Methods for in Vitro Percutaneous Absorption
Francis N. Marzulli, Howard I. Maibach in Dermatotoxicology Methods: The Laboratory Worker’s Vade Mecum, 2019
A few studies are reported in the literature concerning the separation of the epidermis and dermis by incubation of skin in enzyme preparations. The protease dispase was shown to produce an epidermal sheet that could be easily peeled from the dermis of human skin after a 24-h incubation at 4°C (Kitano and Okada, 1983). A crude bacterial collagenase was found effective in the preparation of epidermal sheets at a concentration of 0.1 and 0.2% after a 3-h incubation at 37°C (Hentzer and Kobayasi, 1978). Unlike dispase separation, most of the cells were found to be nonviable following separation, but differences in temperature of the incubations may be responsible. The use of the enzymes pancreatin and trypsin for epidermal-dermal separation has also been described (Omar and Krebs, 1975).
Intestinal Effects of Bacterial Chemotactic Peptides: Induction of Inflammation, Release of Eicosanoids.
William J. Snape, Stephen M. Collins in Effects of Immune Cells and Inflammation on Smooth Muscle and Enteric Nerves, 2020
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
A Peyman MD Gholam, A Meffert MD Stephen, D Conway MD FACS Mandi, Chiasson Trisha in Vitreoretinal Surgical Techniques, 2019
Dispase, a neutral 41 kDa proteinase isolated from culture filtrates of Bacillus polymyxa , selectively cleaves type IV collagen and fibronectin.17 It has been reported that the enzyme facilitates PVD in enucleated porcine and human eyes, and in pig eyes in vivo.18,19 Using freeze–fracture scanning electron microscopy, partial digestion of the ILM was observed in postmortem eyes, exposing the mosaic pattern of Müller cell endfeet.18 In rabbit eyes in vivo and in human eyes 15 minutes before enucleation, intravitreal injection of dispase caused intraretinal hemorrhages and ILM disruption at bleeding sites.20 In this series, no effect of dispase on the induction of PVD was seen.
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].
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
Optisol-GS was obtained from Bausch & Lomb (Irvine, CA). Phosphate-buffered saline (PBS), Dulbecco’s Modified Eagle’s Medium (DMEM), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, Ham’s F12 solution, fetal bovine serum (FBS), sodium bicarbonate, dimethyl sulphoxide (DMSO), human epidermal growth factor, hydrocortisone, insulin-transferrin-sodium selenite medium supplement, gentamicin, and amphotericin B were purchased from Sigma-Aldrich (St. Louis, MO). Dispase II was obtained from Roche Diagnostics (Basel, Switzerland), cholera toxin A subunit from Biomol (Exeter, UK), 6 mm biopsy punches from Kai Industries (Gifu, Japan), 6–0 C-2 monofilament sutures (Ethicon Ethilon) from Johnson & Johnson (New Brunswick, NJ), 24 mm culture plate inserts (74 μm mesh size polyester membrane; Netwell) from Corning Costar (Corning, NY), and vancomycin from Abbott Laboratories (Abbott Park, IL). The calcein-acetoxymethyl ester (CAM)/ethidium homodimer 1 (EH-1) kit was obtained from Invitrogen (Carlsbad, CA). The antibodies used in the study are presented in Table 1.
Correlation between thyroidal and peripheral blood total T cells, CD8+ T cells, and CD8+ T- regulatory cells and T-cell reactivity to calsequestrin and collagen XIII in patients with Graves’ ophthalmopathy
Published in Endocrine Research, 2018
Farah Al-Ansari, Hooshang Lahooti, Leanne Stokes, Senarath Edirimanne, Jack Wall
Thyroid tissue was obtained fresh at thyroidectomy and transferred to the laboratory on ice where the tissue was cut and minced using a sharp razor. Ten mL of DMEM medium (Thermo Fisher Scientific) containing penicillin/streptomycin was added to the thinly cut pieces in a tissue culture plate. Dispase and Collagenase enzymes were added to the thinly cut tissue in the DMEM medium for final concentrations of 0.6 U/mL and 80 µg/mL, respectively. The plate was incubated at 5% CO2 pressure and 37°C temperature for a period of 90 minutes for enzyme digestion. After the incubation period, the suspension was passed through a tissue sieve to filter the tissue and isolate the whole population of thyroid cells. The cell suspension was then centrifuged at 1500 rpm for 6 min. The supernatant was discarded and the cells were suspended in 6 mL of PBS that was layered onto 3 mL of Ficoll–Hypaque and centrifuged as recommended by the manufacturer to isolate the MNCs. The isolated MNCs were washed three times with PBS. For the cell proliferation assay, the cells were used fresh, immediately after isolation.
Related Knowledge Centers
- Bacteria
- Collagen
- Epithelium
- Fibronectin
- Leucine
- Mesenchyme
- Protease
- Trypsin
- Phenylalanine
- Paenibacillus Polymyxa