This systematic review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 statement. The research questions were formulated using the Population, Intervention, Comparison, and Outcome (PICO) framework, in which the population corresponded to the investigated cell type; the intervention entailed TGF-β2 administration; the comparison involved an appropriate control group; and the outcomes of interest were the onset of posterior capsular opacification (PCO), the concentration of TGF-β2 required to induce PCO, and the composition of the cell-culture medium.

We included population-based studies that reported the use of TGF-β2 in posterior capsule opacification (PCO) modeling using lens epithelial cells. Eligibility criteria were predefined and comprised original in-vitro, full-text articles published in English between 1 June 2014 and 30 July 2024. Abstracts, preprints, and non-English publications were excluded. Studies employing wounded capsular-bag models or administering additional treatments prior to TGF-β2 exposure were also excluded, as such models represent a more complex tissue system with intrinsic wound-healing responses that differ from those observed in simplified cell-culture assays. The review protocol was prospectively registered on the Open Science Framework (OSF) (DOI: https://doi.org/10.17605/OSF.IO/YBXVW), because PROSPERO does not accept registrations of systematic reviews limited to in-vitro studies.

Two investigators (LRW and IK) independently extracted study data from the PubMed, ScienceDirect, SpringerLink, Taylor & Francis, and SAGE Journals databases. A comprehensive search strategy based on Medical Subject Headings (MeSH) terms was implemented in each database as follows: (“posterior capsule opacification” OR “posterior capsular opacification” OR “capsule opacifications” OR “secondary cataract” OR “PCO”) AND (“lens epithelial cells”) AND (“TGF-β2” OR “epithelial–mesenchymal transition (EMT)”). The search covered the period from 1 June 2014 to 15 July 2024. Three additional researchers (H.S., S.W., and H.K.P.) resolved any discrepancies. The literature search was finalised at the end of September 2024.

Studies were classified according to their respective research methodologies. Two investigators independently extracted data, recording the following variables: title, authors, year of publication, cell type, culture medium, TGF-β2 dose, PCO timing, procedures employed, and both quantitative and qualitative outcomes.

The data were synthesized qualitatively. Findings from 16 in vitro studies examining TGF-β2–induced posterior capsular opacification (PCO) changes were summarized to highlight molecular and cellular responses associated with the treatment, specifically whether TGF-β2 exposure up-regulated epithelial-to-mesenchymal transition (EMT) markers—α-SMA, fibronectin, collagen I, vimentin, and N-cadherin—and/or down-regulated epithelial markers such as E-cadherin and ZO-1. When available, fold-change values were extracted and mapped to a semi-quantitative EMT score according to predefined criteria: mild (+) = 1–2 markers increased <2-fold with minimal morphological change; moderate (++) = 2–3 markers increased 2–4-fold or clear qualitative EMT features; marked (+++) = ≥3 markers increased ≥4-fold or consistent EMT evidence across multiple assays. Two reviewers (I.K. and L.R.W.) independently applied the scoring criteria, and disagreements were resolved through consensus with a third reviewer.

The methodological quality of the included in-vitro studies was evaluated with the QUIN tool. Although the QUIN tool was originally created for dental in-vitro studies, it was chosen here because, to our knowledge, no validated risk-of-bias instrument exists for ophthalmic cell-culture research. Several of its assessment domains—sample selection, randomisation, blinding, outcome reporting, and statistical analysis—are broadly applicable to laboratory-based experimental studies. For each study, twelve criteria were rated “Yes” (2 points), “Partial” (1 point), or “No” (0 points), yielding a maximum score of 24. For the sampling method, a score of 0 was assigned when the procedure was not reported, 1 when partially described, and 2 when fully described (including cell source, passage number, and handling). For randomisation, all studies received a score of 0, since none explicitly mentioned random allocation of dishes or wells, despite this being technically possible in cell culture. Overall percentage scores were calculated as (Total Score ÷ 24) × 100. Based on these percentages, studies were classified as having low (>70%), medium (50–70%), or high (<50%) risk of bias 6. Detailed domain-level scores for all included studies are presented in Appendix 1 (Supplementary Material). The evaluations were carried out independently by two authors (I.K. and L.R.W.), and discrepancies were resolved through discussion, with a third reviewer (H.S.) acting as arbiter when required.

