Development of an immortalized porcine intramuscular pre-adipocyte cell line to facilitate adipogenic investigation in vitro

Abstract

Intramuscular fat (IMF) deposition critically influences pork quality, necessitating reliable in vitro models to investigate its regulatory mechanisms. However, developing cost-effective porcine intramuscular pre-adipocyte (IMPA) seed cells capable of sustained proliferation and complete adipogenic differentiation remains technically challenging. Here, we established an immortalized porcine IMPA cell line through lentiviral-mediated transduction of simian virus 40 large T antigen (SV40T). This cell line retained differentiation capacity for over 25 passages, addressing limitations in primary cell lifespan. Functional evaluations demonstrated robust proliferative activity via CCK-8 assays, sustained telomerase activity, and minimal apoptosis, confirming stable growth characteristics during long-term culture. Furthermore, adipogenic induction assays revealed preserved differentiation potential, evidenced by lipid droplet accumulation and adipogenic marker expression. Collectively, this immortalized porcine IMPA cell line provides a valuable and reproducible model for elucidating molecular mechanisms underlying IMF deposition, offering significant utility for agricultural and biomedical research.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12917-025-05204-x.

Introduction

With growing consumer demand for high-quality pork driven by quality-of-life considerations, intramuscular fat (IMF) content has emerged as the primary determinant of pork palatability and flavor characteristics in China [1]. However, the genetic integrity of indigenous Chinese pig populations faces significant threats from the widespread introduction of foreign breeds [2] necessitating urgent conservation efforts for native porcine germplasm resources. The establishment of cell banks represents a strategic approach to preserve indigenous livestock genetic diversity while facilitating multiple scientific objectives: safeguarding genetic resources, elucidating species origins and evolutionary mechanisms, enhancing economically valuable traits through breeding programs, developing transgenic models, and providing standardized biological materials for biomedical research [3, 4]. Global initiatives in germplasm conservation have established substantial repositories of animal genetic material, including specialized cell banks for livestock resources [5, 6]. Notably, Chinese native pig breeds such as Putian pigs are contributing critical biological materials to these conservation efforts through the systematic collection of seed cells. This includes specialized cell types like pre-adipocytes, which serve as essential substrates for advancing both livestock conservation strategies and meat quality research [7].

Current scientific consensus posits that adipocytes originate from mesenchymal stem cells (MSCs), sharing a common progenitor with chondrocytes, osteocytes, and myocytes [8]. Adipogenesis occurs through two sequential differentiation processes: initial lineage commitment where MSCs differentiate into pre-adipocytes, followed by terminal differentiation into mature adipocytes [9]. Comparative analyses reveal that subcutaneous pre-adipocytes exhibit significantly higher expression levels of adipogenic-related genes compared to their intramuscular counterparts, correlating with enhanced adipogenic capacity in subcutaneous adipose tissue [10, 11].

In porcine physiology, these distinct adipose depots demonstrate differential impacts on production traits. While excessive subcutaneous fat accumulation negatively impacts carcass yield and meat quality metrics, intramuscular fat deposition positively influences meat sensory characteristics by enhancing tenderness, juiciness, and flavor profiles [12, 13]. Notably, nutritional management strategies employing high-energy feeding regimes promote intramuscular adipocyte hyperplasia, facilitating the development of distinct marbling patterns that serve as critical quality indicators in pork grading systems.

However, an important limiting factor in the in vitro assay of pre-adipocytes is that in order to maintain the proliferation and differentiation ability of cells, cells can only undergo a limited number of passages [14]. This phenomenon is mainly constrained by cellular senescence, which occurs in the normal cells of all organisms in nature, which can control the unrestricted proliferation of cells and prevent them from becoming cancerous. When cells show signs of aging, they cannot be reversed and growth arrest occurs [15]. At present, a variety of technologies can achieve breakthroughs in cell aging, the most important of which is cell immortalization technology. By introducing exogenous genes that can immortalize cells into target cells, it can obtain continuous proliferation without changing its karyotype, and can be passaged for a long time and maintain complete differentiation ability [16].

In this study, the researchers constructed a Putian pig intramuscular pre-adipocyte cell line with high adipogenic potential, which has excellent characteristics for in vitro culture research of porcine adipocytes, which can not only provide seed preservation materials for the protection of Putian pig genetic resources, but also provide stable test cells for subsequent improvement of pork quality, growth efficiency and animal cytogenetic research.

Materials and methods

Experimental animals

Seven-day-old male Putian piglets were commercially purchased from Putian Xianglixiang Black Pig Development Co., Ltd. (Putian, Fujian, China). All experimental protocols involving animals were approved by the Animal Ethics Committee of Fujian Agriculture and Forestry University (Approval No. PZCASFAFU23001) and conducted in accordance with the national standard Guidelines for the Ethical Review of Laboratory Animal Welfare (GB/T 1.1–2009).

