Development of a cell line from skeletal trunk muscle of the fish Labeo rohita.

IF 2 4区 生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Cytotechnology Pub Date : 2023-08-01 Epub Date: 2023-06-01 DOI:10.1007/s10616-023-00581-3
Mukunda Goswami, Nevil Pinto, B S Yashwanth, A Sathiyanarayanan, Reza Ovissipour
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Abstract

Labeo rohita is a widely cultivated tropical freshwater carp and found in rivers of South Asian region. A new cell line, designated LRM, has been developed from the muscle tissue of L. rohita. Muscle cells were subcultured up to 38 passages in a Leibovitz's-15 (L-15) supplemented with 10% FBS (Fetal Bovine Serum) and 10 ng/ml bFGF. The LRM cells exhibited fibroblastic morphology with a doubling time of 28 h, and a plating efficiency of 17%. A maximum growth rate was observed for LRM cells at 28 °C, 10% FBS and 10 ng/ml bFGF. A cytochrome C oxidase subunit I (COI) gene sequence was used to authenticate the developed cell line. Chromosome analysis revealed 50 diploid chromosomes. The fibroblastic characteristics of the of the LRM cells was confirmed by immunocytochemistry. The expression of MyoD gene in LRM cells was analyzed by quantitative PCR in comparison with passages 3, 18 and 32. The expression of MyoD was higher at passage 18 compared to the passages 3 and 32. The LRM cells attached properly onto the 2D scaffold and the expression of the F-actin filament protein was confirmed by phalloidin staining followed by counter staining with DAPI to observe the distribution of the muscle cell nuclei and the cytoskeleton protein. A revival rate of 70-80% was achieved when the LRM cells were cryopreserved at - 196 °C using liquid nitrogen. This study would further contribute to understanding the in vitro myogenesis and progress toward cultivated fish meat production.

Abstract Image

鱼骨骼干肌细胞系的建立。
罗希塔鲤鱼是一种广泛种植的热带淡水鲤鱼,分布于南亚地区的河流中。一种新的细胞系,命名为LRM,已经从L.rohita的肌肉组织中开发出来。肌肉细胞在补充有10%FBS(胎牛血清)和10ng/ml bFGF的Leibovitz-15(L-15)中传代38代。LRM细胞表现出成纤维细胞形态,倍增时间为28小时,铺板效率为17%。在28°C、10%FBS和10ng/ml bFGF条件下观察到LRM细胞的最大生长速率。细胞色素C氧化酶亚基I(COI)基因序列用于鉴定所开发的细胞系。染色体分析显示有50条二倍体染色体。免疫细胞化学证实了LRM细胞的成纤维细胞特性。通过定量PCR分析MyoD基因在LRM细胞中的表达,并与第3代、第18代和第32代进行比较。与第3代和第32代相比,第18代MyoD的表达更高。LRM细胞正确附着在2D支架上,并通过鬼笔肽染色和DAPI反染色来确认F-肌动蛋白丝蛋白的表达,以观察肌肉细胞核和细胞骨架蛋白的分布。当LRM细胞在- 196°C,使用液氮。这项研究将进一步有助于了解体外肌肉生成和养殖鱼肉生产的进展。
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来源期刊
Cytotechnology
Cytotechnology 生物-生物工程与应用微生物
CiteScore
4.10
自引率
0.00%
发文量
49
审稿时长
6-12 weeks
期刊介绍: The scope of the Journal includes: 1. The derivation, genetic modification and characterization of cell lines, genetic and phenotypic regulation, control of cellular metabolism, cell physiology and biochemistry related to cell function, performance and expression of cell products. 2. Cell culture techniques, substrates, environmental requirements and optimization, cloning, hybridization and molecular biology, including genomic and proteomic tools. 3. Cell culture systems, processes, reactors, scale-up, and industrial production. Descriptions of the design or construction of equipment, media or quality control procedures, that are ancillary to cellular research. 4. The application of animal/human cells in research in the field of stem cell research including maintenance of stemness, differentiation, genetics, and senescence, cancer research, research in immunology, as well as applications in tissue engineering and gene therapy. 5. The use of cell cultures as a substrate for bioassays, biomedical applications and in particular as a replacement for animal models.
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