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A technology for invertebrate and cell culture, applied in the field of marine invertebrate cell culture, can solve the problems of late start, lack of stable and reliable cell culture technology solutions for marine invertebrates, etc., and achieve the effect of improving survival rate
Inactive Publication Date: 2018-12-18
ZHEJIANG OCEAN UNIV
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Abstract
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Problems solved by technology
[0004] Due to the late start of marine organism cell culture research and the huge difference between the living environment of marine organisms and terrestrial organisms, there is still a lack of stable and reliable cell culture technology solutions for marine invertebrates, which makes the use of cell biology research methods to explore The physiological regulation mechanism of marine invertebrates and the development of cell engineering application technology have encountered great challenges
Method used
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Embodiment 1
[0036] A method for culturing sea cucumber intestinal cells in this embodiment comprises the following steps:
[0037] 1. Culture medium preparation
[0038] (1) primary culture medium
[0039]
[0040] After the preparation is completed, the 0.22 μm filter membrane is filtered to sterilize and refrigerated for later use.
[0041] (2) Secondary medium
[0042]
[0043] After the preparation is completed, the 0.22 μm filter membrane is filtered to sterilize and refrigerated for later use.
[0044] (3) Tertiary medium
[0045]
[0046]
[0047] After the preparation is completed, the 0.22 μm filter membrane is filtered to sterilize and refrigerated for later use.
[0048] 2. Processing of sea cucumber tissue samples
[0049] Dissect the adult sea cucumber (80-100g) under normal growth state, take out the sea cucumber intestine, remove the contents, soak in 75% ethanol for 5 seconds, take it out quickly and wash it in PBS solution or primary medium At least twic...
Embodiment 2
[0058] In this embodiment, a method for culturing embryonic cells of squid without needles comprises the following steps:
[0059] 1. Culture medium preparation
[0060] (1) primary culture medium
[0061]
[0062] After the preparation is completed, the 0.22 μm filter membrane is filtered to sterilize and refrigerated for later use.
[0063] (2) Secondary medium
[0064]
[0065]After the preparation is completed, the 0.22 μm filter membrane is filtered to sterilize and refrigerated for later use.
[0066] (3) Tertiary medium
[0067]
[0068]
[0069] After the preparation is completed, the 0.22 μm filter membrane is filtered to sterilize and refrigerated for later use.
[0070] 2. Handling of Squid Tissue Samples
[0071] Collect squid squid embryos that develop about 15 days after spawning and fertilization, soak them in 75% ethanol for 5 seconds, take them out quickly and wash them at least twice in PBS solution or primary medium to remove ethanol, and th...
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Abstract
The invention discloses a marine invertebrate cell culture method. The method comprises: (1) dissecting a marine invertebrate, collecting a marine invertebrate tissue sample and cleaning and disinfecting the tissue sample, (2) placing the cleaned and disinfected tissue sample in a primary medium and carrying out enzymatic digestion to obtain a primary cell suspension, (3) dispersing the primary cell suspension into a secondary medium and carrying out pre-culture, (4) replacing the secondary medium with a tertiary medium and carrying out post-culture, and (5) when the primary cell density is 60%, collecting the post-cultured cells, diluting the cells, transferring the cells into the tertiary medium and carrying out subculture. The different culture media in different culture stages of marine invertebrate cells adapt to the different culture requirements of marine invertebrate cells in different culture stages. The method can culture marine invertebrate cells with good viability for a long time and has the characteristics of quick and easy operation, stability and easy repeatability.
Description
technical field [0001] The invention belongs to the technical field of marine biological cell culture, and in particular relates to a marine invertebrate cell culture method. Background technique [0002] With the rapid development of life science, cell engineering has been paid more and more attention in the field of bioengineering technology. In a sense, genetic engineering is the core of modern biotechnology, while cell engineering is the foundation and common platform of modern biotechnology. Cell culture is an essential research method for basic research in cytology, verification of cell structure and function, and cell engineering research. [0003] Primary culture refers to the first culture after removing cells, tissues and organs from the body, also called primary culture. Since the cultured cells have just been separated from the living tissue, they are closer to the living conditions in the organism. As an in vitro expression system, the cell line has good spec...
Claims
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Application Information
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