Current IssueEditorial BoardOnline First ArticlesArchive
Current IssueEditorial BoardOnline First ArticlesArchive

Full Research Article

Development of Primary Cell Culture from Different Tissues of Osteobrama belangeri, a Near Threatened Fish Species of the North-Eastern Region of India

S
Shawna Yadav1
B
Basdeo Kushwaha2
S
S. Murali2
R
Ravindra Kumar2
U
U.K. Sarkar2
H
Himanshu Priyadarshi1
T
Tanmoy Gon Choudhury1
A
Anindya S. Barman1,*
1Department of Fish Genetics and Reproduction, College of Fisheries, CAU (I), Lembucherra-799 210, West Tripura, India.
2Genomics and Computational Resources Division, ICAR-National Bureau of Fish Genetic Resources, Lucknow-226 002, Uttar Pradesh, India.

ABSTRACT

Background: Primary cell culture closely matches the physiological and biochemical features of an in vivo system and it serves as a representative model for studying many key issues of disease, reproduction, genetics and biotechnology. The present study aimed to develop a primary culture system from the Gill, Liver and Kidney tissues of Osteobrama belangeri, which is an important candidate species for aquaculture.

Methods: The primary culture was established from tissues through the explant method. The cultured cells were maintained in Leibovitz’s L-15 Medium supplemented with FBS (Fetal Bovine Serum) and Antibiotic Antimycotic Solution 100X Liquid. The growth optimization of cultured cells was performed at different incubation temperatures (25°C, 28°C, 30°C) and FBS concentrations (5%, 10% and 15%).

Result: The maximum growth rate of cultured gill cells was recorded at 30°C temperature and 15% FBS. The cultured cells were characterized for species authentication using DNA barcoding. The sequence analysis reveals and confirms the species of origin. This primary culture of different tissues provides a foundational in vitro platform for conservation genetics, disease diagnostics and biotechnological applications in O. belangeri.

INTRODUCTION

The North Eastern Region of India harbours a rich and diverse aquatic ecosystem, supporting numerous fish species vital to regional biodiversity and the livelihoods of local communities. Osteobrama belangeri (Pengba), a Near Threatened fish species native to South and Southeast Asia, holds considerable ecological and economic importance (Deepti et al., 2023).  Despite its significance, comprehensive studies on the cellular and molecular biology of Pengba remain scarce, limiting our understanding of its physiology and responses to environmental and biological stressors.
       
Establishing primary cell cultures from different tissues of Pengba provides a valuable approach for understanding its cellular physiology, host-pathogen interactions, toxicological responses and adaptive mechanisms. Primary cell culture offers a robust in vitro system that closely mimics in vivo conditions while allowing controlled experimental manipulation. Although fish cell cultures have been derived from various tissues, fin-derived cultures are most commonly reported because of their high regenerative capacity (Yashwanth, 2019). However, gill, liver and kidney-derived cultures offer distinct advantages because these organs are central to respiration, metabolism, osmoregulation, detoxification and immune function.
       
Gill, liver and kidney tissues are particularly relevant for cellular studies in threatened freshwater fishes. Gills serve as the primary interface with the environment and play key roles in respiration, osmoregulation and pathogen entry (Pampori et al., 2024), while the liver and kidney are critical for detoxification, metabolism and immune regulation. Consequently, primary cell cultures from these tissues enable tissue-specific investigations relevant to environmental stress, disease diagnostics and conservation physiology (Kamble et al., 2023). Fish cell culture systems differ markedly from mammalian models, particularly in terms of incubation temperature and osmotic requirements. Fish cells tolerate a wider temperature range and exhibit lower metabolic rates, enabling prolonged maintenance with minimal intervention (Ern et al., 2023). Consequently, fish cell cultures are widely used for virus isolation and for investigating tissue-and species-specific host-pathogen interactions (Gardenia et al., 2020). Intensification of aquaculture practices, including high stocking densities and uncontrolled inputs, has increased the incidence of viral diseases in cultured carps, often leading to mass mortalities (Nashiruddullah et al., 2021). In this context, well-characterised fish cell cultures are essential tools for advancing research in cellular physiology, immunology, molecular biology and aquaculture biotechnology (Hightower and Renfro, 1988).
       
This study reports the establishment and characterisation of primary cell cultures derived from gill, liver and kidney tissues of Osteobrama belangeri. These cultures provide a valuable platform for future applications in disease diagnostics, environmental toxicology, germplasm conservation and sustainable aquaculture management, contributing to species-specific conservation and comparative fish biology research.

MATERIALS AND METHODS

Maintenance of live fish and collection of tissue samples
 
The study was conducted at the Cell Culture Facility of the National Repository of Fish Cell Lines, ICAR-NBFGR, Lucknow and the Department of Fish Genetics and Reproduction, College of Fisheries, CAU(I), Lembucherra, Tripura. Six live and healthy Pengba (Osteobrama belangeri; 10-15 g) were acclimatised in a 200-L FRP tank with sterile, aerated water for one week. Before tissue collection, six donor fish were fasted for 24 h, anaesthetised with clove oil and externally disinfected with 70% ethanol. Gill, liver and kidney tissues were aseptically excised, rinsed thrice in 1 mL of phosphate-buffered saline (PBS) containing Antibiotic-Antimycotic solution (500 μg/mL streptomycin, 500 IU/mL penicillin and 2.5 μg/mL fungizone) and further cut into 1-2 mm3 explants using sterile scissors, followed by two additional PBS washes.
 
