Supernatant of brown adipocytes, method for preparing same and utilization thereof
A brown fat, cell technology, applied in embryonic cells, animal cells, cell culture medium, etc., can solve problems such as hormone group difficulties
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Embodiment 1
[0360] [Example 1] Hypoglycemic effect of subcutaneous administration of brown adipocyte supernatant derived from human ESCs prepared in a "buffer containing only salts and high-concentration glucose" to mice:
[0361] Using the KhES-3 strain of human ESC (Suemori et al., Biochem Biophys Res Commun 345:926-932, 2006) maintained on mouse fetal fibroblasts (MEF), the method described in [Reference Example 4] was used. Induction of differentiation into brown adipocytes (brownadipocyte: BA). The differentiation medium was removed from the mature BA on the 10th day of differentiation induction (after 8 days of planktonic, and the second day of adherent culture on a 6 cm dish), and Krebs-Ringer-HEPES (KRH) containing 16.8 mM glucose was added. ) buffer (NaCl: 128mM, KCl: 5mM, CaCl 2 2.7mM, MgSO 4 1.2mM,Na 2 HPO 4: 1mM, HEPES (pH7.4): 20mM, glucose 16.8mM) 2ml, in a carbon dioxide gas culture device (37 ℃, 5% CO 2 ) after culturing for 16 hours, the supernatant (BA-SUP) was reco...
Embodiment 2
[0364] [Example 2] Induction of expression of glucose transporter gene Glut4 / GLUT4 when human ESC-derived BA supernatant prepared in "BA differentiation medium" was added to skeletal muscle cells:
[0365] BA was prepared by the method described in [Reference Example 4] using human ESCs (KhES-3 strain) maintained in culture on mouse fetal fibroblasts (MEF). And the differentiation medium was removed from mature BA on day 10 and fresh differentiation medium was added (medium exchange). Next, in a carbon dioxide gas culture device (37°C, 5% CO 2 ) after culturing for 16 hours, the supernatant (BA-SUP) was recovered. For the control group, place fresh differentiation medium, gelatin-coated dishes in a carbon dioxide gas culture device (37°C, 5% CO 2 ), and the supernatant recovered after 16 hours was used as the supernatant of the control group (ControlSUP).
[0366] Using the mouse muscle bud cell line C2C12 obtained from the American Type Culture Collection (ATCC), according...
Embodiment 3
[0370] [Example 3] Insulin secretion-promoting effect of BA supernatant derived from human ESC / iPSC prepared by "buffer containing only salts and high-concentration glucose" on islet β cells:
[0371] In the same manner as in [Example 1], SUP of BA derived from human ESC was prepared. That is, in order to induce BA differentiation from human ESCs maintained and cultured on MEFs (Non-Patent Document 19), the differentiation medium was removed from mature BAs on day 10, and KRH buffer containing 16.8 mM glucose was added, and cultured in carbon dioxide gas. device (37°C, 5% CO 2 ) after culturing for 16 hours and recover the supernatant. On the other hand, MIN6 cells (obtained from Jun Miyazaki, a special professor at Osaka University's Industry-University Cooperation Headquarters (currently)) which are mouse islet β cell lines were treated according to the recommended method (Miyazaki et al., Endocrinology 127:126-132, 1990) in 96-well plates. After it was washed with PBS bu...
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