Table 1.
Quality standard values of the final product.
| Test | Material | Target | Method | Value |
|---|---|---|---|---|
| Specification | ||||
| Morphology | PCFP | Neurosphere | Microscopic observation | No significant mixture of adherent cells |
| Phenotypic marker expression | PCFP | SOX1 | qRT-PCR∗ | ΔCt ≤ 10 |
| FCM | ≥ 90% | |||
| ICC | ≥ 80% | |||
| PAX6 | qRT-PCR∗ | ΔCt ≤ 6 | ||
| PSA-NCAM | FCM | ≥ 90% | ||
| GD2 | FCM | ≥ 90% | ||
| Terminal neural differentiation | PCFP following 4 weeks of differentiation culture | ELAVL3/4 (HuC/D) | ICC | ≥ 50% |
| β–III–tubulin | ICC | Positive | ||
| GFAP | ICC | Positive | ||
| Impurities | ||||
| Phenotypic marker expression | PCFP | OCT4 | qRT-PCR∗∗ | ΔΔCt ≥ 7.643 |
| FCM | ≤ 0.01% | |||
| ICC | ≤ 0.0005% | |||
| TRA-1-60 | FCM | ≤ 0.01% | ||
| SSEA3 | FCM | ≤ 0.01% | ||
| Back culture | PCFP following 1 week of additional culture | OCT4-positive colony | ICC | none |
| Chromosomal analysis | ||||
| Karyotyping | PCFP following 1 week of additional culture | Rate of normal karyotype results | Giemsa staining (50 cells or more) | normal karyotype ≥90% |
| Identical abnormal karyotype | G-banding (20 cells or more) | Clonal abnormality (≥3 cells having the same chromosomal loss, or ≥ 2 cells having the same conformational abnormality or extra chromosomes）is not detected. | ||
| Rate of normal karyotype results | G-banding (20 cells or more) | ≥ 90% | ||
| Tetraploid | G-banding + Giemsa staining (Percentage among 100 metaphase cells or more) | ≤ 4% | ||
| Microbiological quality control | ||||
| Sterility test | CFP | Bacteria and fungi | JP | negative |
| Endotoxin test | Endotoxin | JP | ≤ 25 EU/mL | |
| Mycoplasma test | NAT + Culture | JP | Below the detection sensitivity | |
| Virus test | HBV, HCV, HIV, HTLV-1, and Parvovirus B19 | PCR | Below the detection sensitivity | |
| Genomic analysis | ||||
| CNV | PCFP | De novo CNV | Microarray | ≤ 5 amplification or deletion of 10 kb or more compared to the cells at the end of passage 3 NS/PCs |
| Highly frequent gene mutations causally implicated in hereditary tumor, and cancer (OMIM, CGC, and Shibata list) | WGS + WES (SNV, indel) | No CNV detected in areas where the risk of tumorigenesis is reported | ||
Abbreviations: PCFP, pre-cryopreserved final product; CFP, cryopreserved final product; qRT-PCR, quantitative reverse transcription polymerase chain reaction (comparative Ct); ∗, ΔCt value; ∗∗, ΔΔCt value; FCM, flow cytometry; ICC, immunocytochemistry; NAT, nucleic acid amplification technique; Culture, methods for mycoplasma expansion in broth culture and detection by colony formation on nutrient agar plates; JP, Japanese Pharmacopoeia; CNV, copy number variation; SNV, single nucleotide variation; indel, insertion/deletion; OMIM, Online Mendelian Inheritance in Man; CGC, The Cancer Gene Census; WGS, whole genome sequencing; WES, whole exome sequencing.