Long-term in-vitro culture and subculture of the hemocytes of swimming crab <italic style="font-style: italic">Portunus trituberculatus</italic>

Liwen GUO; Yaqi ZHAO; Huarong GUO

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Long-term in-vitro culture and subculture of the hemocytes of swimming crab Portunus trituberculatus

Biology | Updated：2024-10-14

- Long-term in-vitro culture and subculture of the hemocytes of swimming crab Portunus trituberculatus
- Journal of Oceanology and Limnology Vol. 41, Issue 5, Pages: 1918-1939(2023)
- Affiliations：
  
  1.Key Laboratory of Marine Genetics and Breeding, Ministry of Education, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China
  2.Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao 266003, China
- Author bio：
  
  huarongguo@ouc.edu.cn
- Funds：
- DOI：
  CLC：
- Received：18 March 2022，
  
  Accepted：04 May 2022，
  
  Online First：26 June 2022，
  
  Published：01 September 2023
- Accepted：
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GUO Liwen,ZHAO Yaqi,GUO Huarong.Long-term in-vitro culture and subculture of the hemocytes of swimming crab Portunus trituberculatus[J].Journal of Oceanology and Limnology,2023,41(05):1918-1939.
DOI：

GUO Liwen,ZHAO Yaqi,GUO Huarong.Long-term in-vitro culture and subculture of the hemocytes of swimming crab Portunus trituberculatus[J].Journal of Oceanology and Limnology,2023,41(05):1918-1939. DOI：

摘要

Abstract

Crab cell line

especially continuous crab cell line

can provide us a useful tool for studies on the virology

immunology

and molecular biology of crabs. However

no continuous crab cell line has been available due to the lacking of suitable medium and the occurrence of mitosis-ar

rest. In this study

long-term in vitro culture conditions for both two- (2D) and three-dimensions (3D) were successfully developed for the circulating hemocytes of swimming crab

Portunus trituberculatus

designated as PTH cells. In 2D culture

a novel crab basic medium in osmolarity of 990–1 100 mOsm/kg was optimized for the first time

which is different from Leibovitz’s L-15 medium in mainly the components of amino acids

containing double strengths of the contents of free amino acid mixture in the crab serum. Then an optimal crab growth medium was developed by supplementing 5% fetal bovine serum

50-g/L yeast extract powder

20-µg/L basic fibroblast growth factor and epidermal growth factor into the optimal crab basic medium

and found that it could support a long-term survival of PTH cells in a healthy monolayer up to 347 days and partially break through the mitosis-arrest of crab cells evidenced by the obvious increase of proliferating potential detected in the 10-d primarily cultured PTH cells. These 2D cultured PTH cells could be successfully sub-cultured for 11 times by physical flushing method and well cryopreserved in liquid nitrogen. In 3D culture

using the same crab growth medium

the PTH cell aggregates could be easily formed and healthily maintained on the surface of solidified Matrigel or in the ultra-low-attachment plate with a survival rate of 50%–60% on Day 103. This work largely improved the primary culture and subculture of crab cells and will facilitate the establishment of continuous crab cell line.

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Long-term in-vitro culture and subculture of the hemocytes of swimming crab Portunus trituberculatus

DOI：

摘要

Abstract

关键词

Keywords

references