A Simple Method for Establishing AdherentEx VivoExplant Cultures from Human Eye Pathologies for Use in Subsequent Calcium Imaging and Inflammatory Studies

A novel, simple, and reproducible method for cultivating pathological tissues obtained from human eyes during surgery was developed using viscoelastic material as a tissue adherent to facilitate cell attachment and expansion and calcium imaging of cultured cells challenged by mechanical and acetylcholine (ACh) stimulation as well as inflammatory studies. Anterior lens capsule-lens epithelial cells (aLC-LECs) from cataract surgery and proliferative diabetic retinopathy (PDR) fibrovascular epiretinal membranes (fvERMs) from human eyes were used in the study. We hereby show calcium signaling in aLC-LECs by mechanical and acetylcholine (ACh) stimulation and indicate presence of ACh receptors in these cells. Furthermore, anex vivostudy model was established for measuring the inflammatory response in fvERMs and aLC-LECs upon TNFα treatment.

Download Full-text

A simple method for ex vivo honey bee cell culture capable of in vitro gene expression analysis

PLoS ONE ◽

10.1371/journal.pone.0257770 ◽

2021 ◽

Vol 16 (9) ◽

pp. e0257770

Author(s):

Kazuyo Watanabe ◽

Mikio Yoshiyama ◽

Gaku Akiduki ◽

Kakeru Yokoi ◽

Hiroko Hoshida ◽

...

Keyword(s):

Gene Expression ◽

Cell Culture ◽

Honey Bee ◽

Cell Lines ◽

Fluorescent Protein ◽

Cultured Cells ◽

Ex Vivo ◽

Plasmid Vector ◽

Simple Method

Cultured cells are a very powerful tool for investigating biological events in vitro; therefore, cell lines have been established not only in model insect species, but also in non-model species. However, there are few reports on the establishment of stable cell lines and development of systems to introduce genes into the cultured cells of the honey bee (Apis mellifera). We describe a simple ex vivo cell culture system for the honey bee. Hemocyte cells obtained from third and fourth instar larvae were cultured in commercial Grace’s insect medium or MGM-450 insect medium for more than two weeks maintaining a normal morphology without deterioration. After an expression plasmid vector bearing the enhanced green fluorescent protein (egfp) gene driven by the immediate early 2 (IE2) viral promoter was transfected into cells, EGFP fluorescence was detected in cells for more than one week from one day after transfection. Furthermore, double-stranded RNA corresponding to a part of the egfp gene was successfully introduced into cells and interfered with egfp gene expression. A convenient and reproducible method for an ex vivo cell culture that is fully practicable for gene expression assays was established for the honey bee.

Download Full-text

P 229 Growth of human lens epithelial cells on the anterior lens capsule in vitro

Vision Research ◽

10.1016/0042-6989(95)90545-6 ◽

1995 ◽

Vol 35 ◽

pp. S199

Author(s):

J.H. Meyer ◽

J. Schmidt ◽

F. Eppinger ◽

B. Flügel ◽

K.U. Löffler ◽

...

Keyword(s):

Epithelial Cells ◽

Lens Capsule ◽

Human Lens ◽

Lens Epithelial Cells ◽

Human Lens Epithelial Cells ◽

Anterior Lens Capsule

Download Full-text

Immortalization and Characterization of Rat Lingual Keratinocytes in a High-Calcium and Feeder-Free Culture System Using ROCK Inhibitor Y-27632

International Journal of Molecular Sciences ◽

10.3390/ijms22136782 ◽

2021 ◽

Vol 22 (13) ◽

pp. 6782

Author(s):

Zixing Chen ◽

Wenmeng He ◽

Thomas Chun Ning Leung ◽

Hau Yin Chung

Keyword(s):