Initiation and maintenance of primary culture
 
Primary cell cultures were established from gill, liver and kidney tissues using the explant culture method under aseptic conditions. For each tissue, 15-20 explants were prepared and seeded to establish a primary culture. Explants were placed in T-25 (25 cm2) culture flasks and allowed to attach undisturbed. Afterwards, 50 µL of fetal bovine serum (FBS) (Gibco, USA) was gently added to enhance adhesion. Cultures were incubated at 25, 28 and 30°C with three FBS concentrations (5%, 10% and 15%). After 24 h, 4 mL of Leibovitz’s L-15 (HiMedia, India) medium supplemented with the respective FBS concentrations and Antibiotic-Antimycotic solution (500 μg/mL streptomycin, 500 IU/mL penicillin and 2.5 μg/mL fungizone) was added. Cultures were maintained under static conditions, with 50% medium replacement every 3-5 days until a confluent monolayer developed.
 
Growth evaluation
 
Cell growth was assessed under varying incubation temperatures (25, 28 and 30°C) and FBS concentrations (5%, 10% and 15%) to determine optimal conditions for primary cell proliferation. Cultures were maintained in L-15 medium until a confluent monolayer formed and cellular morphology, attachment and proliferation were routinely monitored using an inverted phase-contrast microscope (Nikon TS100, Japan). At confluency, adherent cells were detached using 1× trypsin-EDTA solution (Himedia, India) (0.25% trypsin, 0.001% EDTA in PBS) and viable cells were quantified with a haemocytometer. Total cell numbers were calculated as the mean count across chambers multiplied by the dilution and correction factors (105).
 
Cell characterisation by DNA barcoding
 
Genomic DNA was isolated from cultured cells and fin tissue of Osteobrama belangeri using the Sambrook and Russell (2001) method. Species identity was confirmed by PCR amplification of the mitochondrial cytochrome c oxidase subunit I (COI) gene using universal fish primers FishF1 and FishR1 (Ward et al., 2005). PCR was performed in a 15 μL reaction containing dNTPs (2.5 mM each), Primer (10 ìM) and Taq. Polymerase (0.75 U), Template DNA (100 ng/μl). The cycling conditions included initial denaturation at 94°C for 3 min, followed by 35 cycles of denaturation (94°C, 30 s), annealing (54°C, 30 s) and extension (72°C, 1 min), with a final extension at 72°C for 7 min. Amplicons were resolved on 2.0% agarose gels, visualised using a gel documentation system and sequenced on an ABI 3700 automated sequencer (Fig 1). Sequence assembly and editing were performed using BioEdit and species confirmation was achieved using BLASTn. The validated COI sequence was deposited in NCBI GenBank.

Fig 1: Agarose gel electrophoresis showing amplified COI gene fragment.


 
Statistical analysis
 
All experiments were conducted using a completely randomised factorial design in triplicate (n = 3). Data were expressed as mean ± standard error (SE). Statistical analysis was performed using MS Excel and SPSS software (version 23.0). Cell proliferation data (cell counts) were expressed as mean ± standard error (SE). For each tissue type (gill, liver and kidney), the effects of temperature (25, 28 and 30°C) and fetal bovine serum (FBS) concentration (5%, 10% and 15%) on cell proliferation were analyzed separately using two-way analysis of variance (ANOVA), with temperature and FBS concentration treated as fixed factors. The interaction effect between temperature and FBS concentration was also evaluated. When significant main or interaction effects were detected (p<0.05), post hoc multiple comparison tests were performed using Tukey’s honestly significant difference (HSD) test to identify statistically significant differences among individual treatment means. Differences were considered statistically significant at p<0.05.

RESULTS AND DISCUSSION

Development of primary cultures from gill, liver and kidney tissue
 
The present study successfully established primary cell cultures from gill, liver and kidney tissues of O. belangeri, a Near Threatened freshwater fish species of the North Eastern Region of India, using the explant method. In the case of gill tissue, approximately 75% of explants successfully attached and proliferated. The explants adhered firmly to the culture flask surface within 24 h of initiation. Cellular outgrowth from the margins of the explants was observed after approximately 36 h (Fig 2a) and a confluent monolayer was formed within 10 days (Fig 2b). For liver tissue, approximately 86% of explants successfully developed primary cultures. Attachment of explants occurred within 24 h and the initiation of cellular outgrowth was observed after 120 h (Fig 3a). A confluent monolayer was achieved after 12 days of culture (Fig 3b). Similarly, kidney tissue explants exhibited a successful attachment rate of approximately 71% within 24 h of seeding. Cellular outgrowth from the explant edges was observed after approximately 72 h and a confluent monolayer was formed after 12 days of incubation (Fig 4a, b).

Fig 2: Primary cell culture development from the gill tissue of Osteobrama belangeri.



Fig 3: Primary cell culture development from liver tissue of Osteobrama belangeri.



Fig 4: Primary cell culture development from kidney tissue of Osteobrama belangeri.