Cell isolation, culture and differentiation

Longissimus dorsi muscle samples were aseptically collected from freshly slaughtered piglets and rinsed in ice-cold phosphate-buffered saline (PBS) supplemented with 2% penicillin-streptomycin (v/v). Tissue digestion was performed using 1 mg/mL collagenase type I (Invitrogen, Carlsbad, CA, USA) in a 37 °C shaking water bath (50 rpm) for 120 min. The enzymatic reaction was neutralized by adding an equal volume of complete growth medium containing 10% fetal bovine serum (FBS), 89% DMEM/F12 (Dulbecco’s Modified Eagle Medium/Ham’s F-12, DMEM/F12), and 1% penicillin-streptomycin (v/v). The cell suspension was sequentially filtered through 200-µm and 100-µm nylon mesh filters to remove undigested tissue fragments. Following dual centrifugation cycles (room temperature, 2,000 × g, 10 min) in 15 mL conical tubes, pelleted intramuscular pre-adipocytes were re-suspended in fresh growth medium and maintained at 37 °C under 5% CO₂. Medium replacement was performed at 48-hour intervals until 80% confluency was achieved.

Following treatment, the cells were induced to differentiate using induction medium consisting of high-glucose DMEM (4.5 g/L) supplemented with 10% FBS, 2.5 µM dexamethasone (DEX), 5 µg/mL insulin, 0.1 mM 3-isobutyl-1-methylxanthine (IBMX), and 2% penicillin-streptomycin (v/v). Fresh induction medium was replaced every 48 h, and differentiation was induced for 10 days.

RNA isolation and quantitative analysis

Total RNA was isolated from cultured cells using the Cell/Tissue Total RNA Isolation Kit (YEASEN, Shanghai, China), with RNA integrity verified by spectrophotometric quantification (A260/A280 ratio > 1.8). First-strand cDNA synthesis was performed using 1 µg total RNA per reaction following the manufacturer’s specifications (Reverse Transcription Kit, YEASEN). Quantitative reverse transcription PCR (qRT-PCR) was conducted using Hieff UNICON^® Universal Blue SYBR Green Master Mix (YEASEN) on a QuantStudio 5 Real-Time PCR System (Applied Biosystems), with thermal cycling parameters set as: 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. Relative gene expression quantification was calculated using the comparative 2^−ΔΔCt method, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serving as the endogenous control. All reactions were performed in technical triplicates with no-template controls included in each assay. Primer sequences for target genes are detailed in Supplementary Table S1.

Cell counting kit-8 (CCK-8) assay

When intramuscular pre-adipocytes cultured in 96-well plates reached a density of 3,000 cells per well, CCK-8 reagent (Herui Biotechnology Co., Ltd., Fuzhou, China) was added to each well and incubated with cells for 24 h. Subsequently, the cells were further incubated for 2 h at 37 °C under 5% CO₂ atmosphere. Absorbance at 450 nm was measured using an EPOCH microplate reader (BioTek Instruments, Inc., USA). Cell viability was calculated by subtracting the optical density (OD) values of blank controls from those of treated groups. All experiments were performed with nine independent biological replicates, with each experimental group containing ten technical replicates (n = 10).

Lentivirus packaging

HEK 293 T cells were seeded in 6-well plates 24 h prior to transfection to achieve approximately 80% confluency at the time of transfection. The transfection complex was prepared by mixing 2 µg of pLenti-SV40T-p2a-BSD plasmid, 1.5 µg of pSPAX2 packaging plasmid, and 0.5 µg of pMD2.G envelope plasmid with 10 µL Lipofectamine 3000 reagent in 0.25 mL Opti-MEM medium (Gibco, Invitrogen, Carlsbad, CA, USA) [17]. The mixture was incubated at room temperature for 20 min to allow complex formation. Subsequently, the transfection complexes were diluted with additional Opti-MEM medium to a final volume of 2 mL per well and added to the pre-seeded cells. Following 8 h of incubation at 37 °C with 5% CO₂, the transfection medium was replaced with 2 mL fresh DMEM/F12 complete medium supplemented with 10% FBS. Viral supernatants were harvested at 24-hour intervals over three consecutive day post-transfection. The collected supernatants were filtered through 0.45-µm pore-size membranes and aliquoted for storage at −80 °C until subsequent use.

Lentivirus titer staining

Following the manufacturer’s protocol, lentivirus titer in freshly prepared or thawed solutions was quantified using the qPCR Lentivirus Titer Kit & ViralEntry™ Bundle (abm, Richmond, VA, USA). The viral titer (expressed as infectious units per milliliter, IU/mL) was calculated using the exponential regression formula: IU/mL = e [(Ct - b)/m], where Ct represents the threshold cycle value, m denotes the slope of the standard curve, and b corresponds to the y-intercept of the standard curve.

