. The aim of our study was to determine the precise method and time frame for the in vitro production of human dermal equivalent and to investigate the effects of ratio of structural elements and vitamin C on characteristics of the engineered tissue. Material and methods. Primary isolation of the foreskin fibroblasts was performed by explant method and enzymatic dissociation. Various collagen gels were obtained by mixing cells (from 25x103 to 200x103/ml) and neutralized collagen type I (from 2 to 4 mg/ml), with or without vitamin C. The routine histological and morphometrical examination was performed. Results. Enzymatic dissociation of the foreskin proved to be a faster method for production of desired number of fibroblasts (7.5x105 for 4 days). The contraction of collagen-gels started from day one through day seven and was dependent on cell and collagen concentration with higher density gels being contracted to a greater extent, except for the lowest/highest values. The best result was achieved with 100x103 cells and 2 mg/ml collagen. Vitamin C at 50 ?g/ml had no effect on speed of tissue formation. Conclusion. A precise approach that mimic the in vivo conditions is needed for the in vitro production of the dermal equivalent suitable for the possible treatment of tissue defects. Nearly ten days are necessary from the donor tissue dissociation to the final product.

scholarly journals Distinct purinergic signaling pathways in prepubescent mouse spermatogonia

2016 ◽  
Vol 148 (3) ◽  
pp. 253-271 ◽  
Author(s):  
David Fleck ◽  
Nadine Mundt ◽  
Felicitas Bruentgens ◽  
Petra Geilenkirchen ◽  
Patricia A. Machado ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Purinergic Signaling ◽  
Cell Communication ◽  
Atp Release ◽  
Cell Types ◽  
Control Male ◽  
Electrophysiological Recordings ◽  
Unique Approach

Spermatogenesis ranks among the most complex, yet least understood, developmental processes. The physiological principles that control male germ cell development in mammals are notoriously difficult to unravel, given the intricate anatomy and complex endo- and paracrinology of the testis. Accordingly, we lack a conceptual understanding of the basic signaling mechanisms within the testis, which control the seminiferous epithelial cycle and thus govern spermatogenesis. Here, we address paracrine signal transduction in undifferentiated male germ cells from an electrophysiological perspective. We identify distinct purinergic signaling pathways in prepubescent mouse spermatogonia, both in vitro and in situ. ATP—a dynamic, widespread, and evolutionary conserved mediator of cell to cell communication in various developmental contexts—activates at least two different spermatogonial purinoceptor isoforms. Both receptors operate within nonoverlapping stimulus concentration ranges, display distinct response kinetics and, in the juvenile seminiferous cord, are uniquely expressed in spermatogonia. We further find that spermatogonia express Ca2+-activated large-conductance K+ channels that appear to function as a safeguard against prolonged ATP-dependent depolarization. Quantitative purine measurements additionally suggest testicular ATP-induced ATP release, a mechanism that could increase the paracrine radius of initially localized signaling events. Moreover, we establish a novel seminiferous tubule slice preparation that allows targeted electrophysiological recordings from identified testicular cell types in an intact epithelial environment. This unique approach not only confirms our in vitro findings, but also supports the notion of purinergic signaling during the early stages of spermatogenesis.

Polarized Light Microscopy for Analyzing Tissue Mechanics During In Vitro Acupuncture

2008 ◽  
Author(s):  
Lowell Taylor Edgar ◽  
Margaret Julias ◽  
David I. Shreiber ◽  
Helen M. Buettner
Keyword(s):  
Connective Tissue ◽  
Light Microscopy ◽  
Type I Collagen ◽  
Polarized Light ◽  
Tissue Mechanics ◽  
Polarized Light Microscopy ◽  
Tissue Response ◽  
Type I ◽  
Collagen Gels

Acupuncture is a traditional therapy originating in China almost 2000 years ago. Acupuncture has slowly been growing in popularity in the West, and clinical evidence has shown the potential for acupuncture as a low-cost ‘alternative’ therapy for an assortment of ailments [1]. The practice of acupuncture involves inserting fine needles into the skin followed by needle manipulation, usually by rotation. Recent studies by Langevin et al demonstrate that this rotation causes the subcutaneous connective tissue to couple to and wind around the needle [2–4], which suggests that mechanotransduction in the connective tissue might play a role in the therapeutic mechanisms that underlay acupuncture [2, 3]. To begin to decompose and quantify this complex mechanism at the tissue level in a controlled setting, we have simulated acupuncture in type I collagen gels in vitro, and have developed algorithms to quantify the tissue response following imaging with polarized light microscopy (PLM).

