scholarly journals Ex vivo expansion and characterization of human corneal endothelium for transplantation: a review

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ingrida Smeringaiova ◽  
Tor Paaske Utheim ◽  
Katerina Jirsova
Keyword(s):  
Corneal Endothelium ◽  
Culture Media ◽  
Ex Vivo ◽  
Morphological Changes ◽  
Storage Conditions ◽  
Lamellar Keratoplasty ◽  
Mesenchymal Transition ◽  
Induced Pluripotent Cells ◽  
Ex Vivo Culture ◽  
Culture Protocol

AbstractThe corneal endothelium plays a key role in maintaining corneal transparency. Its dysfunction is currently treated with penetrating or lamellar keratoplasty. Advanced cell therapy methods seek to address the persistent global deficiency of donor corneas by enabling the renewal of the endothelial monolayer with tissue-engineered grafts. This review provides an overview of recently published literature on the preparation of endothelial grafts for transplantation derived from cadaveric corneas that have developed over the last decade (2010–2021). Factors such as the most suitable donor parameters, culture substrates and media, endothelial graft storage conditions, and transplantation methods are discussed. Despite efforts to utilize alternative cellular sources, such as induced pluripotent cells, cadaveric corneas appear to be the best source of cells for graft preparation to date. However, native endothelial cells have a limited natural proliferative capacity, and they often undergo rapid phenotype changes in ex vivo culture. This is the main reason why no culture protocol for a clinical-grade endothelial graft prepared from cadaveric corneas has been standardized so far. Currently, the most established ex vivo culture protocol involves the peel-and-digest method of cell isolation and cell culture by the dual media method, including the repeated alternation of high and low mitogenic conditions. Culture media are enriched by additional substances, such as signaling pathway (Rho-associated protein kinase, TGF-β, etc.) inhibitors, to stimulate proliferation and inhibit unwanted morphological changes, particularly the endothelial-to-mesenchymal transition. To date, this promising approach has led to the development of endothelial grafts for the first in-human clinical trial in Japan. In addition to the lack of a standard culture protocol, endothelial-specific markers are still missing to confirm the endothelial phenotype in a graft ready for clinical use. Because the corneal endothelium appears to comprise phenotypically heterogeneous populations of cells, the genomic and proteomic expression of recently proposed endothelial-specific markers, such as Cadherin-2, CD166, or SLC4A11, must be confirmed by additional studies. The preparation of endothelial grafts is still challenging today, but advances in tissue engineering and surgery over the past decade hold promise for the successful treatment of endothelial dysfunctions in more patients worldwide.

scholarly journals Mouse Embryonic Tooth Germ Dissection and Ex vivo Culture Protocol

BIO-PROTOCOL ◽  
2020 ◽  
Vol 10 (3) ◽  
Author(s):  
Xue Han ◽  
Keigo Yoshizaki ◽  
Tian Tian ◽  
Kanako Miyazaki ◽  
Ichiro Takahashi ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Tooth Germ ◽  
Ex Vivo Culture ◽  
Culture Protocol

scholarly journals Dysregulation of FGFR signalling by a selective inhibitor reduces germ cell survival in human fetal gonads of both sexes and alters the somatic niche in fetal testes

2019 ◽  
Vol 34 (11) ◽  
pp. 2228-2243 ◽  
Author(s):  
K Harpelunde Poulsen ◽  
J E Nielsen ◽  
H Frederiksen ◽  
C Melau ◽  
K Juul Hare ◽  
...  
Keyword(s):  
Germ Cell ◽  
Cell Survival ◽  
Germ Cells ◽  
Sertoli Cells ◽  
Culture Media ◽  
Ex Vivo ◽  
Gonadal Development ◽  
Fetal Testis ◽  
Ex Vivo Culture

