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In vitro cultivation method for insect mesenteron cells

A technology for in vitro culture and enterocytes, which is applied in the field of insect cell research and culture, and can solve the problems of unsatisfactory separation effect and cell growth status

Inactive Publication Date: 2013-10-16
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, at home and abroad, the midgut tissue is mainly treated with enzymatic hydrolysis methods such as collagenase or trypsin to separate midgut cells, but the separation effect and cell growth status are not ideal.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0010] Example 1: Isolation effect of dispase II from midgut cells of silkworm (Bomyx mori) larvae

[0011] (1) Take healthy fourth instar larvae of silkworm, disinfect them with 10% sodium hypochlorite for 5 min, 75% alcohol for 5 min, and rinse with sterile water for 3 times in an ultra-clean workbench. Transfer the larvae into a Petri dish (33 mm in diameter) filled with 10 mL of sterilized LPS buffer, and dissect the insect to obtain the midgut tissue.

[0012] (2) Put the midgut tissue of the silkworm larvae in a sterilized petri dish, and use collagenase Collagenase (Roche Company), trypsin (Thermo Company), and dispase II (Roche Company) at a concentration of 1 mg / mL respectively. ) and HyQTase enzyme (Thermo Company) and other 4 enzymes were treated, and they were left standing at 28°C, and samples were taken at 15 min, 30 min, 60 min, 90 min and 120 min, respectively, and the midgut tissue and Add the cells to a filter with a pore size of 100 μm (BD Falcon Company) f...

Embodiment 2

[0016] Embodiment 2: In Vitro Culture of Silkworm Larval Midgut Cells

[0017] (1) Screening of the most suitable medium for midgut cells: Take the midgut cells separated by Dispase II enzymatic method, count them on a hemocytometer, and add the cells to TNM-FH (Thermo Company) and TC-100 ( GIBCO Company), Grace (GIBCO Company) and IPL-41 (GIBCO Company) and other 4 different media, according to 1.0×10 per well 5 Cells were seeded into 24-well cell culture plates (BD Falcon Company), 1 mL per well, and cultured in a 28°C incubator. Samples were taken out every 7 days, the cell density was counted with a hemocytometer, and the viability of the cells was determined by trypan blue staining.

[0018] Bombyx mori midgut cells were cultured in 4 kinds of media including TNM-FH, TC-100, Grace and IPL-41. The initial cultured midgut cells were in good condition. With the prolongation of culture time, the cell density decreased and the cell survival rate also decreased. A downward tr...

Embodiment 3

[0023] Example 3: In vitro virus infection of silkworm larvae midgut cells

[0024] (1) Bombyx mori nuclear polyhedrosis virus (BmNPV) infected silkworm embryonic cell line Bm-Em-1 (Li Miaomiao et al., "Establishment and characterization of silkworm embryonic cell line Bm-Em-1", Acta Entomology, 2011, 54(12): 1341-1347) to propagate and make virus suspension.

[0025] (2) The silkworm midgut cells cultured for 7 days in vitro were taken, counted on a hemocytometer, and counted according to 1.0×10 5 The amount of cells / well was transferred to a 24-well cell culture plate and repeated three times; cultured at 28°C for 2 h, sucked off the medium, added BmNPV virus (MOI=10) to each well, cultured at 28°C and observed for 4 days Then check the virus infection results.

[0026] (3) After inoculation with BmNPV, the cell morphology gradually changed, the number of round cells increased, the nucleus enlarged, the chromatin in the nucleus condensed into blocks, and the cells swelled....

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PUM

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Abstract

The invention discloses an in vitro cultivation method for insect mesenteron cells; the vitro cultivation method adds TNM-FH culture medium which contains conditional medium and fetal calf serum into the obtained mesenteron cells; the invention provides a simple and effective cultivation method for the insect mesenteron cells, which is suitable for in vitro cultivation of the insect mesenteron cells, and physiological and pathological research of mesenteron. The cultivation method comprises enzyme liquid used for dissociating mesenteron cells and suitable culture medium used for maintaining in vitro state of the cells, as well as serum concentration and additive, so that the survival rate of the mesenteron cells just separated is more than 80%, the survival rate of the cells after 60 d of in vitro cultivation is more than 20%; the mesenteron cells under in vitro state can be infected by karyotype polyhedral body virus, and is capable of secreting alkaline phosphatase; the in vitro cultivation method lays a foundation for researching physiological function of insect mesenteron.

Description

[0001] This application is a divisional application of the previous application with the application number 2012104754988, the title of the invention "a method for the isolation and in vitro culture of insect midgut cells", and the application date is November 29, 2012. technical field [0002] The invention belongs to the technical field of insect cell research and cultivation, in particular to a method for isolating and culturing midgut cells from the midgut tissue of silkworm (Bomyx mori) larvae. Background technique [0003] The midgut is the main part of the digestive tract of insects. The midgut cells are composed of columnar cells, goblet cells and stem cells, which play an important role in the life activities of insects. The midgut of insects is the main part of secreting digestive enzymes and other enzymes, which plays a role in digesting food and absorbing nutrients. At the same time, the midgut is also the target of various unfavorable factors including pathogeni...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/07
Inventor 李长友郑桂玲周洪旭吴艳蕾童丹丹
Owner QINGDAO AGRI UNIV
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