       
Microscopic examination revealed all cultures contained a heterogeneous population of epithelial and fibroblast-like cells, with fibroblastic cells predominating (approximately 70-80%). A similar predominance has been widely reported in fish primary cultures and cell line development (Bejar et al., 1997; Lai et al., 2003; Lakra et al., 2006; Babu et al., 2011; Lakra and Goswami, 2011; Goswami et al., 2012), likely reflecting the faster attachment and proliferation of fibroblast cells under serum-supplemented conditions, whereas epithelial cells generally require more specific growth factors and proliferate more slowly in vitro (Bols et al., 2005). The explant technique offers advantages over enzymatic dissociation, including procedural simplicity, better preservation of cell-cell interactions and reduced cellular damage (Avella et al., 1994; Tyagi and Mani, 2023). Consistent with the present findings, earlier studies have reported efficient attachment and culture establishment using explant methods in teleosts and shellfish, such as Cirrhinus mrigala (Nanda et al., 2014), Labeo calbasu (Yadav et al., 2022) and other aquatic species (Toullec et al., 1996; Rathore et al., 2007; Wick et al., 2018; Khurshid et al., 2022).
 
Growth studies
 
Cell growth at different temperatures and FBS concentrations
 
Microscopic examination revealed tissue-specific growth responses in primary explant cultures derived from gill, liver and kidney tissues of O. belangeri under different incubation temperatures (25, 28 and 30°C) and fetal bovine serum (FBS) concentrations (5, 10 and 15%). Cell attachment and proliferation were significantly influenced by tissue type, temperature, serum concentration and their interactions (p<0.05).
       
Gill-derived cultures exhibited significantly higher growth at 30°C, followed by 28°C and 25°C, indicating a higher thermal optimum for gill cells. In contrast, liver and kidney cultures showed maximum proliferation at 28°C, with reduced growth at 30°C and 25°C (Fig 5-7). These findings suggest tissue-specific thermal preferences, likely reflecting differences in metabolic activity and physiological roles of individual tissues. Similar temperature-dependent growth patterns have been reported in other fish cell cultures, where optimal growth typically occurs between 24 and 32°C depending on species and tissue origin (Wolf, 1973; Tong et al., 1997; Lakra et al., 2006; Goswami et al., 2012; Dubey et al., 2014; Han et al., 2022). The observed variation in optimal temperature is consistent with reports that cells derived from warm-water fishes generally grow well between 25 and 30°C, whereas cold-water species exhibit lower optima (Gabridge, 1985; Goswami et al., 2013; Murali et al., 2020).

Fig 5: Cell growth of gill tissue at different temperatures (25°C, 28°C and 30°C) and FBS concentration (5, 10 and 15%) observed under inverted microscope (10X).



Fig 6: Cell growth of liver tissue at different temperatures (25°C, 28°C and 30°C) and FBS concentration (5, 10 and 15%) observed under inverted microscope (10X).



Fig 7: Cell growth of kidney tissue at different temperatures (25°C, 28°C and 30°C) and FBS concentration (5, 10 and 15%) observed under inverted microscope (10X).


       
Serum concentration also significantly influenced cell proliferation across all tissues. Gill cultures showed maximum growth at 15% FBS combined with 30°C, whereas liver and kidney cultures exhibited optimal proliferation at 15% FBS and 28°C. In all cases, growth at 15% FBS was significantly higher than at 10% and 5% FBS (Fig 8-10). This trend highlights the critical role of serum supplementation during the early stages of primary culture establishment. FBS provides essential growth factors, hormones, lipids, attachment-promoting proteins and anti-trypsin activity, which collectively enhance cell adhesion and proliferation (Freshney, 2005). Comparable serum-dependent growth responses have been widely documented in fish primary cultures and cell lines (Bols et al., 1994; Freshney, 2005; Hameed et al., 2006; Babu et al., 2011).

Fig 8: Quantitative analysis of cell growth in gill tissue-derived primary cultures of Osteobrama belangeri under different temperature and FBS conditions.



Fig 9: Quantitative analysis of cell growth in liver tissue-derived primary cultures of Osteobrama belangeri under different temperature and FBS conditions.



Fig 10: Quantitative analysis of cell growth in kidney tissue-derived primary cultures of Osteobrama belangeri under different temperature and FBS conditions.


       
The requirement of higher serum concentrations (15-20%) during initial culture establishment has been widely reported in fish cell culture studies (Bols et al., 1994; Hameed et al., 2006; Babu et al., 2011; Beaulah et al., 2017). Similar observations of maximum growth at 15% FBS have been documented in fin and organ-derived fish cell lines, including Clarias batrachus and other teleost species (Babu et al., 2011; Lakra et al., 2010). The present findings further support that serum requirements and thermal optima are both tissue- and species-specific, emphasising the importance of optimising culture conditions for each tissue type.
 
Cell counting
 
Cell counts obtained using a haemocytometer were consistent with microscopic observations and revealed distinct tissue-specific growth responses under varying temperature and FBS conditions (Table 1). Gill-derived cultures exhibited a marked increase in cell density with rising temperature and serum concentration, reaching a maximum of 1.20 × 105  cells ml-1 at 15% FBS and 30°C. In contrast, liver and kidney cultures showed optimal proliferation at 28°C with 15% FBS, attaining peak densities of 0.76 × 105  and 0.90 × 105 cells ml-1, respectively.

Table 1: Effect of temperature and fetal bovine serum (FBS) concentration on cell proliferation in primary cultures derived from gill, liver and kidney tissues of Osteobrama belangeri.


       
Two-way ANOVA revealed that temperature and FBS concentration had significant effects on cell proliferation in gill-derived cultures and a significant temperature × FBS interaction (Table 2). Gill cells exhibited maximum proliferation at 30°C, particularly at 15% FBS, whereas significantly lower cell counts were recorded at 25°C across all serum concentrations. Post hoc comparisons further confirmed that each temperature and FBS level differed significantly from one another, indicating a strong temperature-dependent growth response in gill tissue.