Oil-red O staining

Following adipogenic differentiation induction, differentiated adipocytes were washed three times with phosphate-buffered saline (PBS, 1×) and fixed with 4% paraformaldehyde (PFA) for 30 min at room temperature. After PFA removal, cells were rinsed three times with deionized water and subsequently stained with Oil Red O working solution (60% saturated, Yuanye Bio-Technology, Shanghai, China) for 30 min at 37 °C. Morphological observations were performed using a Nikon Eclipse Ti inverted microscope (Nikon Instruments, Shanghai, China) with digital image acquisition. Following staining, cells underwent three additional PBS washes before quantitative analysis. For lipid quantification, Oil Red O was eluted with isopropanol and absorbance measurements were conducted at 510 nm using an EPOCH 2 microplate reader (BioTek Instruments, Winooski, VT, USA).

Apoptotic flow cytometry analysis

When cells reached approximately 85% confluency, they were washed twice with PBS and subsequently detached using 0.25% trypsin-EDTA solution. The cell suspension was centrifuged at 300 × g for 5 min to collect cell pellets. Apoptosis detection was performed using the FITC-Annexin V/PI Apoptosis Detection Kit (UElandy Biotechnology, Suzhou, China) according to manufacturer specifications. Briefly, 1 × 10⁵ cells were resuspended in 100 µL binding buffer and stained with 5 µL FITC-Annexin V and 5 µL propidium iodide (PI) for 15 min at room temperature in darkness. Cellular fluorescence was immediately analyzed using a NovoCyte 3000 flow cytometer (Agilent Technologies, Hangzhou, China) with 488 nm excitation. Data acquisition and analysis were performed using NovoExpress software (v1.6.1).

Telomerase activity quantification

Telomerase enzymatic activity was determined using a porcine-specific Telomerase ELISA Kit (LAMI Biotechnology, Shanghai, China) following optimized protocol. Cell lysates were prepared using ice-cold lysis buffer containing 0.5% CHAPS. Microplate wells pre-coated with anti-telomerase capture antibodies were sequentially incubated with 100 µL samples and horseradish peroxidase (HRP)-conjugated detection antibodies for 60 min at 37 °C. After five washes with PBST (PBS containing 0.05% Tween-20), 100 µL 3,3’,5,5’-tetramethylbenzidine (TMB) substrate solution was added for chromogenic development. The enzymatic reaction was terminated after 15 min with 50 µL stop solution, and absorbance at 450 nm (reference wavelength 630 nm) was measured using an EPOCH2 microplate reader (BioTek Instruments, Vermont, USA). Telomerase activity was calculated against a standard curve generated with recombinant porcine telomerase (0–100 mIU/mL).

Senescence-associated β-galactosidase staining

Cellular senescence was assessed using the Senescence β-Galactosidase Staining Kit (Beyotime Biotechnology, Nanjing, China) following manufacturer’s protocol. Briefly, cells cultured in 6-well plates at 80% confluence were washed twice with PBS. Subsequently, 1 mL of β-galactosidase staining fixative solution was added and maintained at room temperature for 15 min. Following three PBS washes (3 min/wash), cells were incubated with 1 mL of freshly prepared staining working solution at 37 °C in a CO₂-free incubator for 12–16 h (overnight). Senescence-associated β-galactosidase (SA-β-gal) activity was quantified by counting positively stained cells (exhibiting blue cytoplasmic precipitates) under an inverted optical microscope (200× magnification) using ImageJ software (v1.53).

Chromosomal karyotype analysis

For metaphase chromosome preparation, exponentially growing cells were treated with 0.3 µg/mL colchicine (Sigma-Aldrich) for 6 h at 37 °C. After trypsinization and centrifugation (300 × g, 5 min), cell pellets were resuspended in pre-warmed 0.075 M KCl hypotonic solution and incubated at 37 °C for 15 min. Cells were fixed using ice-cold methanol: acetic acid (3:1 penicillin-streptomycin) fixative through three sequential treatments: initial fixation for 3 min at room temperature, followed by two 20-minutes fixation cycles with fresh fixative. The final cell suspension in residual fixative (approximately 1 mL) was dropped onto pre-chilled microscope slides and air-dried overnight. Chromosomal spreads were stained with 10% Giemsa working solution (Sigma-Aldrich) for 30 min, rinsed gently with distilled water, and air-dried completely. Karyotypic evaluation was performed using phase-contrast microscopy (Olympus BX53) with metaphase chromosome counting and structural analysis conducted via Cytovision software (v3.93).