Abstract STUDY QUESTION Does experimental manipulation of fibroblast growth factor 9 (FGF9)-signalling in human fetal gonads alter sex-specific gonadal differentiation? SUMMARY ANSWER Inhibition of FGFR signalling following SU5402 treatment impaired germ cell survival in both sexes and severely altered the developing somatic niche in testes, while stimulation of FGF9 signalling promoted Sertoli cell proliferation in testes and inhibited meiotic entry of germ cells in ovaries. WHAT IS KNOWN ALREADY Sex-specific differentiation of bipotential gonads involves a complex signalling cascade that includes a combination of factors promoting either testicular or ovarian differentiation and inhibition of the opposing pathway. In mice, FGF9/FGFR2 signalling has been shown to promote testicular differentiation and antagonize the female developmental pathway through inhibition of WNT4. STUDY DESIGN, SIZE, DURATION FGF signalling was manipulated in human fetal gonads in an established ex vivo culture model by treatments with recombinant FGF9 (25 ng/ml) and the tyrosine kinase inhibitor SU5402 (10 μM) that was used to inhibit FGFR signalling. Human fetal testis and ovary tissues were cultured for 14 days and effects on gonadal development and expression of cell lineage markers were determined. PARTICIPANTS/MATERIALS, SETTING, METHODS Gonadal tissues from 44 male and 33 female embryos/fetuses from first trimester were used for ex vivo culture experiments. Tissues were analyzed by evaluation of histology and immunohistochemical analysis of markers for germ cells, somatic cells, proliferation and apoptosis. Culture media were collected throughout the experimental period and production of steroid hormone metabolites was analyzed in media from fetal testis cultures by liquid chromatography–tandem mass spectrometry (LC-MS/MS). MAIN RESULTS AND THE ROLE OF CHANCE Treatment with SU5402 resulted in near complete loss of gonocytes (224 vs. 14 OCT4+ cells per mm2, P < 0.05) and oogonia (1456 vs. 28 OCT4+ cells per mm2, P < 0.001) in human fetal testes and ovaries, respectively. This was a result of both increased apoptosis and reduced proliferation in the germ cells. Addition of exogenous FGF9 to the culture media resulted in a reduced number of germ cells entering meiosis in fetal ovaries (102 vs. 60 γH2AX+ germ cells per mm2, P < 0.05), while in fetal testes FGF9 stimulation resulted in an increased number of Sertoli cells (2503 vs. 3872 SOX9+ cells per mm2, P < 0.05). In fetal testes, inhibition of FGFR signalling by SU5402 treatment altered seminiferous cord morphology and reduced the AMH expression as well as the number of SOX9-positive Sertoli cells (2503 vs. 1561 SOX9+ cells per mm2, P < 0.05). In interstitial cells, reduced expression of COUP-TFII and increased expression of CYP11A1 and CYP17A1 in fetal Leydig cells was observed, although there were no subsequent changes in steroidogenesis. LARGE SCALE DATA N/A LIMITATIONS, REASONS FOR CAUTION Ex vivo culture may not replicate all aspects of fetal gonadal development and function in vivo. Although the effects of FGF9 were studied in ex vivo culture experiments, there is no direct evidence that FGF9 acts in vivo during human fetal gonadogenesis. The FGFR inhibitor (SU5402) used in this study is not specific to FGFR2 but inhibits all FGF receptors and off-target effects on unrelated tyrosine kinases should be considered. WIDER IMPLICATIONS OF THE FINDINGS The findings of this study suggest that dysregulation of FGFR-mediated signalling may affect both testicular and ovarian development, in particular impacting the fetal germ cell populations in both sexes. STUDY FUNDING/COMPETING INTEREST(S) This work was supported in part by an ESPE Research Fellowship, sponsored by Novo Nordisk A/S to A.JØ. Additional funding was obtained from the Erichsen Family Fund (A.JØ.), the Aase and Ejnar Danielsens Fund (A.JØ.), the Danish Government’s support for the EDMaRC programme (A.JU.) and a Wellcome Trust Intermediate Clinical Fellowship (R.T.M., Grant no. 098522). The Medical Research Council (MRC) Centre for Reproductive Health (R.T.M.) is supported by an MRC Centre Grant (MR/N022556/1). The authors have no conflict of interest to disclose.