Table 2: Two-way ANOVA showing the effects of Temperature and FBS concentration on cell number for gill tissue.


       
In liver-derived cultures, both temperature and FBS concentration significantly influenced cell growth, with a significant temperature×FBS interaction (Table 3). Maximum proliferation was observed at 28°C combined with 15% FBS, while growth declined at 30°C despite higher serum supplementation. These findings indicate a narrower optimal thermal range for liver cells compared to gill cells.

Table 3: Two-way ANOVA showing the effects of Temperature and FBS concentration on cell number for liver tissue.


       
Similarly, kidney-derived cultures showed significant effects of temperature, FBS concentration and their interaction on cell proliferation (Table 4). Optimal growth occurred at 28°C with 15% FBS, whereas both lower (25°C) and higher (30°C) temperatures resulted in significantly reduced cell counts. The moderate interaction effect suggests that kidney cells are particularly sensitive to deviations from optimal culture conditions.

Table 4: Two-way ANOVA showing the effects of temperature and FBS concentration on cell number for kidney tissue.


       
These differential responses highlight inherent tissue-specific metabolic and physiological adaptations, wherein gill tissue, being directly exposed to environmental fluctuations, exhibits greater tolerance to elevated temperatures, while internal organs such as liver and kidney maintain narrower thermal growth ranges. Similar tissue-dependent temperature preferences have been reported in teleost primary cell culture systems (Wolf, 1973; Tong et al., 1997; Lakra et al., 2006; Goswami et al., 2012; de Almeida et al., 2022; Lahnsteiner, 2024).
 
Molecular characterization for species authentication
 
The origin of the cultured cells was confirmed using DNA barcoding based on the mitochondrial cytochrome c oxidase subunit I (COI) gene. The amplified COI fragment (~540 bp) was sequenced and deposited in the NCBI GenBank under accession number OR539611. Sequence homology analysis revealed >99% similarity with reference Osteobrama belangeri COI sequences available in GenBank, thereby confirming the species authenticity of the cultured cells.
       
Species authentication is an essential quality control step in cell culture-based studies, particularly in conservation and biodiversity research, as it prevents misidentification and cross-contamination (Petit-Marty et al., 2021). The COI gene is widely recognised as a universal DNA barcode for animal identification (Hebert et al., 2003) and has been extensively used to authenticate fish cell lines and primary cultures (Cooper et al., 2007; Barman et al., 2014; Yashwanth et al., 2020). The standardised COI barcode region, typically amplified using universal primers, has proven effective for reliable species-level identification across diverse teleost taxa, including cultured fish cells.
       
The successful establishment of primary cell cultures from multiple tissues of O. belangeri provides a validated in vitro system with broad applicability in conservation genetics, environmental toxicology, disease diagnostics, viral susceptibility studies and germplasm conservation. The availability of gill, liver and kidney-derived cultures offers a controlled platform for tissue-specific investigations of cellular physiology, stress responses, immune function and host-pathogen interactions that are difficult to elucidate solely in vivo. In view of the species’ increasing vulnerability to habitat degradation, climate variability and emerging diseases, these cultures constitute a non-lethal, sustainable tool for conservation-oriented research.
       
This study thus establishes a foundational framework for the future development of continuous cell lines. It highlights the importance of species-specific cell culture systems for long-term applications in conservation biology, disease management and aquaculture biotechnology of O. belangeri. Nonetheless, long-term subculture stability and detailed characterisation of the heterogeneous cell populations were not assessed. Future work should prioritise the development and characterisation of continuous cell lines to support immunocytochemical profiling, cryopreservation and advanced applications in aquaculture and conservation research.

CONCLUSION

The present study successfully established primary cell cultures from gill, liver and kidney tissues of Osteobrama belangeri and defined tissue-specific optimal growth conditions. Gill-derived cultures exhibited maximal proliferation at 30°C, whereas liver and kidney cultures showed significantly higher growth at 28°C. Among the serum levels tested, 15% fetal bovine serum consistently supported the most significant proliferation across all tissues, followed by 10% and 5%. Species authentication of the cultured cells was verified by DNA barcoding of the mitochondrial cytochrome c oxidase subunit I (COI) gene, which showed >99% sequence identity with reference O. belangeri entries in the NCBI database, thereby confirming the species origin and reliability of the culture system. This work provides the first tissue-specific in vitro cellular resource for O. belangeri, a Near Threatened freshwater fish of conservation concern. The authenticated primary cultures offer a non-lethal, sustainable platform for investigating species-specific cellular physiology, thermal tolerance, toxicological responses, disease susceptibility and host-pathogen interactions without imposing additional pressure on wild stocks. Moreover, these cultures constitute a critical foundation for the development of continuous cell lines, enabling long-term applications in conservation biology, disease management and aquaculture biotechnology for O. belangeri.

ACKNOWLEDGEMENT

The authors are thankful to the Honorable Vice Chancellor, Central Agricultural University, Imphal, Director, ICAR-NBFGR, Lucknow and Dean, College of Fisheries, Lembucherra, Tripura, for supporting and providing the necessary facilities to carry out this work.
 
Disclaimers
 
The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Data availability
 
The data generated during the current study will be available upon request.
 
Informed consent
 
All the experiments were conducted following the animal ethics guidelines and the study was approved by the Institutional Animal Ethics Committee (IAEC) of ICAR-NBFGR, Lucknow and College of Fisheries (Central Agricultural University, Imphal), India.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article.