The study selection process was conducted in accordance with the PRISMA 2020 flowchart (Figure 1). Database searching yielded 287 records: PubMed (n = 202), ScienceDirect (n = 85), SpringerLink (n = 0), Taylor & Francis (n = 2), and SAGE Journals (n = 3). After removal of 139 duplicates and 3 conference abstracts, 145 records remained. Title and abstract screening eliminated 128 records. During full-text assessment, 3 articles were excluded because their samples originated from injured capsular tissue, and 3 were excluded because the cells had received additional treatments before TGF-β₂ exposure. Detailed reasons for exclusion are provided in Supplementary Table S1. Ultimately, 16 studies met the inclusion criteria and were incorporated into the review (Table 2).

The selected studies were further analyzed by extracting key information related to PCO modelling, as presented in Table 1. Sixteen studies met the inclusion criteria; four employed lens epithelial cells (LECs) from the SRA01/04 cell line, whereas others utilised primary human lens epithelial (HLE) cells. Marked variation was observed in the culture media: nine investigations utilised Dulbecco’s modified Eagle’s medium (DMEM), whereas the remainder adopted modified formulations. With respect to transforming growth factor-β2 (TGF-β2), three studies applied 5 ng/mL to induce epithelial-to-mesenchymal transition (EMT), seven applied 10 ng/mL, and the others used alternative concentrations. Eight studies incubated cells for 24 h post-TGF-β2 exposure; the remainder reported 24–48 h, 2–4 days, or did not specify the duration. Most studies verified TGF-β2 efficacy by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and Western blotting. Expression of α-smooth-muscle actin (α-SMA), fibronectin (FN), collagen I (Col I), vimentin and E-cadherin served as canonical fibrotic markers. Collectively, the literature demonstrates that in-vitro PCO models can be established by culturing lens epithelial cells in defined media supplemented with TGF-β2 2,3,7,8. A concentration of 10 ng/mL TGF-β2 7–13 consistently provoked morphological conversion from cobblestone to elongated, spindle-shaped phenotypes and elicited cataract-like molecular changes 3,12,14, which were confirmed by qRT-PCR and Western blotting. TGF-β2 exposure up-regulated α-SMA, FN, Col I, vimentin and N-cadherin while down-regulating zonula occludens-1 (ZO-1) and E-cadherin 2,3,7,8,11–13,15–18. NEAT1 expression was likewise elevated; however, no significant difference was observed between 5 and 10 ng/mL 19. Transwell assays further showed that TGF-β2 enhanced SRA01/04 cell viability, EdU incorporation, invasion and migratory distance 17. Across studies, EMT markers (α-SMA, FN, Col I, vimentin, N-cadherin) were consistently up-regulated, whereas epithelial markers (E-cadherin, ZO-1) were suppressed. Despite methodological heterogeneity (Western blotting, qRT-PCR, immunofluorescence, Transwell assays), most experiments reported pronounced EMT (++/+++), underscoring the robustness of TGF-β2-induced fibrotic remodelling in these PCO models.

The methodological quality of the 16 included studies was assessed using the QUIN tool, which evaluates 12 domains, including sample selection, randomisation, blinding, outcome measurement, and statistical analysis; each domain is assigned 0–2 points. All investigations were therefore classified as having a low risk of bias. This uniform rating likely reflects the standardised reporting typically observed in in-vitro lens epithelial cell experiments, where core procedures are largely harmonised. Conversely, the clustering of scores also highlights a limitation of the QUIN tool, namely its reduced ability to discriminate between methodological strengths and weaknesses when studies follow similar laboratory workflows. The individual QUIN scoring sheets for each of the 12 domains per study are available in Appendix 1 (Supplementary Material) for transparency.

Table 2 summarises the characteristics of the 16 included studies. The initial section describes investigations employing established human lens epithelial cell lines (e.g., SRA01/04, HLE-B3, FHL124), whereas the final three studies utilised primary lens epithelial cells or capsular explants. The table lists the culture medium, concentration of TGF-β2, incubation period, EMT-associated markers that were up-regulated, epithelial markers that were down-regulated, and the overall strength of the EMT response.