Abstract 18802: Transcriptome Profiles of Chronically Closed or Cycling Aortic Valve Leaflets are Highly Similar Compared to Chronically Open Valves: Distention Drives Leaflet Homeostasis

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Katsuhide Maeda ◽  
Xiaoyuan Ma ◽  
Frank Hanley ◽  
R. Kirk Riemer
Keyword(s):  
Gene Expression ◽  
Aortic Valve ◽  
Culture Media ◽  
Ex Vivo ◽  
Valve Leaflet ◽  
Valve Closure ◽  
Cell Culture Media ◽  
Endothelial Cell Culture ◽  
Flow Through ◽  
Ex Vivo Culture

Introduction: Throughout the valve cycle mechanical forces of multiple types and intensities are exerted on the leaflets, sinus and annulus. The mechanisms through which these forces regulate the homeostatic maintenance of valve leaflet architecture are still poorly understood. Ventricular mechanical support with a continuous flow device results in chronic closure of the outflow valve. Whether chronic valve closure affects valve leaflet homeostasis is a significant but unsettled clinical concern. Hypothesis: We asked if chronic closure of the aortic valve (AV) impairs leaflet homeostasis by comparing the transcriptome profiles of AV cultured under different mechanical force conditions: Normally cycling (Flow); Not cycling but open (Static); or Not cycling but closed (Static-Closed, SC). Methods: Ex vivo culture of native rat aortic valves, (8 valves per bioreactor, 4 valves per condition) was conducted in a flow bioreactor for seven days at 37C in endothelial cell culture media under conditions approximating the normal stroke volume of the rat heart. In each of 4 independent experiments, flow-induced valve cycling (Flow) was compared with either Static or SC condition in paired groups. After culture, leaflets were dissected from 3 valves/condition and pooled, mRNA was extracted and expression was evaluated using whole genome microarrays. Results: Statistical-based unsupervised hierarchical clustering analysis of the leaflet transcriptome profiles revealed only two distinct patterns of leaflet gene expression between the three groups of AV. Flow and SC groups had nearly identical profiles whereas the Static valve group was distinctly different (p<0.05). Conclusions: These results reveal that the stretching of valve leaflets by a filling volume inducing full coaptation is apparently sufficient to largely preserve their architecture and phenotypic gene expression in the seven day culture period tested. In contrast, the absence of valve closure produces a markedly different pattern of gene expression. Therefore, the absence of flow through the closed AV valve does not appear to be inherently destructive compared with conditions in which the valve remains open.

Evaluation of Microbial Load from Canned Soya Milk Drinks in Malaysia

2016 ◽  
Vol 2 (1) ◽  
pp. 22-25
Author(s):  
Nur Amalina binti Mustafa ◽  
Muhammad Ashraf bin Redzuan ◽  
Muhamad Hazim bin Zuraimi ◽  
Muhamad Shuhaimi bin Shuib ◽  
Shahnaz Majeed ◽  
...  
Keyword(s):  
Culture Media ◽  
Storage Condition ◽  
Storage Conditions ◽  
Nutrient Agar ◽  
Microbial Load ◽  
Blood Agar ◽  
Human Consumption ◽  
Processing Unit ◽  
Soya Milk ◽  
Two Samples

Objective: Owing to the habit of consuming ready food among the citizens of Malaysia a study was conducted to evaluate 20 samples of canned soya milk for the presence of possible microbial content. The samples were collected randomly from shopping malls, restaurants and kiosk in Ipoh Malaysia. Methods: All samples collected across Ipoh, were subjected to test for presence bacteria in nutrient agar, blood agar and macConkey media. The possible microbial load was swapped from surface and soya milk content with a sterile cotton and streaked on nutrient agar, blood agar and macConkey culture media. The streaked petri plates were incubated for 48 hours at 37oC. Results: The study revealed negative microbial growth in all except two samples from the surface and soya milk content collected from a restaurant in nutrient agar and blood agar medium. The presence of microbes was conformed as gram positive staphylococcus sp. through gram staining. The positive growth may be imputed to poor storage condition at the restaurant. Conclusion: It can be computed from the study that the majority of the samples were free from bacterial growth, suggesting strong in house quality control mechanism at the processing unit and exquisite storage conditions in malls and kiosk suggesting that soya milk available in malls and kiosk are fit for human consumption.