Soft agar tumorigenicity assay

The tumorigenic potential of cells was evaluated using the Soft Agar Cell Cloning Kit (GENMED, Shanghai, China) following standardized protocols. Briefly, cell suspensions were mechanically dissociated through repetitive pipetting to achieve single-cell suspensions with a single-cell rate exceeding 95%. Based on preliminary proliferation assessments, cells were subsequently diluted to a final density of 2,500 cells per experimental unit. The base agar layer was prepared by mixing 5% agar solution with pre-warmed complete culture medium (1:9 penicillin-streptomycin) maintained at 37 °C, followed by immediate transfer to T25 culture dishes. For the semi-solid agar layer, 2.5 mL cell suspension was combined with an equal volume of 5% agar solution to establish a 0.25% agar-medium matrix. This cell-containing semi-solid mixture was promptly layered onto the pre-solidified base agar in T25 dishes and allowed to polymerize at ambient temperature. All cultures were maintained in a humidified incubator at 37 °C with 5% CO₂. Colony formation was monitored periodically through phase-contrast microscopy, with quantitative analysis performed following established colony counting criteria (≥ 50 cells/colony).

Statistical analysis

All statistical analyses were conducted using SPSS 21.0 (IBM Corporation). Intergroup differences were assessed using one-way analysis of variance (ANOVA) followed by Duncan’s post hoc test. Statistical significance thresholds were established as follows: P < 0.05 for significant differences, P < 0.01 for highly significant differences, and P < 0.001 for extremely significant differences.

Results

Characterization of Porcine intramuscular pre-adipocytes (IMPAs)

The longissimus dorsi muscle tissue underwent enzymatic digestion with collagenase, followed by isolation of primary intramuscular pre-adipocytes (IMPAs) through differential adhesion method in culture dishes. At 24 h post-seeding, adherent cells exhibited uniform attachment with characteristic short-spindle or round morphology (Fig. 1A). Subsequent observation after 3–5 days of culture revealed robust cellular proliferation, achieving confluence exceeding 85%. During this differentiation phase, the cells underwent gradual morphological transformation into elongated fusiform shapes, demonstrating typical monolayer parallel alignment characteristic of mature IMPAs cultures (Fig. 1B). Cell proliferation kinetics of primary IMPAs were assessed using CCK-8 assay. Quantitative analysis revealed distinct growth phases: cells exhibited a lag phase with minimal proliferative activity during the initial 48 h of culture, followed by a transition to exponential growth phase commencing on day 3. This rapid proliferation persisted for approximately 6 days, reaching stationary phase by day 9. The observed growth pattern demonstrated robust proliferative capacity of primary IMPA, with characteristic sigmoidal curve progression (Fig. 1C).

Fig. 1.

Characterization of porcine intramuscular pre-adipocytes (IMPAs). A Morphology of primary intramuscular pre-adipocytes (IMPAs) 24 h post-isolation and culture; (B) Cellular morphology of primary IMPAs at day 3 of culture; (C) Proliferation kinetics of primary IMPAs demonstrated by growth curve analysis; (D) Fifth-passage IMPAs after 16 days of culture; (E) Lipid droplet formation visualized by Oil Red O staining in differentiated IMPAs; (F) Quantitative analysis of lipid accumulation measured by ImageJ software; (G) Relative mRNA expression levels of adipogenic differentiation markers determined by qRT-PCR; (H) Chromosomal stability assessment through karyotype analysis (G-banding). Scale bars: 100 μm (A, B, D, E); 20 μm (H). Data presented as mean ± SEM from three independent biological replicates. Statistical significance denoted as **P < 0.01 and ***P < 0.001 (one-way ANOVA)

To determine the proliferative capacity of primary IMPAs, the cells underwent serial subculture for expansion. Experimental observations revealed that by the fifth passage, these IMPAs exhibited significantly diminished proliferation rates accompanied by distinct morphological alterations. Specifically, the cells developed branched morphologies with a notable increase in both mononucleated and multinucleated cellular phenotypes. Furthermore, the cell population failed to reach confluence even after extended culture periods exceeding 16 days (Fig. 1D). These collective findings demonstrate that primary IMPAs possess a limited effective mitotic capacity, with their proliferative potential not extending beyond five population doublings.

To further assess the differentiation potential of isolated primary IMPAs, third-generation cells were subjected to differentiation induction. The morphological analysis revealed that small lipid droplets began to emerge in the cells starting on day 3 of differentiation. These lipid droplets progressively increased in size by day 6, ultimately coalescing into large lipid droplets by day 9 (Fig. 1E-F). Molecular characterization demonstrated detectable expression of key adipogenic markers in third-generation IMPAs, including C/EBPα, PPARγ, FABP4, and FASN (Fig. 1G). These findings collectively indicate that cultured IMPAs maintain their adipogenic differentiation capacity through multiple passages, as evidenced by both morphological lipid accumulation patterns and sustained expression of adipogenic markers.