To visualize heterogeneity in EMT induction, we constructed a heat-map-style table (Figure 2) summarizing cell type, TGF-β2 dose, incubation time, and EMT outcome strength across the included studies. Each row represents a study, whereas the columns employ categorical color-coding to facilitate rapid visual comparison between models. TGF-β2 doses were classified into three categories: low (0–5 ng/mL) shown in yellow, medium (5–10 ng/mL) shown in orange, and high (15–20 ng/mL) shown in dark orange. Incubation time was similarly categorized as short (≤ 24 h) in light turquoise, intermediate (24–48 h) in mid-blue, and long (> 48 h) in dark blue. EMT strength was depicted using a purple gradient, with weak (+) coded as dark violet, moderate (++) as lavender, and strong (+++) as pink.

This visual synopsis highlights the consistent induction of strong EMT responses (+++) across most studies, particularly at medium to high TGF-β2 doses and incubation periods exceeding 24 h. A minority of investigations employing lower doses or alternative culture systems (e.g., FHL124 cells) exhibited moderate rather than strong EMT outcomes.

The available studies consistently demonstrate that TGF-β2 is a potent inducer of epithelial–mesenchymal transition (EMT) in lens epithelial cells (LECs); three studies administered 5 ng/mL, whereas seven used 10 ng/mL. Importantly, a concentration of 10 ng/mL TGF-β2 induces marked morphological changes in LECs, shifting them from a single-layer, cobblestone-like morphology to elongated, spindle-shaped cells characteristic of the fibrotic remodeling that underlies posterior capsule opacification (PCO)24. Huang et al. (2020) further showed that 10 ng/mL TGF-β2 in Dulbecco’s modified Eagle’s medium (DMEM) increased SRA01/04 cell migration by approximately 2.5-fold through the miR-497-5p/circ-GGA3 axis. However, the cellular response is time-dependent; ~24 h of exposure is sufficient to trigger transcriptional changes, whereas 48–72 h is required for observable phenotypic effects (e.g., stress-fiber formation)13.

The methodologies used to evaluate the impact of TGF-β2 comprised quantitative reverse-transcription PCR (qRT-PCR) and Western blot analyses, which confirmed the up-regulation of key epithelial–mesenchymal transition (EMT) and fibrotic markers, including α-SMA, fibronectin (FN), collagen type I (Col I), vimentin and N-cadherin, together with a concomitant down-regulation of E-cadherin 24,25. These markers are critical to validating the fibrotic cataract model because they mirror the conversion of lens epithelial cells (LECs) into a myofibroblastic phenotype, a hallmark of posterior capsular opacification (PCO) 26. Notably, NEAT1 expression was also elevated in TGF-β2-treated cells, implicating this long non-coding RNA in facilitating EMT. NEAT1 participates in diverse cellular processes—including proliferation, migration and differentiation—all of which are pertinent to the fibrotic changes observed in PCO 19. Beyond morphological and molecular alterations, functional read-outs such as Transwell assays demonstrated that TGF-β2 treatment increased the viability and migratory capacity of SRA01/04 cells, thereby further supporting its role in driving EMT and PCO 12. The crosstalk of multiple signalling cascades, particularly the TGF-β/Smad and Notch pathways, has been implicated in these events, underscoring the complexity of EMT regulation in LECs 3. Collectively, these findings highlight the pivotal role of TGF-β2 in initiating EMT and promoting the development of PCO.

Most studies utilize Dulbecco's Modified Eagle Medium (DMEM) or Minimum Essential Medium (MEM) as the basal medium 27. DMEM is preferred because its high glucose concentration (4.5 g L⁻¹), along with abundant amino acids and vitamins, supports robust cell proliferation 30. Xie et al. (2022) and Zhang et al. (2018) cultured SRA01/04 cells in DMEM supplemented with 10 % fetal bovine serum (FBS), achieving stable growth and reproducible responses to TGF-β₂ stimulation 3,7. Conversely, serum-free formulations have been adopted to minimise the influence of endogenous growth factors; for example, Fan et al. (2024) employed such media during ferroptosis assays 21. Although these findings are noteworthy, they remain preliminary and were therefore excluded from the core model-optimisation synthesis.

Fetal bovine serum (FBS) concentrations typically range from 1 % to 20 %, contingent on the experimental objective. Lower concentrations (1–5 %) preserve the slowly proliferating central-lens epithelial phenotype, whereas higher concentrations (10–20 %) approximate the proliferative zone of the lens. Wang et al. (2021) reported that supplementation with 10 % FBS in DMEM/F-12 optimally maintained HLE-B3 viability, although TGF-β2 concentrations exceeding 10 ng/mL were cytotoxic17. In contrast, serum-free protocols (e.g., Fan et al.) enable the investigation of isolated signaling pathways without interference from serum-derived growth factors21.

