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Intestinal Effects of Bacterial Chemotactic Peptides: Induction of Inflammation, Release of Eicosanoids.
Published in William J. Snape, Stephen M. Collins, Effects of Immune Cells and Inflammation on Smooth Muscle and Enteric Nerves, 2020
Louise E. LeDuc, Cynthia C. Nast, Fergus Shanahan, Alda Vidrich
In preliminary experiments, using techniques successful for the isolation of human colonic lamina propria cells10, we were unable to isolate lamina propria cell from rabbit colon. However these techniques were useful in generating cells from rabbit small intestine. We further modified these techniques as follows: Segments of rabbit small intestine were rapidly excised, trimmed of mesentery and cut longitudinally obverse to the mesentery, the intestine was rinsed and the Peyer’s patches excised. Portions of the intestine were cut into small pieces and were then incubated, shaking, in 1mM EDTA final concentration at 37° for 4h; the medium was changed at 1/2h intervals. Following incubation in EDTA to remove the epithelial cells, the pieces of intestine was incubated for one hour in dispase (BM), and then filtered through a mesh sieve. After a two hour incubation on ice, cells were counted and incubated with or without stimuli for 15 min at 37°. The reaction was terminated by rapid centrifugation, (17,000 × g, 30 s) and the cell-free supernatant frozen immediately for subsequent radioimmunoassay. Aliquots of the unused cells were pelleted onto glass slides in a cytospin centrifuge, fixed, and stained with Giemsa, for a differential cell count. Cells from mesenteric lymph node and excised Peyer’s patches were mechanically separated, washed, and similarly incubated and quantitated.
Pharmacologic vitreolysis
Published in A Peyman MD Gholam, A Meffert MD Stephen, D Conway MD FACS Mandi, Chiasson Trisha, Vitreoretinal Surgical Techniques, 2019
Theoretically, the enzymes described above act on different molecules at the vitreoretinal interface or within the vitreous gel, resulting in PVD, vitreous liquefaction, or a combination. Dispase acts on type IV collagen, the main structural protein of basement membranes and of the ILM, and it is not surprising that morphologic changes of the inner retina have been reported following application of the enzyme.18McCuen and co-workers19 found no abnormalities on electroretinography in young pigs, possibly indicating that these morphologic changes may not affect retinal function. However, retinal hemorrhages have been reported following an intravitreal injection of dispase, and the enzyme has been shown to effectively induce proliferative vitreoretinopathy (PVR) in rabbits in a dose-dependent fashion, known as the dispase model of PVR.20,45 Future studies are needed to investigate the safety of dispase before clinical studies can be considered with this agent.
The Treatment of Burns
Published in Howard Green, Therapy with Cultured Cells, 2019
First we had to have a method of preparing grafts from cultures. The cells could not be simply scraped from the vessel surface because the basal layer, which contains the multiplying cells of the stratified culture, would be destroyed. After failing in several attempts to find a successful method of detachment, we hit upon the enzyme Dispase, a neutral protease discovered in Japan. This enzyme detached a confluent layer of cultured keratinocytes from the surface of the dish without dissociating the cells from each other. The detached cell layer then shrank to half or less of its area and could be picked up and, supported by a backing, be applied as a graft (Fig. 9) (Green et al., 1979).
Optisol-GS Storage of Cultured Human Limbal Epithelial Cells at Ambient Temperature Is Superior to Hypothermic Storage
Published in Current Eye Research, 2020
Catherine Joan Jackson, Lara Pasovic, Sten Raeder, Amer Sehic, Borghild Roald, Maria F. de la Paz, Kim Alexsander Tønseth, Tor Paaske Utheim
The research was conducted in accordance with the Declaration of Helsinki and approved by the Norwegian Regional Committee for Medical and Health Research Ethics. Corneoscleral tissue from three cadaveric donors (five eyes) was shipped from El centro de Oftalmología Barraquer (Barcelona, Spain) after obtaining informed consent from the next of kin for use of the tissue for research purposes. The corneoscleral rings were placed in organ culture medium at ambient temperature for shipment to Oslo University Hospital, Oslo, Norway. Cryopreserved human amniotic membranes13 were thawed and attached to Netwell culture inserts (Corning, New York, USA) using 6–0 non-absorbable sutures. The limbal explants were prepared and cultured on amniotic membrane following the culture method of Utheim et al. 2019.14,15 Briefly, the limbal tissue was rinsed three times with DMEM, placed in a culture dish and exposed for 10 minutes to Dispase II in Mg- and Ca-free Hanks’ balanced salt solution in an incubator (37°C and 5% CO2). Dispase activity was inhibited by rinsing with DMEM containing 10% FBS. The limbal rings were divided into pieces and one explant was placed on amniotic membrane in the center of each insert. The donor tissue was evenly distributed between the two study groups in order to avoid bias.
Corneal epithelial stem cells for corneal injury
Published in Expert Opinion on Biological Therapy, 2018
Dominique Bremond-Gignac, Henri Copin, Moncef Benkhalifa
There are two main methods for producing LPCs. The first is the explant culture technique, in which a small biopsy of limbal epithelium and stroma (from 1 to 6 mm2) is plated on a substrate. These biopsies are often removed from the upper or lower limbal ring region [28]. The second technique is the suspension culture system, in which limbal tissue is treated with enzymes (to separate the stroma from the epithelium) and isolated epithelial cells are then seeded on a substratum. This method usually employs two enzymes: dispase, which digests basement membrane collagen and separates epithelial cells from the stroma, and trypsin, which separates clumps of limbal epithelial cells (LECs) into a suspension of single cells. These enzyme treatment protocols can be performed on a limbal biopsy or a complete limbal ring [28, 29].
Improved protocol for simultaneous analysis of leukocyte subsets and epithelial cells from murine and human lung
Published in Experimental Lung Research, 2018
Christine Happle, Lena Meyer-Decking, Anika Dreier, Martin Wetzke, Stephanie Gläsener, Ruth Grychtol, Peter Braubach, Alexandra Jablonka, Sibylle Haid, Adan Chari Jirmo, Anika Habener, Jelena Skuljec, Gesine Hansen
In brief, the same digestion methods as described for murine samples were applied. Lung specimens (peripheral lung tissue free from tracheal branches and infectious or tumor lesions) were received within maximum 24 h after clinical resection, weighed and split into half (∼0.5 g per sample per digestion) and subjected to either dispase/DNase or collagenase/DNase digestion. For the dispase technique, lung specimens were placed directly into the dispase solution (2 ml of 50 U/ml dispase/PBS in a 15 ml Falcon Tube for a 0.5 g lung slice) and incubated for 15 min at room temperature. Then, lung pieces were minced with a scalpel and processed exactly as described above for the right murine lung. For the analysis of human lung leukocytes, half of the lung specimen was placed into a dissociation tube containing collagenase/DNase in complete RPMI medium (1 mg/ml and 0.1 mg/ml in complete RPMI, respectively) and processed as described above for the left mouse lung. After erylysis and final washing step, human lung cell suspensions were resuspended in flow cytometry buffer (PBS with 0.5% bovine serum albumin) and further analyzed as described below.