Phagocytosis of Liposome Particles by Rat Splenic Immature Monocytes Makes Them Transiently and Highly Immunosuppressive In Ex Vivo Culture Conditions

2011 ◽  
Vol 337 (1) ◽  
pp. 42-49 ◽  
Author(s):  
Daisuke Takahashi ◽  
Hiroshi Azuma ◽  
Hiromi Sakai ◽  
Keitaro Sou ◽  
Daiko Wakita ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Culture Conditions ◽  
Ex Vivo Culture

Modulation effects of hexamethylene bisacetamide on growth and differentiation of cultured human malignant glioma cells

1996 ◽  
Vol 84 (5) ◽  
pp. 831-838 ◽  
Author(s):  
Xiao-Nan Li ◽  
Zi-Wei Du ◽  
Qiang Huang
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Malignant Glioma ◽  
Culture Media ◽  
Morphological Changes ◽  
Control Group ◽  
Cell Cycle Distribution ◽  
Content Type ◽  
Hexamethylene Bisacetamide ◽  
Human Malignant Glioma

✓ The modulation effects of hexamethylene bisacetamide (HMBA), a differentiation-inducing agent, on growth and differentiation of cells from human malignant glioma cell line SHG-44 were studied. At cytostatic doses (2.5 mM, 5 mM, 7.5 mM, and 10 mM for 15 days), HMBA exerted a marked inhibitory effect on cell proliferation. Exposure to HMBA (5 mM and 10 mM for 12 days) also resulted in an accumulation of cells in G0/G1 phase and a decrease of cells in S phase as analyzed by flow cytometry. The reversible effects of 7.5 mM HMBA and 10 mM HMBA on cell proliferation and 10 mM HMBA on disruption of cell cycle distribution were observed when HMBA was removed from culture media on Day 6 and replaced with HMBA-free media. Colony-forming efficiency (CFE) in soft agar was remarkably decreased by HMBA (2.5 mM, 5 mM, 7.5 mM, and 10 mM for 14 days), and in 7.5 mM HMBA— and 10 mM HMBA—treated cells, the CFEs were reduced to 25% and 12.5%, respectively, of that in untreated cells. Cells treated with HMBA (5 mM and 10 mM for 15 days) remained tumorigenic in athymic nude mice, but the growth rates of the xenografts were much slower than those in the control group. The effects of HMBA on cell proliferation, cell cycle distribution, CFE, and growth of xenografts were dose dependent. A more mature phenotype was confirmed by the morphological changes from spindle shape to large polygonal stellate shape and remarkably elevated expression of glial fibrillary acidic protein in cells exposed to HMBA (5 mM, 10 mM for 15 days). Our results showed that a more differentiated phenotype with marked growth arrest was induced in SHG-44 cells by HMBA.

scholarly journals Pharmacological ablation of astrocytes reduces Aβ degradation and synaptic connectivity in an ex vivo model of Alzheimer’s disease

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Nicola Davis ◽  
Bibiana C. Mota ◽  
Larissa Stead ◽  
Emily O. C. Palmer ◽  
Laura Lombardero ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Culture Media ◽  
Ex Vivo ◽  
Wild Type ◽  
Insulin Degrading Enzyme ◽  
Ex Vivo Model ◽  
Model Of Alzheimer’S Disease ◽  
Aβ Clearance ◽  
5Xfad Mice

