Chinese mitten crab blood cell culture medium and culturing method

A technology of Chinese mitten crab and its culture method, which is applied in the culture medium and culture field of Chinese mitten crab hemocytes, and can solve the problems of immature cell culture technology, poor cell morphology, short survival time, etc.

Active Publication Date: 2019-04-12
SHANGHAI OCEAN UNIV
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  • Application Information

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Problems solved by technology

On the other hand, with the continuous development of genomics, transcriptomics, proteomics, metabolomics and other technologies, as well as the continuous improvement of gene editing technology, the research on gene function and molecular breeding of crabs is in great need of its cell lines. At present, the cell culture technology of crabs is immature, and the primary cell culture of Eriocheir sinensis still has many problems such as poor cell shape, low...

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  • Chinese mitten crab blood cell culture medium and culturing method
  • Chinese mitten crab blood cell culture medium and culturing method
  • Chinese mitten crab blood cell culture medium and culturing method

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preparation example Construction

[0035] The second aspect of the present invention provides the preparation method of aforementioned medium, comprising: providing TC-100 basal medium, adding calcium chloride, alkali metal chloride, glucose and buffer solution to described TC-100 basal medium, adjusting Add antibiotic solution after osmolarity.

[0036] In the preparation method of the medium provided by the present invention, the TC-100 base medium is 0.9-1L.

[0037] In the preparation method of the culture medium provided by the present invention, the quality of calcium chloride is 1-2g.

[0038] In the preparation method of the culture medium provided by the present invention, the quality of the alkali metal chloride is 1-2g.

[0039] In the preparation method of the medium provided by the present invention, the mass of glucose is 1.5-2.5 g.

[0040] In the preparation method of the medium provided by the present invention, the osmotic pressure is adjusted to 900-1100mOsm / kg by using an osmometer.

[00...

Embodiment 1

[0059] 1) Preparation method of culture medium

[0060] Weigh 1.5g of calcium chloride, 1.5g of sodium chloride and 2g of glucose respectively. Measure 1L of TC-100 basal medium, put the above-mentioned weighed substances into TC-100 medium, add 5% 1×PBS and mix well, use an osmometer to adjust the osmotic pressure to 1000mOsm / kg, and then Filter through a 0.22 micron bacterial filter. Add 1% penicillin-streptomycin double antibody solution.

[0061] 2) In vitro culture of Chinese mitten crab hemocytes

[0062] Take the Chinese mitten crab used in the experiment, wipe the body surface with 75% alcohol for sterilization, and use a 1000ul sterile syringe to extract 500ul hemolymph from the basement membrane of the fifth step of the crab used in the experiment and mix it with an equal volume of anticoagulant uniform. The blood cells were collected by refrigerated centrifugation at 4°C and 2500r / min for 5 minutes in a refrigerated centrifuge. Discard the supernatant on the ul...

Embodiment 2

[0064] 1) Preparation method of culture medium

[0065] Weigh 1.5g of calcium chloride, 1.5g of sodium chloride and 2g of glucose respectively. Measure 1L of TC-100 basal medium, put the above-mentioned weighed substances into TC-100 medium, add 5% 1×PBS and mix well, use an osmometer to adjust the osmotic pressure to 1000mOsm / kg, and then Filter through a 0.22 micron bacterial filter. Add 1% penicillin-streptomycin double antibody solution and 5% fetal bovine serum.

[0066] 2) In vitro culture of Chinese mitten crab hemocytes

[0067] Take the Chinese mitten crab used in the experiment, wipe the body surface with 75% alcohol for sterilization, and use a 1000ul sterile syringe to extract 500ul hemolymph from the basement membrane of the fifth step of the crab used in the experiment and mix it with an equal volume of anticoagulant uniform. The blood cells were collected by refrigerated centrifugation at 4°C and 2500r / min for 5 minutes in a refrigerated centrifuge. Discard...

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Abstract

The present invention belongs to the technical field of cell culture and particularly relates to a Chinese mitten crab blood cell culture medium and a culturing method. The Chinese mitten crab blood cell culture medium comprises a basic culture medium and added components, wherein the basic culture medium is selected from a TC-100 basic culture medium; and the added components and concentrations thereof are as follows: 1%-10% of a buffer solution, 1g/L-2g/L of calcium chloride, 1g/L-2g/L of alkali metal chloride, 1.5g/L-2.5g/L of glucose and 1%-1.5% of antibiotic. The culture medium significantly improves survival rate of Chinese mitten crab blood cells in in-vitro culture and prolongs duration of the blood cell culture in vitro, and the blood cells cultured in vitro can maintain good cellmorphology.

Description

technical field [0001] The invention belongs to the technical field of cell culture, and in particular relates to a culture medium of Chinese mitten crab hemocytes and a culture method thereof. Background technique [0002] Chinese mitten crab (Eriocheir sinensis), commonly known as "river crab", is widely distributed in my country and is an important cultured crab in my country, with high economic value and deep cultural heritage. In recent years, with the continuous expansion of the scale of Chinese mitten crab breeding, the continuous increase of the breeding area and breeding density, the annual increase in production has caused the continuous occurrence of related diseases and seriously affected the health of the Chinese mitten crab breeding industry. steady development. Hemocytes play an important role in the host immune response, including recognition, phagocytosis, melanization, cytotoxicity, and intercellular information transmission. Blood cells cultured in vitro...

Claims

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Application Information

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IPC IPC(8): C12N5/07
CPCC12N5/0601C12N2500/12C12N2500/14C12N2500/34Y02A40/81
Inventor 杨鹤岳武成陈晓雯王军王成辉
Owner SHANGHAI OCEAN UNIV
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