The choice of medium depends on the research objective. For proliferation and viability studies, a basal medium consisting of DMEM supplemented with 10 % FBS is commonly used 28,29. To enhance proliferation, epidermal growth factor (EGF, 10 ng/mL) or basic fibroblast growth factor (bFGF, 5 ng/mL) may be added 29. Additionally, an extracellular matrix (ECM) component such as gelatin or type IV collagen is frequently included to promote optimal cell adhesion 28. In contrast, for EMT and fibrosis induction, a low-serum medium (DMEM + 1 % FBS) is generally preferred to minimize serum interference 30. TGF-β2 (10 ng/mL) is routinely applied for 24–48 h to induce EMT and fibrosis 31, whereas lapatinib (1 μM) can be employed to inhibit ErbB signalling, if required 32.

Incubation times following TGF-β2 treatment have been reported to range from 24 h to several days, with eight studies specifically employing a 24-h period. This interval is commonly selected because it permits the initial induction of EMT while limiting prolonged exposure that could elicit additional, potentially confounding, alterations in cellular behaviour 33. Standardizing these parameters ensures that the observed effects can be attributed to TGF-β2 per se, rather than to secondary changes induced by extended exposure 24,32.

Epithelial identity begins to deteriorate; the mRNA levels of E-cadherin and ZO-1 decrease by 50–70 % within 6–12 h, weakening intercellular junctions and loosening their tight architecture 31. Concomitantly, fibrotic features emerge, with α-SMA and fibronectin expression increasing 2–4-fold by 12–24 h, driven by Smad2/3 phosphorylation and ERK/MAPK activation 10. This transition also triggers autophagy, evidenced by LC3-II accumulation and p62 degradation, which marks a shift in cellular metabolism 33. Supporting these observations, Huang et al. (2020) demonstrated that exposure of SRA01/04 cells to TGF-β2 (10 ng/mL) for 24 h produced a 3.5-fold increase in α-SMA protein and a 70 % rise in cell migration, even though the cells had not yet undergone complete morphologic transformation 13. These findings suggest that autophagy may function as a regulatory mechanism alongside EMT, although its role appears to be secondary to the optimization of culture conditions.

This review was conceived to highlight the practical considerations involved in establishing robust in vitro posterior capsular opacification (PCO) models. Although epithelial-to-mesenchymal transition (EMT) constitutes the biological foundation of these systems, experimental reproducibility is primarily governed by the technical parameters of the culture milieu. Three variables are particularly influential. First, TGF-β2 concentration: lower concentrations (0–5 ng/mL) induce EMT progressively and preserve cellular viability, whereas higher concentrations (≥10 ng/mL) provoke a rapid, fibrotic-like phenotype. Second, incubation duration: a 24 h exposure is most frequently employed and is generally sufficient to up-regulate canonical EMT markers; nevertheless, several studies have extended incubation to 48–96 h to capture more persistent responses or to evaluate therapeutic interventions. Third, the cellular source and substratum: primary human lens epithelial cells seeded on native capsule explants recapitulate the in vivo environment more faithfully than immortalised cell lines cultured on plastic. Collectively, meticulous optimisation of dose, timing and culture architecture enhances the fidelity of in vitro PCO models and facilitates inter-laboratory comparability. Unlike previous narrative reviews that considered PCO models within broader biological or clinical frameworks, the present review systematically defines the critical experimental parameters and offers actionable guidance for laboratories aiming to optimise and standardise in vitro PCO assays.

Several limitations should be acknowledged. The included studies exhibited substantial variability in cell models, culture conditions, TGF-β2 dosing, incubation times, and EMT markers, which limited comparability and precluded quantitative analysis. The vote-counting and semi-quantitative scoring approaches remain subjective, as differences in reporting detail and assay methods may affect how EMT strength is classified. Although the QUIN tool provides a structured framework, it was not designed for ophthalmic in-vitro research and may not fully capture relevant sources of bias, potentially contributing to the clustering of studies in the “low-risk” category. In addition, the review included only English-language full-text articles, which may have introduced language bias and excluded potentially relevant studies. Nevertheless, the review provides a coherent synthesis of TGF-β2-induced EMT models and highlights consistent patterns that may guide future experimental work.