Establishment of Lymphoblastoid Cell Lines from Iranian Multiple Sclerosis Patients: A Renewable Genetic Resource

Technical Summary: Establishment of Lymphoblastoid Cell Lines from Iranian Multiple Sclerosis Patients

Problem Statement
Multiple sclerosis (MS) is a complex immune-mediated disorder with a significant genetic component. While genome-wide association studies (GWAS) have identified over 200 susceptibility loci, the majority of this data is derived from European populations. There is a critical lack of high-quality, renewable genetic resources for non-European populations, specifically Iranians, who exhibit a moderate-to-high MS burden. Furthermore, relying on fresh peripheral blood for genetic studies is often impractical for longitudinal research or rare variant analysis due to ethical and logistical constraints. Currently, Iran lacks a centralized, publicly accessible biobank of Epstein–Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) from MS patients, limiting multicenter collaboration and independent verification of results.

Methodology
The study aimed to establish a biobank of EBV-transformed LCLs from Iranian MS patients following international biobanking standards.

• Sample Collection: Peripheral blood (8 mL) was collected from 72 clinically diagnosed MS patients (51 female, 21 male; mean age 37.9 years) using sodium heparin tubes. All participants provided written informed consent, and the study was approved by the Royan Institute Ethics Committee. Patients with active infections or positive serology for HIV, HBV, or HCV were excluded.
• Cell Isolation and Transformation: Peripheral blood mononuclear cells (PBMCs) were isolated via Ficoll-Paque density gradient centrifugation. The cells were immortalized using EBV-containing supernatant derived from the B95-8 marmoset cell line. Cultures were maintained in RPMI-1640 medium with 10% fetal bovine serum (FBS) at 37°C with 5% CO₂.
• Expansion and Cryopreservation: Successfully transformed cells, identified by morphological changes (aggregation, enlarged lymphoblastoid cells) and sustained proliferation within 10–14 days, were expanded for 3–5 passages. Cells were cryopreserved at a concentration of 5 × 10⁶ cells/vial in 90% FBS and 10% DMSO, stored initially at -75°C, and subsequently moved to the vapor phase of liquid nitrogen (-196°C).
• Quality Control (QC): A rigorous QC panel was applied to all established lines:
  • Mycoplasma: Detected via Hoechst 33258 staining, multiplex PCR (targeting 7 species), and culture-based PPLO broth/agar methods.
  • Sterility: Tested for aerobic and anaerobic bacteria and fungi using Tryptone Soya Broth and Thioglycolate Medium.
  • Species Authentication: Confirmed via PCR amplification of the mitochondrial Cytochrome C oxidase subunit I (COI) gene.
  • Viral Screening: PCR and RT-PCR were used to screen for HBV, HCV, HIV, and EBV.

Key Results

• Establishment Success: Stable LCLs were successfully established from 48 of the 72 collected samples, yielding a success rate of 66.7%. The 24 failed attempts were attributed to microbial contamination, poor sample quality (e.g., clotting), or transformation failure. Statistical analysis showed no significant difference in age or sex distribution between successful and failed groups.
• Quality Assurance: All 48 established cell lines tested negative for mycoplasma, bacterial, and fungal contamination. Species authentication confirmed the human origin of all lines. Viral screening confirmed the absence of HIV, HBV, and HCV in donor samples, while EBV DNA was detected in all lines, consistent with successful transformation.
• Resource Characterization: The cell lines demonstrated a population doubling time of 24–48 hours. Comprehensive metadata, including de-identified patient demographics and QC results, were documented in a searchable database.

Significance and Claims
The authors claim this study represents the first reported biobank of EBV-transformed LCLs established specifically from Iranian MS patients. The primary significance of this work lies in creating a renewable, well-characterized source of high-molecular-weight genomic DNA for the Iranian population, a group historically underrepresented in global MS genetic studies.

The paper posits that this resource will facilitate:

• Future genetic association studies and biomarker discovery specific to the Iranian genetic background.
• Functional genomic investigations and the validation of risk variants found in Western cohorts.
• The development of personalized medicine approaches for the region.

The authors emphasize that the cell lines are available through the Iranian Biological Resource Centre (IBRC) for qualified research, thereby addressing the gap in accessible, population-specific genetic resources in the Middle East. The study acknowledges limitations, including the relatively small sample size and the lack of transcriptomic or proteomic profiling, framing the work as a foundational step for future systems biology approaches.