Calcium Imaging ◽

Cultured Cells ◽

Rho Kinase ◽

Culture Method ◽

Taste Bud ◽

Label Free ◽

Sprague Dawley ◽

Rock Inhibitor ◽

High Calcium

Cultured keratinocytes are desirable models for biological and medical studies. However, primary keratinocytes are difficult to maintain, and there has been little research on lingual keratinocyte culture. Here, we investigated the effect of Y-27632, a Rho kinase (ROCK) inhibitor, on the immortalization and characterization of cultured rat lingual keratinocyte (RLKs). Three Y-27632–supplemented media were screened for the cultivation of RLKs isolated from Sprague–Dawley rats. Phalloidin staining and TUNEL assay were applied to visualize cytoskeleton dynamics and cell apoptosis following Y-27632 removal. Label-free proteomics, RT-PCR, calcium imaging, and cytogenetic studies were conducted to characterize the cultured cells. Results showed that RLKs could be conditionally immortalized in a high-calcium medium in the absence of feeder cells, although they did not exhibit normal karyotypes. The removal of Y-27632 from the culture medium led to reversible cytoskeletal reorganization and nuclear enlargement without triggering apoptosis, and a total of 239 differentially expressed proteins were identified by proteomic analysis. Notably, RLKs derived from the non-taste epithelium expressed some molecular markers characteristic of taste bud cells, yet calcium imaging revealed that they rarely responded to tastants. Collectively, we established a high-calcium and feeder-free culture method for the long-term maintenance of RLKs. Our results shed some new light on the immortalization and differentiation of lingual keratinocytes.

Download Full-text

The Regularity of Sustained Firing Reveals Two Populations of Slowly Adapting Touch Receptors in Mouse Hairy Skin

Journal of Neurophysiology ◽

10.1152/jn.00810.2009 ◽

2010 ◽

Vol 103 (6) ◽

pp. 3378-3388 ◽

Cited By ~ 48

Author(s):

Scott A. Wellnitz ◽

Daine R. Lesniak ◽

Gregory J. Gerling ◽

Ellen A. Lumpkin

Keyword(s):

Fluorescent Protein ◽

Ex Vivo ◽

Receptive Fields ◽

Receptor Subtypes ◽

Type I ◽

Genetic Dissection ◽

Merkel Cells ◽

Simple Method ◽

Green Fluorescent ◽

Nerve Recordings

Touch is initiated by diverse somatosensory afferents that innervate the skin. The ability to manipulate and classify receptor subtypes is prerequisite for elucidating sensory mechanisms. Merkel cell–neurite complexes, which distinguish shapes and textures, are experimentally tractable mammalian touch receptors that mediate slowly adapting type I (SAI) responses. The assessment of SAI function in mutant mice has been hindered because previous studies did not distinguish SAI responses from slowly adapting type II (SAII) responses, which are thought to arise from different end organs, such as Ruffini endings. Thus we sought methods to discriminate these afferent types. We developed an epidermis-up ex vivo skin–nerve chamber to record action potentials from afferents while imaging Merkel cells in intact receptive fields. Using model-based cluster analysis, we found that two types of slowly adapting receptors were readily distinguished based on the regularity of touch-evoked firing patterns. We identified these clusters as SAI (coefficient of variation = 0.78 ± 0.09) and SAII responses (0.21 ± 0.09). The identity of SAI afferents was confirmed by recording from transgenic mice with green fluorescent protein–expressing Merkel cells. SAI receptive fields always contained fluorescent Merkel cells ( n = 10), whereas SAII receptive fields lacked these cells ( n = 5). Consistent with reports from other vertebrates, mouse SAI and SAII responses arise from afferents exhibiting similar conduction velocities, receptive field sizes, mechanical thresholds, and firing rates. These results demonstrate that mice, like other vertebrates, have two classes of slowly adapting light-touch receptors, identify a simple method to distinguish these populations, and extend the utility of skin–nerve recordings for genetic dissection of touch receptor mechanisms.

Download Full-text

Transscleral optical coherence tomography?An experimental study in ex-vivo human eyes

Lasers in Surgery and Medicine ◽

10.1002/lsm.10031 ◽

2002 ◽

Vol 30 (3) ◽

pp. 209-215 ◽

Cited By ~ 7

Author(s):

Hans Hoerauf ◽

J�rg Winkler ◽

Christian Scholz ◽

Christopher Wirbelauer ◽

Roswitha S. Gordes ◽

...