Electroneutral H+ secretion in distal tubule of Amphiuma

1987 ◽  
Vol 252 (4) ◽  
pp. F691-F699 ◽  
Author(s):  
B. Stanton ◽  
A. Omerovic ◽  
B. Koeppen ◽  
G. Giebisch
Keyword(s):  
Basolateral Membrane ◽  
Apical Cell ◽  
Distal Tubule ◽  
Cell Types ◽  
Disulfonic Acid ◽  
Type I ◽  
Cellular Mechanisms ◽  
Apical Cell Membrane ◽  
Membrane Type

This study examines the cellular mechanisms of acid secretion by the in vitro perfused late distal tubule of Amphiuma kidney. Acidification of tubule fluid occurred against an electrochemical gradient of 16 mV; thus H+ secretion was active. Amiloride (1 mM) or a reduction of sodium in the perfusion fluid (from 83.7 to 7.7 mM) partially reduced acidification. Amiloride, in the presence of low sodium, completely inhibited acidification. Furthermore, acetazolamide and ouabain in the bath solution (0.1 mM) also inhibited acidification. Conductive properties of the epithelium and of individual cell membranes were determined by means of cable analysis of the tubule and intracellular voltage recordings. The transepithelial voltage and resistance averaged -0.4 +/- 0.4 mV, lumen negative, and 7,147 +/- 845 omega X cm, respectively. Two functionally different cell types were identified by intracellular microelectrodes. Type I cells had a basolateral membrane voltage (Vbl) of -67.7 mV. As determined by ion substitution experiments, the basolateral membrane was conductive to K+ and Cl-. This cell also had a 4-acetamido-4'-isothiocyanostilbene-2-2'-disulfonic acid (SITS)-sensitive Na+-dependent HCO3- exit pathway in the basolateral membrane. Type II cells had a Vbl of -76.1 mV (P less than 0.05 vs. type I) and the basolateral membrane was conductive to K+ and Cl- but not to HCO3-. HCO3- movement across the basolateral membrane in this cell may occur by electroneutral Cl- -HCO3- exchange. The apical cell membrane of both cell types did not contain measurable ionic conductances, as evidenced by a high value of apical membrane fractional resistance (0.98 +/- 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Hydroxylamine and Carboxymethoxylamine Can Inhibit Toxoplasma gondii Growth through an Aspartate Aminotransferase-Independent Pathway

2020 ◽  
Vol 64 (3) ◽  
Author(s):  
Jixu Li ◽  
Huanping Guo ◽  
Eloiza May Galon ◽  
Yang Gao ◽  
Seung-Hun Lee ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Drug Targets ◽  
Protozoan Parasite ◽  
Cell Types ◽  
Lytic Cycle ◽  
Type I ◽  
Content Type ◽  
Mechanism Of Inhibition

ABSTRACT Toxoplasma gondii is an obligate intracellular protozoan parasite and a successful parasitic pathogen in diverse organisms and host cell types. Hydroxylamine (HYD) and carboxymethoxylamine (CAR) have been reported as inhibitors of aspartate aminotransferases (AATs) and interfere with the proliferation in Plasmodium falciparum. Therefore, AATs are suggested as drug targets against Plasmodium. The T. gondii genome encodes only one predicted AAT in both T. gondii type I strain RH and type II strain PLK. However, the effects of HYD and CAR, as well as their relationship with AAT, on T. gondii remain unclear. In this study, we found that HYD and CAR impaired the lytic cycle of T. gondii in vitro, including the inhibition of invasion or reinvasion, intracellular replication, and egress. Importantly, HYD and CAR could control acute toxoplasmosis in vivo. Further studies showed that HYD and CAR could inhibit the transamination activity of rTgAAT in vitro. However, our results confirmed that deficiency of AAT in both RH and PLK did not reduce the virulence in mice, although the growth ability of the parasites was affected in vitro. HYD and CAR could still inhibit the growth of AAT-deficient parasites. These findings indicated that HYD and CAR inhibition of T. gondii growth and control of toxoplasmosis can occur in an AAT-independent pathway. Overall, further studies focusing on the elucidation of the mechanism of inhibition are warranted. Our study hints at new substrates of HYD and CAR as potential drug targets to inhibit T. gondii growth.

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