To validate the suitability of isolated Putian IMPAs for lentiviral treatment applications, karyotypic analysis was performed to confirm the diploid status of the established cell line. Following colchicine-mediated mitotic arrest, microscopic examination of metaphase spreads revealed that 46 out of 50 analyzed cells (92%) exhibited the diploid chromosome number (2 N = 38), meeting the criterion for cell line establishment requiring > 80% diploid cell predominance (Fig. 1H). These results demonstrate the successful establishment of a diploid Putian IMPAs cell population, which satisfies the essential cytogenetic requirements for subsequent lentiviral infection experiments.

Identification of IMPAs after lentiviral infection

To generate lentiviral particles, 293 T packaging cells were co-transfected with lentiviral packaging plasmid (pSPAX2 and pMD2.G) and transfer plasmid (pLenti-SV40T-p2a-BSD) using lipid-based transfections. As neither the packaging plasmids nor the transfer plasmid contained a fluorescent marker, a separate control transfection was performed. In this control, the packaging plasmids were co-transfected with a plasmid encoding GFP (pCDH-CMV-MCS-EF1-GFP Puro) to enable assessment of transfection efficiency and determination of the optimal time window for harvesting lentiviral supernatant. Fluorescence analysis revealed approximately 80% transfection efficiency at 24 h post-transfection, prompting initial viral supernatant harvest. Subsequent observation at 48 h post-transfection showed substantial cell mortality in the transfected population, with surviving cells exhibiting near-complete (≈ 100%) transfection efficiency, followed by secondary supernatant collection and subsequent viral concentration (Fig. 2A).

Fig. 2.

Identification of IMPAs after lentiviral infection. A Fluorescence images illustrating plasmid transfection efficiency at various time points; (B) Representative micrographs demonstrating microbial contamination status through simultaneous detection of infected cells, bacterial colonies, and mycoplasma particles; (C) Electrophoresis analysis identifying mycoplasma marker genes in infected cell cultures; (D) Selective screening outcomes of IMPAs under 8 µg/mL blasticidin (BSD) pressure over 5 days; (E) 5-day cell killing curves after treatment with increasing concentrations of BSD; (F) Post-infection screening results of SV40T-IMPAs maintained under 8 µg/mL BSD conditions for 5 days; (G) Comparative analysis of SV40T relative expression levels in IMPAs across infection statuses and successive passages (SV40T-IMPAs-P5 to P25). Scale bar: 100 μm. Data presented as mean ± SEM from three independent biological replicates. Statistical significance denoted as **P < 0.01, was determined by one-way ANOVA with reference to SV40T-P5 group

During the lentiviral co-transfection process, the supernatant was screened for bacterial and mycoplasma contamination through microscopic observation and quantitative PCR (qPCR) analysis. Microscopic examination revealed no turbidity or particulate matter in the culture medium of transfected cells, with no observable bacterial or fungal contamination (Fig. 2B). Concurrent mycoplasma-specific qPCR detection demonstrated negative results in both experimental samples and negative control groups (Fig. 2C), indicating that the lentiviral supernatant was suitable for subsequent infection experiments.

The optimal screening concentration of blasticidin (BSD) for infected immortalized intramuscular pre-adipocytes (IMPAs) was initially determined by utilizing the BSD resistance conferred by the SV40T vector prior to lentiviral transduction. Quantitative analysis demonstrated complete cell death in all IMPAs following 5 days of treatment with 8 µg/mL BSD, establishing this concentration as the optimal selection threshold for subsequent experiments (Fig. 2D-E).

Following 48-hour exposure to lentiviral supernatant (3.63 × 10⁶ IU/mL), transduced cells underwent selection with 8 µg/mL BSD. Initial viability assessment at day 5 post-treatment revealed limited surviving populations. Continuous antibiotic pressure was maintained until day 10, at which point viable colonies were isolated using cloning rings and subsequently expanded in 24-well plates through serial passaging (Fig. 2F).

qPCR analysis of SV40T expression in SV40T-infected IMPAs across successive passages demonstrated that expression was undetectable in negative controls. Stable SV40T expression was observed in infected IMPAs from passage 5 (SV40T-IMPAS-P5), P15(SV40T-IMPAS-P25), through P25 (SV40T-IMPAS-P25), with expression levels demonstrating progressive elevation correlating with increased passage number (Fig. 2G).

These findings confirm successful generation of an IMPA cell line exhibiting stable exogenous SV40T expression and sustained viral function across long-term culture.

