Primary culture method used for maintaining proliferation and differentiation of fish intestinal cells
A technology of primary culture, proliferation and differentiation, applied in the field of animal intestinal cell separation and culture, which can solve the problems of difficult practical use, complicated culture operation process, and easy to be polluted.
Inactive Publication Date: 2012-03-21
SICHUAN AGRI UNIV
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Problems solved by technology
[0004] (2) The existing fish skin collagen smear to promote intestinal cell adhesion is the existing fish skin collagen culture method. Due to the complicated production procedure of fish skin collagen and no commercial reagents to buy, it is difficult to use in practice
[0005] (3) The existing primary culture method of fish intestinal cells adopts a carbon dioxide incubator for cultivation, which is easily polluted, making the cultivation process more complicated
Method used
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Embodiment 1
[0039] Embodiment 1: A kind of fish enterocyte separation and primary culture method, carry out the following steps successively:
[0040] 1. Remove the heads of 2 carp weighing about 50 grams and destroy the spinal cord. The body surface was routinely wiped and sterilized with 75% alcohol cotton balls, the abdominal cavity was cut open with an ophthalmologist, the whole internal organs were taken out, and put into a petri dish filled with 30ml Hanks solution.
[0041] 2. Separate the intestinal tract with stainless steel tweezers, cut out all parts of the intestinal tract from the back of the enteric bulb to the front of the last natural fold of the intestinal tract, put it into a petri dish filled with 30ml of Hanks solution, and carefully remove the system on the surface of the intestinal tract. Membranous tissue, and then rinse the intestinal lumen with a syringe.
[0042] 3. Weigh the flushed intestinal tissue, and then cut it into 2-3 mm tissue pieces with ophthalmic sc...
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The invention discloses a primary culture method used for maintaining proliferation and differentiation of fish intestinal cells. The method comprises the following steps: 1) disinfecting the body surface of fish by using a conventional method, taking out the whole viscus under an aseptic condition, removing mesenteric tissue on the surface of an intestinal tract, and clearing away content in the intestinal tract; 2) cutting the intestinal tract into small tissue pieces with a length of 2 to 3 mm; 3) digesting and separating intestinal cells by using mixed liquor so as to obtain fish intestinal cells and cell mass; 4) carrying out separation and purification by using the method of centrifugation so as to obtain fish intestinal cell mass; 5) inoculating the fish intestinal cell mass obtained after separation and purification onto a culture plate coated by collagen for culture, with the number of the fish intestinal cell mass counted; 6) carrying out primary culture on the inoculated fish intestinal cell mass at a temperature of 24 to 26 DEG C and allowing cells to attachedly grow and to proliferate and differentiate. In the invention, an optimum variety of separase for fish intestinal cells, optimum utilization concentration of the separase and an optimum purifying method for intestinal cell mass are tentatively found out based on the structural characteristics of fish intestinal tract tissue; and optimum inoculation and closed culture conditions for fish intestinal cells are tentatively found out by employing the method of convenient coating of a cell culture vessel with the commodity of collagen.
Description
technical field [0001] The invention relates to a method for isolating and culturing animal intestinal cells, in particular to a method for isolating, purifying and primary culturing fish intestinal cells. Background technique [0002] Fish enterocytes are the main functional cells of the intestine, and participate in various important physiological processes such as digestion and absorption of intestinal nutrients, immune barrier and stress response. At the same time, enterocytes are the fastest proliferating and differentiated cells, which are very important for maintaining the normal function of the intestinal mucosal epithelium. Studying enterocytes in vivo is difficult due to the extensive interactions between nerves, humoral cells, and many cell types. The in vitro culture research model of enterocytes provides an accurate means for studying the proliferation, differentiation and apoptosis of enterocytes, the absorption, utilization and transformation of nutrients. I...
Claims
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IPC IPC(8): C12N5/071
Inventor 周小秋姜俊冯琳刘扬胡凯姜维丹
Owner SICHUAN AGRI UNIV