Plant molecular farming-derived epidermal growth factor revolutionizes hydrogels for improving glandular epithelial organoid biofabrication

• Published in: SLAS technology 2023-08, Vol.28 (4), p.278-291
• Main Authors: Phan, Toan V., Oo, Yamin, Rodboon, Teerapat, Nguyen, Truc T., Sariya, Ladawan, Chaisuparat, Risa, Phoolcharoen, Waranyoo, Yodmuang, Supansa, Ferreira, Joao N.
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate; Drug delivery; Epidermal growth factor; Epidermal Growth Factor - pharmacology; Exocrine gland; Hyaluronic Acid - pharmacology; Hydrogel; Hydrogels; Molecular Farming; Organoids; Plant molecular farming
• ISSN: 2472-6303; 2472-6311
• DOI: 10.1016/j.slast.2023.03.002

Epidermal growth factor (EGF) is a known signaling cue essential towards the development and organoid biofabrication particularly for exocrine glands. This study developed an in vitro EGF delivery platform with Nicotiana benthamiana plant-produced EGF (P-EGF) encapsulated on hyaluronic acid/alginate (HA/Alg) hydrogel to improve the effectiveness of glandular organoid biofabrication in short-term culture systems. Primary submandibular gland epithelial cells were treated with 5 – 20 ng/mL of P-EGF and commercially available bacteria-derived EGF (B-EGF). Cell proliferation and metabolic activity were measured by MTT and luciferase-based ATP assays. P-EGF and B-EGF 5 – 20 ng/mL promoted glandular epithelial cell proliferation during 6 culture days on a comparable fashion. Organoid forming efficiency and cellular viability, ATP-dependent activity and expansion were evaluated using two EGF delivery systems, HA/Alg-based encapsulation and media supplementation. Phosphate buffered saline (PBS) was used as a control vehicle. Epithelial organoids fabricated from PBS-, B-EGF-, and P-EGF-encapsulated hydrogels were characterized genotypically, phenotypically and by functional assays. P-EGF-encapsulated hydrogel enhanced organoid formation efficiency and cellular viability and metabolism relative to P-EGF supplementation. At culture day 3, epithelial organoids developed from P-EGF-encapsulated HA/Alg platform contained functional cell clusters expressing specific glandular epithelial markers such as exocrine pro-acinar (AQP5, NKCC1, CHRM1, CHRM3, Mist1), ductal (K18, Krt19), and myoepithelial (α-SMA, Acta2), and possessed a high mitotic activity (38–62% Ki67 cells) with a large epithelial progenitor population (∼70% K14 cells). The P-EGF encapsulation strikingly upregulated the expression of pro-acinar AQP5 cells through culture time when compared to others (B-EGF, PBS). Thus, the utilization of Nicotiana benthamiana in molecular farming can produce EGF biologicals amenable to encapsulation in HA/Alg-based in vitro platforms, which can effectively and promptly induce the biofabrication of exocrine gland organoids. [Display omitted]