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    Full Research Article

    Development of Primary Cell Culture from Different Tissues of Osteobrama belangeri, a Near Threatened Fish Species of the North-Eastern Region of India

    S
    Shawna Yadav1
    B
    Basdeo Kushwaha2
    S
    S. Murali2
    R
    Ravindra Kumar2
    U
    U.K. Sarkar2
    H
    Himanshu Priyadarshi1
    T
    Tanmoy Gon Choudhury1
    A
    Anindya S. Barman1,*
    1Department of Fish Genetics and Reproduction, College of Fisheries, CAU (I), Lembucherra-799 210, West Tripura, India.
    2Genomics and Computational Resources Division, ICAR-National Bureau of Fish Genetic Resources, Lucknow-226 002, Uttar Pradesh, India.

    ABSTRACT

    Background: Primary cell culture closely matches the physiological and biochemical features of an in vivo system and it serves as a representative model for studying many key issues of disease, reproduction, genetics and biotechnology. The present study aimed to develop a primary culture system from the Gill, Liver and Kidney tissues of Osteobrama belangeri, which is an important candidate species for aquaculture.

    Methods: The primary culture was established from tissues through the explant method. The cultured cells were maintained in Leibovitz’s L-15 Medium supplemented with FBS (Fetal Bovine Serum) and Antibiotic Antimycotic Solution 100X Liquid. The growth optimization of cultured cells was performed at different incubation temperatures (25°C, 28°C, 30°C) and FBS concentrations (5%, 10% and 15%).

    Result: The maximum growth rate of cultured gill cells was recorded at 30°C temperature and 15% FBS. The cultured cells were characterized for species authentication using DNA barcoding. The sequence analysis reveals and confirms the species of origin. This primary culture of different tissues provides a foundational in vitro platform for conservation genetics, disease diagnostics and biotechnological applications in O. belangeri.

    INTRODUCTION

    The North Eastern Region of India harbours a rich and diverse aquatic ecosystem, supporting numerous fish species vital to regional biodiversity and the livelihoods of local communities. Osteobrama belangeri (Pengba), a Near Threatened fish species native to South and Southeast Asia, holds considerable ecological and economic importance (Deepti et al., 2023).  Despite its significance, comprehensive studies on the cellular and molecular biology of Pengba remain scarce, limiting our understanding of its physiology and responses to environmental and biological stressors.
           
    Establishing primary cell cultures from different tissues of Pengba provides a valuable approach for understanding its cellular physiology, host-pathogen interactions, toxicological responses and adaptive mechanisms. Primary cell culture offers a robust in vitro system that closely mimics in vivo conditions while allowing controlled experimental manipulation. Although fish cell cultures have been derived from various tissues, fin-derived cultures are most commonly reported because of their high regenerative capacity (Yashwanth, 2019). However, gill, liver and kidney-derived cultures offer distinct advantages because these organs are central to respiration, metabolism, osmoregulation, detoxification and immune function.
           
    Gill, liver and kidney tissues are particularly relevant for cellular studies in threatened freshwater fishes. Gills serve as the primary interface with the environment and play key roles in respiration, osmoregulation and pathogen entry (Pampori et al., 2024), while the liver and kidney are critical for detoxification, metabolism and immune regulation. Consequently, primary cell cultures from these tissues enable tissue-specific investigations relevant to environmental stress, disease diagnostics and conservation physiology (Kamble et al., 2023). Fish cell culture systems differ markedly from mammalian models, particularly in terms of incubation temperature and osmotic requirements. Fish cells tolerate a wider temperature range and exhibit lower metabolic rates, enabling prolonged maintenance with minimal intervention (Ern et al., 2023). Consequently, fish cell cultures are widely used for virus isolation and for investigating tissue-and species-specific host-pathogen interactions (Gardenia et al., 2020). Intensification of aquaculture practices, including high stocking densities and uncontrolled inputs, has increased the incidence of viral diseases in cultured carps, often leading to mass mortalities (Nashiruddullah et al., 2021). In this context, well-characterised fish cell cultures are essential tools for advancing research in cellular physiology, immunology, molecular biology and aquaculture biotechnology (Hightower and Renfro, 1988).
           
    This study reports the establishment and characterisation of primary cell cultures derived from gill, liver and kidney tissues of Osteobrama belangeri. These cultures provide a valuable platform for future applications in disease diagnostics, environmental toxicology, germplasm conservation and sustainable aquaculture management, contributing to species-specific conservation and comparative fish biology research.

    MATERIALS AND METHODS

    Maintenance of live fish and collection of tissue samples
     
    The study was conducted at the Cell Culture Facility of the National Repository of Fish Cell Lines, ICAR-NBFGR, Lucknow and the Department of Fish Genetics and Reproduction, College of Fisheries, CAU(I), Lembucherra, Tripura. Six live and healthy Pengba (Osteobrama belangeri; 10-15 g) were acclimatised in a 200-L FRP tank with sterile, aerated water for one week. Before tissue collection, six donor fish were fasted for 24 h, anaesthetised with clove oil and externally disinfected with 70% ethanol. Gill, liver and kidney tissues were aseptically excised, rinsed thrice in 1 mL of phosphate-buffered saline (PBS) containing Antibiotic-Antimycotic solution (500 μg/mL streptomycin, 500 IU/mL penicillin and 2.5 μg/mL fungizone) and further cut into 1-2 mm3 explants using sterile scissors, followed by two additional PBS washes.
     