Keyword(s):

Experimental Study ◽

Optical Coherence Tomography ◽

Ex Vivo ◽

Optical Coherence ◽

Human Eyes

Download Full-text

Quantitative Studies of Fibronectin Adsorption on Submicron Scaffolds

ASME 2012 Summer Bioengineering Conference, Parts A and B ◽

10.1115/sbc2012-80633 ◽

2012 ◽

Author(s):

Shawn Regis ◽

Manisha Jassal ◽

Sina Youssefian ◽

Nima Rahbar ◽

Sankha Bhowmick

Keyword(s):

Surface Functionalization ◽

Cultured Cells ◽

Cell Attachment ◽

Md Simulations ◽

Cell Types ◽

Biological Processes ◽

Integrin Receptors ◽

Tissue Engineering Scaffolds ◽

Quantitative Studies

Fibronectin plays a crucial role in adhesion of several cell types, mainly due to the fact that it is recognized by at least ten different integrin receptors. Since most cell types can bind to fibronectin, it becomes involved in many various biological processes. The interaction of cells with ECM proteins such as fibronectin provides the signals affecting morphology, motility, gene expression, and survival of cells [1]. Fibronectin exists in both soluble and insoluble forms; soluble fibronectin is secreted by cells and exits in cell media or body fluids, whereas insoluble fibronectin exists in tissues or the extracellular matrix of cultured cells [2]. The ability to control adsorption of fibronectin on tissue engineering scaffolds would therefore play a huge role in controlling cell attachment and survival in vivo. This can be achieved through surface functionalization of the scaffolds. The goal of these studies is to use molecular dynamics (MD) simulations to mechanistically understand how fibronectin adsorption is enhanced by surface functionalization of submicron scaffolds.

Download Full-text

A Simple Method for High Temporal Resolution Calcium Imaging with Dual Excitation Dyes

Biophysical Journal ◽

10.1016/s0006-3495(98)77644-1 ◽

1998 ◽

Vol 75 (4) ◽

pp. 2025-2029 ◽

Cited By ~ 17

Author(s):

Luc Leybaert ◽

James Sneyd ◽

Michael J. Sanderson

Keyword(s):

Temporal Resolution ◽

Calcium Imaging ◽

High Temporal Resolution ◽

Simple Method

Download Full-text

Impact of freeze-thaw cytoablation on aqueous outflow patterns in ex vivo anterior chamber perfusion cultures and whole eyes

F1000Research ◽

10.12688/f1000research.53572.1 ◽

2021 ◽

Vol 10 ◽

pp. 525

Author(s):

Raoul Verma-Fuehring ◽

Mohamad Dakroub ◽

Alicja Strzalkowska ◽

Piotr Strzalkowski ◽

Hong Han ◽

...

Keyword(s):

Trabecular Meshwork ◽

Structural Changes ◽

Ex Vivo ◽

Anterior Segment ◽

Large Change ◽

Invasive Technique ◽

Freeze Thaw ◽

Before And After ◽

Human Eyes ◽

Porcine Eyes

Background: Porcine eyes have been widely used as ex vivo models in glaucoma research, as they share similar features with human eyes. Freeze-thawing is a non-invasive technique that has been used to obliterate living cells in anterior segment ex vivo cultures, to prepare them for further research such as cellular repopulation. This technique has previously been shown to reduce the intraocular pressure (IOP) in porcine eyes. The aim of this study was to investigate whether freeze-thaw cytoablation causes corresponding canalogram outflow changes in perfused anterior segment cultures (AFT) and whole porcine eyes (WFT). We hypothesized that the known IOP drop in AFT after trabecular meshwork ablation by freeze-thaw would be accompanied by a similarly large change in the distal outflow pattern. Methods: Two-dye (fluorescein and Texas red) reperfusion canalograms were used to compare the outflow time before and after two -80°C cycles of freeze-thaw. We assigned 28 freshly enucleated porcine eyes to four groups: perfused anterior segment dye controls (ACO, n = 6), perfused whole eye dye controls (WCO, n = 6), freeze-thaw treated anterior segment cultures (AFT, n = 10), and freeze-thaw treated whole eyes (WFT, n = 6). Results: In control groups ACO and WCO, the two different dyes had similar filling times. In AFT, the outflow pattern and filling times were unchanged. In WFT, the temporal superior quadrant filled more slowly (p = 0.042) while all others remained unchanged. The qualitative appearance of distal outflow spaces was altered only in some eyes. Conclusions: Freeze-thaw cytoablation caused neither loss nor leakage of distal outflow structures. Surprisingly, the loss of an intact trabecular meshwork over the entire circumference did not result in a general acceleration of quadrant outflow times. The results validate freeze-thawing as a method to generate an extracellular matrix without major structural changes.

Download Full-text