SV40T-IMPAs overcome the mitotic division limitation observed in primary IMPAs while maintaining sustained proliferative capacity

To assess whether lentiviral infection induced chromosomal abnormalities, SV40T-IMPAs underwent colchicine treatment followed by karyotypic analysis. Results demonstrated that 42 out of 50 analyzed cells exhibited the normal diploid chromosome number (2 N = 38), confirming the absence of lentivirus-induced chromosomal aberrations in SV40T-IMPAs (Fig. 3A). CCK-8 proliferation assays demonstrated that tenth-passage SV40T-IMPAs (P10) retained robust proliferative capacity post-lentiviral transduction (Fig. 3B). Furthermore, telomerase activity analysis using a commercial detection kit revealed significantly enhanced enzymatic activity in SV40T-IMPAs compared to primary IMPAs controls (P < 0.001), with sustained activity levels maintained through successive cell passages (Fig. 3C).

Fig. 3.

SV40T-IMPAs overcame the mitotic number limitation of primary IMPAs and maintained complete proliferative capacity. A Karyotype analysis of SV40T-immortalized porcine intramuscular pre-adipocytes (SV40T-IMPAs); (B) Growth curves of 25th-generation SV40T-IMPAs (SV40T-IMPAs-P25); (C) Telomerase activity detection in SV40T-IMPAs at primary culture and successive passages; (D) Soft agar colony formation assay for tumorigenicity evaluation. Scale bars: 20 μm (A), 100 μm (D). Data are presented as mean ± SEM (n = 3 independent biological experiments). Statistical significance was determined by one-way ANOVA (***P < 0.001)

To circumvent potential tumorigenesis resulting from the unlimited proliferative capacity conferred by IMPAs infection, we conducted soft agar colony formation assays to evaluate malignant transformation in three experimental groups: uninfected IMPA-P5 cells (negative control), HeLa cells (positive control), and SV40T-IMPA-P15 cells (experimental group). The results demonstrated that while prominent cell colonies formed in the HeLa positive control group, neither the SV40T-IMPA-P15 experimental group nor the IMPA-P5 negative control group exhibited significant colony formation (Fig. 3D). These findings indicate that SV40T-IMPAs maintain genomic stability without undergoing malignant transformation, despite their extended passage history.

These findings collectively demonstrate that SV40T-IMPAs effectively circumvent replicative senescence limitations inherent to primary IMPAs while maintaining genomic integrity and proliferative capacity, and importantly, do not undergo malignant transformation.

Immortalized IMPAs overcome the natural apoptotic barrier of the cell

To investigate the senescence resistance in immortalized cells, we performed β-galactosidase (β-gal) staining on SV40T-immortalized IMPAs (SV40T-IMPAs). This analysis revealed distinctive senescence patterns at different cell passages. Uninfected IMPAs exhibited significantly higher senescence levels than SV40T-IMPAs-P3 (passage 3) after only three subcultures (P < 0.01). This trend intensified with subsequent passages, as IMPAs-P5 (passage 5) demonstrated markedly increased senescence compared to IMPAs-P3 (P < 0.01). In contrast, SV40T-IMPAs maintained stable senescence profiles through successive passages (P5, P15, and P25), with senescence levels in SV40T-IMPAs-P5 being significantly lower than those in non-immortalized IMPAs-P3 (P < 0.01) (Fig. 4A-F).

Fig. 4.

Immortalized IMPAs overcome the natural apoptotic barrier of the cell. A-K β-Galactosidase staining analysis in IMPAs (A-E) and quantitative assessment of senescence-associated β-galactosidase-positive cells (F). G-K Flow cytometric evaluation (G-J) with corresponding quantitative data (K) comparing cellular characteristics among different passages of IMPA cells. Experimental groups included: non-immortalized IMPA cells at passage 3 (IMPA-P3), and SV40T antigen-immortalized IMPA cells at passages 5, 15, and 25 (designated as SV40T-P5, SV40T-P15, and SV40T-P25, respectively). Quantification of β-galactosidase staining was performed using ImageJ software. Scale bars represent 100 μm. Data represent mean ± SEM from three independent biological replicates. Statistical significance was determined by one-way ANOVA (***P < 0.001)

Apoptotic analysis using dual-parameter flow cytometry with Annexin V/PI staining showed substantial reduction in cell death parameters for immortalized cells. SV40T-IMPAs at later passages (P15, P20, and P25) exhibited significantly lower percentages of apoptotic cells (early and late apoptosis) and necrotic cells compared to non-immortalized IMPAs-P3 (P < 0.01) (Fig. 4G-K). These findings collectively demonstrate that SV40T-mediated immortalization effectively bypasses the intrinsic apoptotic checkpoint in IMPAs while maintaining replicative stability through successive passages.