    Initiation and maintenance of primary culture
     
    Primary cell cultures were established from gill, liver and kidney tissues using the explant culture method under aseptic conditions. For each tissue, 15-20 explants were prepared and seeded to establish a primary culture. Explants were placed in T-25 (25 cm2) culture flasks and allowed to attach undisturbed. Afterwards, 50 µL of fetal bovine serum (FBS) (Gibco, USA) was gently added to enhance adhesion. Cultures were incubated at 25, 28 and 30°C with three FBS concentrations (5%, 10% and 15%). After 24 h, 4 mL of Leibovitz’s L-15 (HiMedia, India) medium supplemented with the respective FBS concentrations and Antibiotic-Antimycotic solution (500 μg/mL streptomycin, 500 IU/mL penicillin and 2.5 μg/mL fungizone) was added. Cultures were maintained under static conditions, with 50% medium replacement every 3-5 days until a confluent monolayer developed.
     
    Growth evaluation
     
    Cell growth was assessed under varying incubation temperatures (25, 28 and 30°C) and FBS concentrations (5%, 10% and 15%) to determine optimal conditions for primary cell proliferation. Cultures were maintained in L-15 medium until a confluent monolayer formed and cellular morphology, attachment and proliferation were routinely monitored using an inverted phase-contrast microscope (Nikon TS100, Japan). At confluency, adherent cells were detached using 1× trypsin-EDTA solution (Himedia, India) (0.25% trypsin, 0.001% EDTA in PBS) and viable cells were quantified with a haemocytometer. Total cell numbers were calculated as the mean count across chambers multiplied by the dilution and correction factors (105).
     
    Cell characterisation by DNA barcoding
     
    Genomic DNA was isolated from cultured cells and fin tissue of Osteobrama belangeri using the Sambrook and Russell (2001) method. Species identity was confirmed by PCR amplification of the mitochondrial cytochrome c oxidase subunit I (COI) gene using universal fish primers FishF1 and FishR1 (Ward et al., 2005). PCR was performed in a 15 μL reaction containing dNTPs (2.5 mM each), Primer (10 ìM) and Taq. Polymerase (0.75 U), Template DNA (100 ng/μl). The cycling conditions included initial denaturation at 94°C for 3 min, followed by 35 cycles of denaturation (94°C, 30 s), annealing (54°C, 30 s) and extension (72°C, 1 min), with a final extension at 72°C for 7 min. Amplicons were resolved on 2.0% agarose gels, visualised using a gel documentation system and sequenced on an ABI 3700 automated sequencer (Fig 1). Sequence assembly and editing were performed using BioEdit and species confirmation was achieved using BLASTn. The validated COI sequence was deposited in NCBI GenBank.

    Fig 1: Agarose gel electrophoresis showing amplified COI gene fragment.


     
    Statistical analysis
     
    All experiments were conducted using a completely randomised factorial design in triplicate (n = 3). Data were expressed as mean ± standard error (SE). Statistical analysis was performed using MS Excel and SPSS software (version 23.0). Cell proliferation data (cell counts) were expressed as mean ± standard error (SE). For each tissue type (gill, liver and kidney), the effects of temperature (25, 28 and 30°C) and fetal bovine serum (FBS) concentration (5%, 10% and 15%) on cell proliferation were analyzed separately using two-way analysis of variance (ANOVA), with temperature and FBS concentration treated as fixed factors. The interaction effect between temperature and FBS concentration was also evaluated. When significant main or interaction effects were detected (p<0.05), post hoc multiple comparison tests were performed using Tukey’s honestly significant difference (HSD) test to identify statistically significant differences among individual treatment means. Differences were considered statistically significant at p<0.05.

    RESULTS AND DISCUSSION

    Development of primary cultures from gill, liver and kidney tissue
     
    The present study successfully established primary cell cultures from gill, liver and kidney tissues of O. belangeri, a Near Threatened freshwater fish species of the North Eastern Region of India, using the explant method. In the case of gill tissue, approximately 75% of explants successfully attached and proliferated. The explants adhered firmly to the culture flask surface within 24 h of initiation. Cellular outgrowth from the margins of the explants was observed after approximately 36 h (Fig 2a) and a confluent monolayer was formed within 10 days (Fig 2b). For liver tissue, approximately 86% of explants successfully developed primary cultures. Attachment of explants occurred within 24 h and the initiation of cellular outgrowth was observed after 120 h (Fig 3a). A confluent monolayer was achieved after 12 days of culture (Fig 3b). Similarly, kidney tissue explants exhibited a successful attachment rate of approximately 71% within 24 h of seeding. Cellular outgrowth from the explant edges was observed after approximately 72 h and a confluent monolayer was formed after 12 days of incubation (Fig 4a, b).

    Fig 2: Primary cell culture development from the gill tissue of Osteobrama belangeri.



    Fig 3: Primary cell culture development from liver tissue of Osteobrama belangeri.



    Fig 4: Primary cell culture development from kidney tissue of Osteobrama belangeri.


           
    Microscopic examination revealed all cultures contained a heterogeneous population of epithelial and fibroblast-like cells, with fibroblastic cells predominating (approximately 70-80%). A similar predominance has been widely reported in fish primary cultures and cell line development (Bejar et al., 1997; Lai et al., 2003; Lakra et al., 2006; Babu et al., 2011; Lakra and Goswami, 2011; Goswami et al., 2012), likely reflecting the faster attachment and proliferation of fibroblast cells under serum-supplemented conditions, whereas epithelial cells generally require more specific growth factors and proliferate more slowly in vitro (Bols et al., 2005). The explant technique offers advantages over enzymatic dissociation, including procedural simplicity, better preservation of cell-cell interactions and reduced cellular damage (Avella et al., 1994; Tyagi and Mani, 2023). Consistent with the present findings, earlier studies have reported efficient attachment and culture establishment using explant methods in teleosts and shellfish, such as Cirrhinus mrigala (Nanda et al., 2014), Labeo calbasu (Yadav et al., 2022) and other aquatic species (Toullec et al., 1996; Rathore et al., 2007; Wick et al., 2018; Khurshid et al., 2022).
     
