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Preparation method of silkworm decellularization hemolymph liquid

A technology of hemolymph and decellularization, applied in animal cells and other directions, can solve problems such as poisoning, unsatisfactory culture conditions, affecting the pharmacological effects of products, etc.

Inactive Publication Date: 2012-12-19
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The presence of serum is a major obstacle for purification and may even affect the pharmacological effect of the product
Especially for the cell culture of invertebrates, because its nutritional requirements and living conditions are fundamentally different from those of vertebrates, applying the existing culture conditions of vertebrates in the primary culture of invertebrate cells cannot meet the requirements. The serum of higher animals May play a toxic role in the living environment of lower animals
There is no relevant research report

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] (1) Disinfection of silkworm body surface

[0022] In the aseptic operating table, 200 healthy silkworms in the dormancy stage of the 2nd instar were taken, and placed in a sodium hypochlorite solution with a mass percentage concentration of 2% for body surface disinfection for 5 minutes, and then sterilized with ethanol with a volume ratio concentration of 75% for 5 minutes, and then disinfected with sterile water for 5 minutes. After the residual sodium hypochlorite and ethanol are rinsed with bacterial water, silkworms are placed on sterile absorbent paper to absorb moisture;

[0023] (2) Preparation of silkworm hemolymph

[0024] The silkworm was punctured with a sterile needle in the abdomen and feet to take blood, and the silkworm hemolymph was obtained;

[0025] (3) Antioxidant treatment

[0026] In the obtained silkworm hemolymph, add vitamin C by 10g / L;

[0027] (4) Decellularization treatment

[0028] Centrifuge at 12000g for 20min at 4°C, take the superna...

Embodiment 2

[0035] (1) Disinfection of silkworm body surface

[0036] In the aseptic operating table, 100 healthy silkworms in the 3rd instar dormancy stage were taken, and placed in a sodium hypochlorite solution with a mass percentage concentration of 2% for body surface disinfection for 5 min, and then sterilized with ethanol with a volume ratio concentration of 75% for 5 min. After the residual sodium hypochlorite and ethanol are rinsed with bacterial water, silkworms are placed on sterile absorbent paper to absorb moisture;

[0037] (2) Preparation of silkworm hemolymph

[0038] The silkworm was punctured with a sterile needle in the abdomen and feet to take blood, and the silkworm hemolymph was obtained;

[0039] (3) Antioxidant treatment

[0040] In the obtained silkworm hemolymph, add vitamin C by 20g / L;

[0041] (4) Decellularization treatment

[0042] Centrifuge at 12000g for 20min at 4°C, take the supernatant after centrifugation, dialyze in PBS buffer at 4°C for 24 hours, ...

Embodiment 3

[0049] (1) Disinfection of silkworm body surface

[0050] In the aseptic operating table, 400 healthy silkworms in the dormancy stage of the 2nd instar were taken, and placed in a sodium hypochlorite solution with a mass percentage concentration of 2% for body surface disinfection for 5 min, and then sterilized with ethanol with a volume ratio concentration of 75% for 5 min. After the residual sodium hypochlorite and ethanol are rinsed with bacterial water, silkworms are placed on sterile absorbent paper to absorb moisture;

[0051] (2) Preparation of silkworm hemolymph

[0052] The silkworm was punctured with a sterile needle in the abdomen and feet to take blood, and the silkworm hemolymph was obtained;

[0053] (3) Antioxidant treatment

[0054] In the obtained silkworm hemolymph, add vitamin C by 15g / L;

[0055] (4) Decellularization treatment

[0056] Centrifuge at 12000g for 20min at 4°C, take the supernatant after centrifugation, dialyze in PBS buffer at 4°C for 24 ...

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PUM

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Abstract

The invention relates to a preparation method of silkworm decellularization hemolymph liquid. The preparation method comprises a series of steps of disinfecting the body surfaces of silkworms, preparing the hemolymph liquid of the silkworms, carrying out antioxidant treatment and cell extraction on the liquid, removing bacteria from the liquid and the like. The method takes the hemolymph liquid of the silkworms as materials to prepare a nutrient substance applied to the cell culture of insects. Vitamin C is used for inhibiting the activity of tyrosine oxidase in the hemolymph liquid, so that the hemolymph liquid does not get black by oxidation when being contacted with the outside, the composition and the content of main nutrient substances in the hemolymph liquid are not changed, and the cell culture is not influenced. Compared with serum, the hemolymph liquid of the silkworms is more suitable for the cell culture of the insects, and is low in price.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to cell culture and the preparation of cell culture liquid, in particular to a method for preparing silkworm decellularized hemolymph. Background technique [0002] Serum is the most widely used natural medium in in vitro culture. The growth factors contained in serum can promote the proliferation of cells, and the attachment factors can promote cell adhesion. It is also the source of minerals, lipids and hormones. The primary culture of invertebrate cells requires the addition of certain components to the basal medium to form a complete medium. The added ingredients are usually 5% to 20% fetal bovine serum, hydrolyzed milk protein and yeast extract, etc., in order to meet the requirements of different invertebrate cell culture. [0003] However, the use of serum has been questioned in many ways, mainly because there are many problems with serum: [0004] The main components in serum,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/07
Inventor 朱勇赵诏徐水周婵肖欢
Owner SOUTHWEST UNIVERSITY
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