Immortalized cells maintain an efficient adipogenic differentiation ability

To assess the adipogenic differentiation capacity of SV40T-immortalized intramuscular pre-adipocytes (SV40T-IMPAs) while excluding potential interference from lentiviral infection, we performed in vitro differentiation assays coupled with molecular characterization. Oil red O staining demonstrated that SV40T-IMPAs maintained robust adipogenic potential following differentiation induction, as evidenced by substantial lipid droplet accumulation (Fig. 5A-B). Complementary quantitative real-time PCR (qPCR) analysis revealed significant upregulation of adipogenic marker genes during differentiation progression. Specifically, the expression levels of C/EBPα, PPARγ, FABP4, and FASN showed marked elevation (P < 0.01) in day 12 differentiated SV40T-IMPAs compared with undifferentiated controls (Fig. 5C-F). These findings collectively indicate that the immortalized IMPAs retain full adipogenic differentiation competence while preserving the essential biological characteristics of primary IMPAs.

Fig. 5.

Immortalized cells maintain an efficient adipogenic differentiation ability. A-B Representative Oil Red O staining images (A) and corresponding quantitative analysis (B) of lipid accumulation in SV40T-immortalized porcine intramuscular preadipocytes (SV40T-IMPAs) at day 12 post-differentiation induction. C-F mRNA expression profiles of adipogenic marker genes in differentiated SV40T-IMPAs at day 12. Scale bars: 100 μm. Quantitative analysis of Oil Red O staining was conducted using ImageJ software. Data are presented as mean ± SEM (n = 3 independent biological replicates). Statistical significance was determined by one-way ANOVA (*P < 0.05, ***P < 0.001)

Discussion

As a crucial experimental cellular material for adipogenesis research, primary pre-adipocytes are typically isolated from adipose tissue or ectopic adipose deposition tissues (e.g., skeletal muscle tissue) across various species. This approach enables researchers to obtain tissue-specific and species-matched primary pre-adipocyte models for investigating adipogenic mechanisms [18, 19]. However, experimental applications of primary pre-adipocytes face technical limitations due to the Hayflick limit inherent in primary cell cultures, necessitating repeated isolation procedures [20]. Significant inter-batch variability in cellular physiological characteristics frequently occurs due to donor-dependent factors including animal physiological status and genetic heterogeneity [21]. To address these challenges, cellular immortalization has emerged as an effective strategy for establishing stable pre-adipocyte models. This technique has been successfully implemented across multiple cell types, such as white adipocytes, brown adipocytes, fibroblasts, fibro-adipogenic progenitors (FAPs), skeletal muscle satellite cells (SCs), intestinal epithelial cell (IECs), etc., and diverse species, including humans, mice, swine, bovines, and chicken, etc [22–26]. Immortalized cell lines demonstrate particular utility in overcoming the limitations of primary cell cultures while maintaining species- and tissue-specific biological characteristics essential for adipogenesis studies.

Intramuscular fat (IMF) content is a critical determinant of pork quality, as it directly influences the flavor and palatability of the meat [27]. Within skeletal muscle tissue, intramuscular adipocytes play a pivotal role in IMF deposition by mediating fat accumulation through adipogenic differentiation pathways, thereby regulating overall IMF levels in swine [28]. Notably, within the adipocyte developmental hierarchy, pre-adipocytes serve as progenitor cells with the capacity for terminal differentiation into mature adipocytes [29]. During the course of this investigation, researchers elucidated the molecular mechanisms underlying adipogenesis through the isolation of pre-adipocyte. The biological characteristics of mature adipocytes necessitated this experimental approach. These cells are terminally differentiated and have completed adipose storage. While mature adipocytes may serve as phenotypic evidence in adipogenesis studies, their utility for mechanistic exploration at the molecular level remains limited [30]. Consequently, establishing porcine intramuscular pre-adipocyte (IMPA) cell line carries substantial scientific significance and economic potential. Notably, commercial porcine IMPA cell lines remain unavailable to date, underscoring the exploratory nature of this preliminary investigation. The experimental methodology employed standard cell isolation protocols that demonstrated robust efficacy, enabling successful cultivation of target cells. Subsequent characterization confirmed the isolated primary cells as porcine longissimus dorsi-derived pre-adipocytes, as substantiated by morphological and molecular analyses (Fig. 1).

Currently, various methods exist for constructing immortalized cell lines, including radiation exposure and chemical carcinogen treatment to induce malignant transformation. However, these approaches not only exhibit low efficiency but also risk causing genomic damage that compromises the integrity of cellular genetic material [31]. Consequently, a common methodology involves viral-mediated random integration of exogenous oncogenes (such as SV40T) into primary cell genomes, as the constructs lack species-specific homologous sequences for targeted recombination. This strategy enables sustained telomerase activation and maintenance of mitotic capacity, thereby achieving cellular immortalization [32]. In the present study, we employed the simian virus 40 large T antigen (SV40T), a pivotal oncoprotein in SV40 viral pathogenesis, for cellular immortalization. Mechanistically, SV40T exerts dual regulatory effects: first, it inhibits cellular senescence pathways by disrupting the tumor suppressor functions of retinoblastoma protein (pRb) and p53 through direct protein-protein interactions; second, it activates telomerase activity to maintain telomere length homeostasis, thereby preserving replicative potential [33]. Our experimental data quantitatively confirmed the telomerase-activating effect of SV40T in IMPAs through telomerase activity quantification, with results presented in Figs. 2 and 3.

