A kit for marine mollusk primary cell separation and culture and uses thereof

A technology for marine molluscs and primary cells, applied in animal cells, invertebrate cells, cell dissociation methods, etc., can solve the slow progress of marine mollusc cell culture work, and there is no universal method for marine mollusk cell culture , culture medium and kits, etc., to reduce the probability of microbial contamination, ensure high activity, low pollution, and low microbial contamination rate

Active Publication Date: 2017-07-04
XIAMEN MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although researchers have carried out a lot of exploratory research work on marine mollusk cell culture, there are still no universal methods, media and kits suitable for marine mollusc cell culture, and the work on marine mollusk cell culture has made great progress. slow

Method used

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  • A kit for marine mollusk primary cell separation and culture and uses thereof
  • A kit for marine mollusk primary cell separation and culture and uses thereof
  • A kit for marine mollusk primary cell separation and culture and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] The marine mollusc primary cell isolation and culture kit consists of packing box, 3 100mL lotion I, 3 100mL lotion II, 50mL lotion III, 15mL solution A, 50mL solution B, 10mL solution C, and instructions for use composition.

[0046] Wherein the lotion I is sterile-filtered seawater containing 100 μg / mL ampicillin, 100 μg / mL streptomycin, 60 μg / mL penicillin, 8 μg / mL nystatin, and 50 μg / mL gentamicin. Take a 100mL sterile sealed bottle, label it, and store it at -20-4°C.

[0047] Lotion II is sterile filtered seawater containing 200 μg / mL ampicillin, 200 μg / mL streptomycin, 120 μg / mL penicillin, 16 μg nystatin, and 100 μg / mL gentamicin. Seal the bottle, label it, and store it at -20-4°C.

[0048] Lotion III is prepared with sterile ultrapure water and contains 20.8g / L glucose, 22.5g / L sodium chloride, 3.36g / L EDTA-2Na, 8g / L sodium citrate pH=7.25 and seawater isotonic chelate Combine the buffer solution, divide it into 50mL sterile sealed bottles, label it, and stor...

Embodiment 2

[0054] First pour 15mL of lotion I and lotion II into six 50mL sterile centrifuge tubes respectively, then pour 5mL of lotion III into the other two 15mL sterile centrifuge tubes, and press it from lotion I Sequence numbered 1, 2, 3, 4, 5, 6, 7, 8 to Wash Solution III and capped immediately.

[0055] Use sterile ophthalmic surgical scissors and tweezers to remove about 200 mg of relatively clean tissue pieces from molluscs, cut them into 1 cm2 size, carefully put them into a sterile test tube containing 10 mL of lotion I, vortex and shake for 30 seconds, repeat 3 times Second-rate.

[0056] Carefully remove the tissue block, put it into a sterile test tube containing 10 mL of washing solution II, vortex and shake it thoroughly for 30 seconds, and repeat 3 times.

[0057] Carefully remove the tissue block, clamp it into a 15 mL sterile test tube containing 5 mL of lotion III, vortex and shake it thoroughly for 10 seconds, and repeat 2 times.

[0058] Take out the tissue block...

Embodiment 3

[0062] Oyster tissue specimens were taken, and the operation steps were the same as in Example 2, to finally obtain suspended spherical oyster gill cells. For the cell density, cell activity trend, and cell micrographs of continuous primary culture, subculture, and subculture for 21 days, please refer to Figure 2-4 .

[0063] Table 1 shows the changes in the cell activity of the isolated gill cells of abalone variegated by the present invention after continuous primary culture in vitro for 30 days.

[0064] Table 1

[0065]

[0066] Use the present invention to isolate the variegated abalone gill cells in vitro for continuous primary culture for 30 days. The micrograph of the cell state (100×) can be found in Figure 5 , using the present invention to carry out the adherent cell clone (200×) produced after the primary culture of the mantle of abalone versicolor for 8 days. Figure 6 .

[0067] After using the variegated abalone cells prepared by the present invention f...

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Abstract

The invention provides a kit for marine mollusk primary cell separation and culture and uses thereof, and relates to marine mollusk cell culture. The kit is provided with a packing box, washing liquid I bottles, washing liquid II bottles, washing liquid III bottles, a solution A bottle, a solution B bottle, a solution C bottle and an operating instruction. The washing liquid I bottles, the washing liquid II bottles, the washing liquid III bottles, the solution A bottle, the solution B bottle, the solution C bottle and the operating instruction are in the packing box. The kit is applied in sterile separation and in-vitro primary culture of a plurality of somatic cells of a plurality of marine mollusks. Through optimizing conditions for separation and primary culture of marine mollusk cells, proper conditions for separation and primary culture of the marine mollusk cells are acquired. A problem that rapid construction of marine mollusk cell rapid separation and primary culture sterile culture systems cannot be achieved in the prior art is overcome. A method for marine mollusk primary cell rapid separation and primary cell sterile culture is established. The kit for marine mollusk primary cell separation and culture is provided.

Description

technical field [0001] The invention relates to marine mollusc cell culture, in particular to a marine mollusc primary cell isolation and culture kit and application thereof. Background technique [0002] Cell culture is one of the important technologies and commonly used methods in modern bioengineering technology. It is widely used in research fields such as cell metabolism, gene transcription, protein expression, cell environment emergency and immune mechanism, drug screening and biological drug expression and production. In the research of plants, mammals, fish and insects, cell and tissue culture has become an indispensable research method, especially in related gene expression regulation mechanisms, cell signal transduction pathways, cellular environmental stress mechanisms, and genetic Cell culture has become an irreplaceable means for mutation research and other aspects, and it is also an important technology to promote the rapid development of modern biotechnology. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/07
CPCC12N5/0601C12N2509/00
Inventor 罗联忠许莉庄玲萍孔雪柯仙童刘钟晓
Owner XIAMEN MEDICAL COLLEGE
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