Thailand harbors a remarkable diversity of betta (Siamese fighting fish); however, their wild populations are threatened by habitat loss and genetic pollution from released ornamental fish. Therefore, effective conservation strategies are needed to extend traditional in situ and ex situ methods, which are resource-intensive and prone to external threats. This study established a practical approach for cultivating cells from ornamental bettas and applying it to 11 wild species collected from their specific type localities in Thailand. Additionally, relevant available data on water properties were collected. Cell isolation was achieved from larval caudal fin bud and adult caudal fin tissues, with larval specimens exhibiting a significantly higher isolation success rate (83.3%) and a shorter initial cultivation period (13.27 ± 3.2 days) compared to adult cells (38.8% success, 24.33 ± 7.8 days initial period). After overcoming senescence, the cell doubling time (DT) was approximately 2 days, and cryopreservation assays demonstrated post-thaw viability over 80%, ensuring the long-term viability. While larval isolation is challenging, adult fin biopsy provides a superior alternative by bypassing breeding and euthanasia. This non-lethal, repeatable sampling method allows for precise species identification via external morphology, offering a highly efficient strategy for conservation. Cell isolation was achieved for 10 of the 11 species, 9 of which resulted in established, cryopreserved cell lines, representing the first maintained cell lines from larval and adult tissues of Betta species and providing a novel, valuable resource for both biotechnology and the crucial biodiversity conservation of this important freshwater fish of Thailand.

Background and Aim: Canine mammary cancer (CMC) is the most frequently diagnosed malignancy in female dogs, sharing significant pathological and molecular similarities with human breast cancer (HBC). Despite the availability of various CMC cell lines, most represent triple-negative or epidermal growth factor receptor 2 (ErbB2)-enriched subtypes, which limit research on hormone receptor-positive cancers. This study aimed to establish and characterize novel CMC cell lines representing luminal A and B subtypes. Materials and Methods: Between 2020 and 2021, 31 canine mammary tumors (CMTs) were collected from clinical cases. Tumor tissues were processed for primary culture, and two cell lines – CMGT_071020 and CMGT_180321 – were successfully established. Immunohistochemistry (IHC) was used to assess expression of estrogen receptor alpha (ERα), progesterone receptor (PR), ErbB2, Ki-67, vimentin, and multi-cytokeratin. Functional assays (wound-healing and transwell migration) assessed metastatic behavior. Gene expression (EGFR, TP53, Bcl-2, PTEN, SNAIL, N-cadherin, and E-cadherin) was analyzed using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Cell line authentication was confirmed through short tandem repeat (STR) profiling and mycoplasma testing. Results: The CMGT_071020 (luminal B) and CMGT_180321 (luminal A) cell lines were derived from malignant epithelial tumors and maintained stable growth over 30 passages. IHC confirmed molecular subtype classifications. CMGT_071020 exhibited a fibroblast-like morphology, a high Ki-67 index (67%), and superior migratory capacity compared to CMGT_180321 and the commercial ErbB2-enriched REM134 cell line. E-cadherin expression was significantly elevated in CMGT_071020 (p < 0.05), whereas the expression levels of other genes were comparable. STR analysis verified their genetic uniqueness, and both lines were free from mycoplasma contamination. Conclusion: This study successfully established and characterized two novel hormone receptor-positive CMC cell lines, representing luminal A and luminal B subtypes. The CMGT_071020 line exhibited higher metastatic potential, offering a promising model for aggressive hormone-responsive CMC. These cell lines provide valuable tools for comparative oncology and may facilitate subtype-specific therapeutic research. Keywords: canine mammary cancer, cell line establishment, comparative oncology, immunohistochemistry, luminal A, luminal B, metastasis, molecular subtype, reverse transcription quantitative real-time polymerase chain reaction.

The isolation of fibroblast-like cells from crocodile skeletal muscle provides a valuable platform for studies in comparative physiology, reptilian biology, regenerative medicine, and tissue engineering. In this article, we present an optimized protocol for isolating and characterizing fibroblast-like cells derived from the embryonic skeletal muscle of the Siamese crocodile (Crocodylus siamensis). The procedure improved cell yields and viability while maintaining phenotypic and genetic stability. Dorsal and tail skeletal muscle tissue was cultured in flasks pre-coated with collagen. The cells attached and began migrating from the explants within one day. Optimal culture conditions were achieved using Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 or Minimum Essential Medium Alpha supplemented with 10% fetal bovine serum, 2% crocodile serum, and growth-promoting factors, incubated at 28 °C without CO₂. These conditions supported a shorter population doubling time and enhanced cell proliferation. The established cells displayed a spindle-shaped fibroblastic morphology, expressed the fibroblast-specific marker fibronectin, and maintained a stable karyotype through passage 20. The cell line remained stable and proliferative for at least 30 passages under standard culture conditions. Our study established the first standardized fibroblast-like cell line derived from C. siamensis, thus providing a foundation for future studies in reptilian physiology, cytogenetics, and regenerative biotechnology.