Immortalized cell lines established through SV40T lentiviral transduction require rigorous tumorigenicity assessment due to the inherent oncogenic risk associated with p53 tumor suppressor inactivation [34]. Our tumor formation assay demonstrated that the engineered SV40T-IMPAs cell line maintains controlled proliferative capacity without exhibiting tumorigenic transformation (Fig. 3), fulfilling essential criteria for stable immortalization while preserving cellular functionality appropriate for experimental applications.

Current viral immortalization strategies primarily employ three oncogenic agents: SV40 large T antigen, human papillomavirus E6/E7 proteins, and Epstein-Barr virus-derived elements, each demonstrating distinct efficiency in establishing permanent cell lines through targeted interference with cellular senescence pathways [35, 36]. Notably, alternative non-viral approaches have emerged, including telomerase reverse transcriptase (TERT) activation. This technique has been successfully implemented in generating immortalized porcine intramuscular preadipocytes from Yorkshire × Landrace hybrids [37], contrasting with SV40T-based immortalization protocols employed for subcutaneous adipose-derived precursor cells [38]. These established models share functional similarities with the current system but differ in immortalization strategies and tissue origins, thereby expanding the methodological repertoire for adipocyte research.

Notably, as upstream progenitor cells of adipocytes, pre-adipocytes exhibit a defining cellular characteristic of adipogenic differentiation potential. Adipogenesis represents an energy storage process characterized by progressive intracellular lipid droplet accumulation and fusion during differentiation, ultimately culminating in triglyceride storage during mid-to-late differentiation stages [39]. This adipogenic differentiation process is governed by stage-specific transcriptional regulators: C/EBPα and PPARγ serve as key mid-early period of adipogenic markers, while FABP4 and FASN emerge as predominant late period of adipogenic markers. Our experimental results demonstrate that SV40T-immortalized porcine pre-adipocytes (SV40T-IMPAs) retained robust adipogenic differentiation capacity. However, it is important to note that the level of adipogenic differentiation was slightly reduced in SV40T-IMPAs compared to primary pre-adipocytes. In contrast, no significant difference in differentiation capacity was observed between different passages of SV40T-IMPAs (results not shown). This partial impairment in differentiation may be attributed to lentivirus-mediated random insertion of the exogenous SV40T sequences, potentially causing genomic disruption and thereby affecting the adipogenic potential of the pre-adipocytes. A similar reduction in both osteogenic and adipogenic differentiation potential has been observed in lentivirally transduced (mesenchymal stem cells, MSCs) compared to primary MSCs in other studies [40, 41]. Nevertheless, the overall data confirm the successful establishment of an immortalized porcine IMPAs system in this investigation (Fig. 5).

It is noteworthy that the colony selection method employed in this study, utilizing cloning rings to isolate viable colonies following lentiviral transduction and blasticidin selection, represents a standard and effective approach for establishing stable cell lines. However, this technique, while ensuring the isolation of robustly growing colonies, does not strictly guarantee a single-cell origin for each expanded population. Therefore, the potential limitation of clonality should be acknowledged, as the resulting cell population, while homogeneously expressing the transgene and exhibiting stable characteristics, might potentially originate from a small number of progenitor cells rather than a single founder cell. Nevertheless, the functional consistency observed across multiple passages and batches in our study—including stable proliferation, sustained adipogenic potential, and genomic integrity—supports the utility and reliability of the established SV40T-IMPA cell line as a valuable in vitro model.

Authors’ contributions

Weimin Lin, Chongfan Du, and Yongjie Huang: Conceptualization, Methodology, Software, Investigation; Weimin Lin and Chongfan Du: Formal Analysis, Writing - Original Draft; Rui Cao and Chao Li: Visualization, Investigation; Tianfang Xiao and Weimin Lin: Funding Acquisition; Weimin Lin: Resources, Supervision; Ruiyi Lin: Writing - Review & Editing.

Funding

This project was supported by Natural Science Foundation of Fujian Province (2023J01446), Modern Agricultural Technology System Program of Fujian Province (2019 − 144), Fujian Province Young and Middle-Aged Teacher Education Research Project (JAT220059).

Data availability

The research data used to support the findings of this study are included within the article.

Declarations

Ethics approval and consent to participate

All animal experiments in this study were approved by the Animal Ethics Committee of Fujian Agriculture and Forestry University (Approval No. PZCASFAFU23001) and conducted in accordance with the national standard Guidelines for the Ethical Review of Laboratory Animal Welfare (GB/T 1.1–2009).

Consent for publication

Not Applicable.

Competing interests

The authors declare no competing interests.