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A separation method, a technology of islet cells, which is applied in the field of cell biology, can solve the problems of fragile islets and unsuitability for islet separation, and achieve the effects of cheap price, less cumbersome gradient solution preparation process, and simple and easy operation
Pending Publication Date: 2022-06-28
ZHONGSHAN HOSPITAL FUDAN UNIV
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This separation method is fast and thorough, but the isolated islets
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Embodiment 1
[0062] The method for isolating islet cells of the present embodiment (this method is a method for non-disease diagnosis and treatment purposes) includes the following steps:
[0063] (1) Take the isolated pancreatic tissue block, about 3×3×3 cm, 10 g, and put it into a sterile 50 mL centrifuge tube filled with 20 mL of pre-cooled PBS buffer.
[0064] (2) In the ultra-clean bench, pour the pancreatic tissue into a 10cm cell culture dish, remove the fat around the pancreas with tissue scissors and vascular forceps, rinse it with pre-cooled PBS, and put it into a new cell culture dish for treatment. passed pancreatic tissue.
[0065] (3) Prepare a collagenase solution with a concentration of 0.4 mg / mL, and use a 1 mL syringe needle to perfuse multiple parts of the pancreatic tissue to fully expand the pancreatic tissue, with a total amount of 2 mL / g.
[0066] (4) Preheat the water bath in advance, put the swollen pancreatic tissue into a 50 mL centrifuge tube containing 20 mL o...
Embodiment 2
[0075] Functional assay of pancreatic islet cells
[0076] (1), prepare Krebs buffer
[0077] 129mmol / L NaCl, 4.8mmol / L KCl, 5mmol / L NaHCO 3 , 1.2mmol / L KH 2 PO 4 , 2.5mmol / LCaCl 2 , 1.2mmol / L MgSO 4 , 2mmol / L glucose, pH 7.4.
[0078] (2), pick out islet cells of similar size under the microscope. Islet cells were cultured in RPMI-1640 (10% FBS, 1% PS and 50 μmol / L β-mercaptoethanol) for 48 h. Each group had at least 4-5 samples, and each sample had at least 5-6 islet cells.
[0079] (3) Transfer the islet cells to a 15 mL tube, and centrifuge twice with Krebs buffer at 1000 rpm and 5 min.
[0080] (4), then the islet cells were treated with Krebs buffer containing 2 mmol / L glucose and 0.1% fatty acid-free BSA in CO 2 Incubate in the incubator for 120 min.
[0081] (5) Centrifuge at 1000 rpm for 5 min, and discard the supernatant.
[0082] (6) Resuspend the islet cells with 1300 μL Krebs buffer containing 2 mmol / L glucose and 0.1% fatty acid-free BSA in each tube. ...
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Abstract
The invention provides islet cells and a separation method thereof, and the separation method comprises the following steps: putting an isolated pancreatic tissue into a phosphate buffer salt solution, and pouring a collagenase solution into the treated pancreatic tissue at multiple parts, so that the pancreatic tissue is fully expanded to obtain a swollen pancreatic tissue; the method comprises the following steps: putting swollen pancreatic tissues into a collagenase-containing solution, centrifuging and digesting for multiple times, adding a buffer solution to terminate digestion, centrifuging, discarding supernatant, and resuspending to obtain a resuspension solution; and filtering, re-suspending the filtrate for multiple times to obtain a minced digestion end product, filtering to obtain a precipitate, blowing the precipitate into a culture dish, and collecting. When collagenase pancreas perfusion is carried out, multi-part slow injection is adopted, so that pancreatic tissues can be fully filled, the operation time is shortened, the operation is simple and convenient, the success rate is high, and pancreas islet injury caused by the fact that a collagenase solution is difficult to reach the interiors of tissue blocks and the digestion speeds inside and outside the tissues are inconsistent in the conventional chopping method is avoided; and the requirements of in-vitro basic experiments can be met.
Description
technical field [0001] The invention belongs to the technical field of cell biology, and particularly relates to a pancreatic islet cell and a separation method thereof. Background technique [0002] Diabetes mellitus is the decline or failure of pancreatic β-cell function caused by a variety of reasons, resulting in absolute or relative insufficiency of insulin. Islet transplantation is a new method for the treatment of insulin-dependent diabetes mellitus, and the isolation, purification and culture of islets in vitro are the premise and key to effective treatment. The two key steps for human islet isolation are islet perfusion and islet isolation. Collagenase perfusion is a key step to ensure the quality and quantity of islet isolation. The traditional tissue shredding and digestion method was first pioneered in 1965, but it is difficult for the collagenase solution to reach the inside of the tissue block, resulting in inconsistent digestion rates inside and outside the t...
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