    Growth studies
     
    Cell growth at different temperatures and FBS concentrations
     
    Microscopic examination revealed tissue-specific growth responses in primary explant cultures derived from gill, liver and kidney tissues of O. belangeri under different incubation temperatures (25, 28 and 30°C) and fetal bovine serum (FBS) concentrations (5, 10 and 15%). Cell attachment and proliferation were significantly influenced by tissue type, temperature, serum concentration and their interactions (p<0.05).
           
    Gill-derived cultures exhibited significantly higher growth at 30°C, followed by 28°C and 25°C, indicating a higher thermal optimum for gill cells. In contrast, liver and kidney cultures showed maximum proliferation at 28°C, with reduced growth at 30°C and 25°C (Fig 5-7). These findings suggest tissue-specific thermal preferences, likely reflecting differences in metabolic activity and physiological roles of individual tissues. Similar temperature-dependent growth patterns have been reported in other fish cell cultures, where optimal growth typically occurs between 24 and 32°C depending on species and tissue origin (Wolf, 1973; Tong et al., 1997; Lakra et al., 2006; Goswami et al., 2012; Dubey et al., 2014; Han et al., 2022). The observed variation in optimal temperature is consistent with reports that cells derived from warm-water fishes generally grow well between 25 and 30°C, whereas cold-water species exhibit lower optima (Gabridge, 1985; Goswami et al., 2013; Murali et al., 2020).

    Fig 5: Cell growth of gill tissue at different temperatures (25°C, 28°C and 30°C) and FBS concentration (5, 10 and 15%) observed under inverted microscope (10X).



    Fig 6: Cell growth of liver tissue at different temperatures (25°C, 28°C and 30°C) and FBS concentration (5, 10 and 15%) observed under inverted microscope (10X).



    Fig 7: Cell growth of kidney tissue at different temperatures (25°C, 28°C and 30°C) and FBS concentration (5, 10 and 15%) observed under inverted microscope (10X).


           
    Serum concentration also significantly influenced cell proliferation across all tissues. Gill cultures showed maximum growth at 15% FBS combined with 30°C, whereas liver and kidney cultures exhibited optimal proliferation at 15% FBS and 28°C. In all cases, growth at 15% FBS was significantly higher than at 10% and 5% FBS (Fig 8-10). This trend highlights the critical role of serum supplementation during the early stages of primary culture establishment. FBS provides essential growth factors, hormones, lipids, attachment-promoting proteins and anti-trypsin activity, which collectively enhance cell adhesion and proliferation (Freshney, 2005). Comparable serum-dependent growth responses have been widely documented in fish primary cultures and cell lines (Bols et al., 1994; Freshney, 2005; Hameed et al., 2006; Babu et al., 2011).

    Fig 8: Quantitative analysis of cell growth in gill tissue-derived primary cultures of Osteobrama belangeri under different temperature and FBS conditions.



    Fig 9: Quantitative analysis of cell growth in liver tissue-derived primary cultures of Osteobrama belangeri under different temperature and FBS conditions.



    Fig 10: Quantitative analysis of cell growth in kidney tissue-derived primary cultures of Osteobrama belangeri under different temperature and FBS conditions.


           
    The requirement of higher serum concentrations (15-20%) during initial culture establishment has been widely reported in fish cell culture studies (Bols et al., 1994; Hameed et al., 2006; Babu et al., 2011; Beaulah et al., 2017). Similar observations of maximum growth at 15% FBS have been documented in fin and organ-derived fish cell lines, including Clarias batrachus and other teleost species (Babu et al., 2011; Lakra et al., 2010). The present findings further support that serum requirements and thermal optima are both tissue- and species-specific, emphasising the importance of optimising culture conditions for each tissue type.
     
    Cell counting
     
    Cell counts obtained using a haemocytometer were consistent with microscopic observations and revealed distinct tissue-specific growth responses under varying temperature and FBS conditions (Table 1). Gill-derived cultures exhibited a marked increase in cell density with rising temperature and serum concentration, reaching a maximum of 1.20 × 105  cells ml-1 at 15% FBS and 30°C. In contrast, liver and kidney cultures showed optimal proliferation at 28°C with 15% FBS, attaining peak densities of 0.76 × 105  and 0.90 × 105 cells ml-1, respectively.

    Table 1: Effect of temperature and fetal bovine serum (FBS) concentration on cell proliferation in primary cultures derived from gill, liver and kidney tissues of Osteobrama belangeri.


           
    Two-way ANOVA revealed that temperature and FBS concentration had significant effects on cell proliferation in gill-derived cultures and a significant temperature × FBS interaction (Table 2). Gill cells exhibited maximum proliferation at 30°C, particularly at 15% FBS, whereas significantly lower cell counts were recorded at 25°C across all serum concentrations. Post hoc comparisons further confirmed that each temperature and FBS level differed significantly from one another, indicating a strong temperature-dependent growth response in gill tissue.

    Table 2: Two-way ANOVA showing the effects of Temperature and FBS concentration on cell number for gill tissue.