Abstract Background Astrocytes provide a vital support to neurons in normal and pathological conditions. In Alzheimer’s disease (AD) brains, reactive astrocytes have been found surrounding amyloid plaques, forming an astrocytic scar. However, their role and potential mechanisms whereby they affect neuroinflammation, amyloid pathology, and synaptic density in AD remain unclear. Methods To explore the role of astrocytes on Aβ pathology and neuroinflammatory markers, we pharmacologically ablated them in organotypic brain culture slices (OBCSs) from 5XFAD mouse model of AD and wild-type (WT) littermates with the selective astrocytic toxin L-alpha-aminoadipate (L-AAA). To examine the effects on synaptic circuitry, we measured dendritic spine number and size in OBCSs from Thy-1-GFP transgenic mice incubated with synthetic Aβ42 or double transgenics Thy-1-GFP/5XFAD mice treated with LAAA or vehicle for 24 h. Results Treatment of OBCSs with L-AAA resulted in an increased expression of pro-inflammatory cytokine IL-6 in conditioned media of WTs and 5XFAD slices, associated with changes in microglia morphology but not in density. The profile of inflammatory markers following astrocytic loss was different in WT and transgenic cultures, showing reductions in inflammatory mediators produced in astrocytes only in WT sections. In addition, pharmacological ablation of astrocytes led to an increase in Aβ levels in homogenates of OBCS from 5XFAD mice compared with vehicle controls, with reduced enzymatic degradation of Aβ due to lower neprilysin and insulin-degrading enzyme (IDE) expression. Furthermore, OBSCs from wild-type mice treated with L-AAA and synthetic amyloid presented 56% higher levels of Aβ in culture media compared to sections treated with Aβ alone, concomitant with reduced expression of IDE in culture medium, suggesting that astrocytes contribute to Aβ clearance and degradation. Quantification of hippocampal dendritic spines revealed a reduction in their density following L-AAA treatment in all groups analyzed. In addition, pharmacological ablation of astrocytes resulted in a decrease in spine size in 5XFAD OBCSs but not in OBCSs from WT treated with synthetic Aβ compared to vehicle control. Conclusions Astrocytes play a protective role in AD by aiding Aβ clearance and supporting synaptic plasticity.

scholarly journals Cbl-transforming Variants Trigger a Cascade of Molecular Alterations That Lead to Epithelial Mesenchymal Conversion

2000 ◽  
Vol 11 (10) ◽  
pp. 3397-3410 ◽  
Author(s):  
Tanya M. Fournier ◽  
Louie Lamorte ◽  
Christiane R. Maroun ◽  
Mark Lupher ◽  
Hamid Band ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epithelial Cells ◽  
Hepatocyte Growth Factor ◽  
Epithelial Mesenchymal Transition ◽  
Morphological Changes ◽  
Cell Spreading ◽  
Mesenchymal Transition ◽  
Amino Terminal ◽  
Src Homology ◽  
Hepatocyte Growth

Dispersal of epithelial cells is an important aspect of tumorigenesis, and invasion. Factors such as hepatocyte growth factor induce the breakdown of cell junctions and promote cell spreading and the dispersal of colonies of epithelial cells, providing a model system to investigate the biochemical signals that regulate these events. Multiple signaling proteins are phosphorylated in epithelial cells during hepatocyte growth factor–induced cell dispersal, including c-Cbl, a protooncogene docking protein with ubiquitin ligase activity. We have examined the role of c-Cbl and a transforming variant (70z-Cbl) in epithelial cell dispersal. We show that the expression of 70z-Cbl in Madin-Darby canine kidney epithelial cells resulted in the breakdown of cell–cell contacts and alterations in cell morphology characteristic of epithelial–mesenchymal transition. Structure–function studies revealed that the amino-terminal portion of c-Cbl, which corresponds to the Cbl phosphotyrosine-binding/Src homology domain 2 , is sufficient to promote the morphological changes in cell shape. Moreover, a point mutation at Gly-306 abrogates the ability of the Cbl Src homology domain 2 to induce these morphological changes. Our results identify a role for Cbl in the regulation of epithelial–mesenchymal transition, including loss of adherens junctions, cell spreading, and the initiation of cell dispersal.

Sign in / Sign up

Export Citation Format

Share Document