           
    In liver-derived cultures, both temperature and FBS concentration significantly influenced cell growth, with a significant temperature×FBS interaction (Table 3). Maximum proliferation was observed at 28°C combined with 15% FBS, while growth declined at 30°C despite higher serum supplementation. These findings indicate a narrower optimal thermal range for liver cells compared to gill cells.

    Table 3: Two-way ANOVA showing the effects of Temperature and FBS concentration on cell number for liver tissue.


           
    Similarly, kidney-derived cultures showed significant effects of temperature, FBS concentration and their interaction on cell proliferation (Table 4). Optimal growth occurred at 28°C with 15% FBS, whereas both lower (25°C) and higher (30°C) temperatures resulted in significantly reduced cell counts. The moderate interaction effect suggests that kidney cells are particularly sensitive to deviations from optimal culture conditions.

    Table 4: Two-way ANOVA showing the effects of temperature and FBS concentration on cell number for kidney tissue.


           
    These differential responses highlight inherent tissue-specific metabolic and physiological adaptations, wherein gill tissue, being directly exposed to environmental fluctuations, exhibits greater tolerance to elevated temperatures, while internal organs such as liver and kidney maintain narrower thermal growth ranges. Similar tissue-dependent temperature preferences have been reported in teleost primary cell culture systems (Wolf, 1973; Tong et al., 1997; Lakra et al., 2006; Goswami et al., 2012; de Almeida et al., 2022; Lahnsteiner, 2024).
     
    Molecular characterization for species authentication
     
    The origin of the cultured cells was confirmed using DNA barcoding based on the mitochondrial cytochrome c oxidase subunit I (COI) gene. The amplified COI fragment (~540 bp) was sequenced and deposited in the NCBI GenBank under accession number OR539611. Sequence homology analysis revealed >99% similarity with reference Osteobrama belangeri COI sequences available in GenBank, thereby confirming the species authenticity of the cultured cells.
           
    Species authentication is an essential quality control step in cell culture-based studies, particularly in conservation and biodiversity research, as it prevents misidentification and cross-contamination (Petit-Marty et al., 2021). The COI gene is widely recognised as a universal DNA barcode for animal identification (Hebert et al., 2003) and has been extensively used to authenticate fish cell lines and primary cultures (Cooper et al., 2007; Barman et al., 2014; Yashwanth et al., 2020). The standardised COI barcode region, typically amplified using universal primers, has proven effective for reliable species-level identification across diverse teleost taxa, including cultured fish cells.
           
    The successful establishment of primary cell cultures from multiple tissues of O. belangeri provides a validated in vitro system with broad applicability in conservation genetics, environmental toxicology, disease diagnostics, viral susceptibility studies and germplasm conservation. The availability of gill, liver and kidney-derived cultures offers a controlled platform for tissue-specific investigations of cellular physiology, stress responses, immune function and host-pathogen interactions that are difficult to elucidate solely in vivo. In view of the species’ increasing vulnerability to habitat degradation, climate variability and emerging diseases, these cultures constitute a non-lethal, sustainable tool for conservation-oriented research.
           
    This study thus establishes a foundational framework for the future development of continuous cell lines. It highlights the importance of species-specific cell culture systems for long-term applications in conservation biology, disease management and aquaculture biotechnology of O. belangeri. Nonetheless, long-term subculture stability and detailed characterisation of the heterogeneous cell populations were not assessed. Future work should prioritise the development and characterisation of continuous cell lines to support immunocytochemical profiling, cryopreservation and advanced applications in aquaculture and conservation research.

    CONCLUSION

    The present study successfully established primary cell cultures from gill, liver and kidney tissues of Osteobrama belangeri and defined tissue-specific optimal growth conditions. Gill-derived cultures exhibited maximal proliferation at 30°C, whereas liver and kidney cultures showed significantly higher growth at 28°C. Among the serum levels tested, 15% fetal bovine serum consistently supported the most significant proliferation across all tissues, followed by 10% and 5%. Species authentication of the cultured cells was verified by DNA barcoding of the mitochondrial cytochrome c oxidase subunit I (COI) gene, which showed >99% sequence identity with reference O. belangeri entries in the NCBI database, thereby confirming the species origin and reliability of the culture system. This work provides the first tissue-specific in vitro cellular resource for O. belangeri, a Near Threatened freshwater fish of conservation concern. The authenticated primary cultures offer a non-lethal, sustainable platform for investigating species-specific cellular physiology, thermal tolerance, toxicological responses, disease susceptibility and host-pathogen interactions without imposing additional pressure on wild stocks. Moreover, these cultures constitute a critical foundation for the development of continuous cell lines, enabling long-term applications in conservation biology, disease management and aquaculture biotechnology for O. belangeri.

    ACKNOWLEDGEMENT

    The authors are thankful to the Honorable Vice Chancellor, Central Agricultural University, Imphal, Director, ICAR-NBFGR, Lucknow and Dean, College of Fisheries, Lembucherra, Tripura, for supporting and providing the necessary facilities to carry out this work.
     
    Disclaimers
     
    The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
     
    Data availability
     
    The data generated during the current study will be available upon request.
     
    Informed consent
     
    All the experiments were conducted following the animal ethics guidelines and the study was approved by the Institutional Animal Ethics Committee (IAEC) of ICAR-NBFGR, Lucknow and College of Fisheries (Central Agricultural University, Imphal), India.

    CONFLICT OF INTEREST

    The authors declare that there are no conflicts of interest regarding the publication of this article.

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