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

Toan V Phan; Yamin Oo; Teerapat Rodboon; Truc T Nguyen; Ladawan Sariya; Risa Chaisuparat; Waranyoo Phoolcharoen; Supansa Yodmuang; Joao N Ferreira

doi:10.1016/j.slast.2023.03.002

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

SLAS Technol. 2023 Aug;28(4):278-291. doi: 10.1016/j.slast.2023.03.002. Epub 2023 Mar 24.

Authors

Toan V Phan¹, Yamin Oo², Teerapat Rodboon², Truc T Nguyen³, Ladawan Sariya⁴, Risa Chaisuparat⁵, Waranyoo Phoolcharoen⁶, Supansa Yodmuang⁷, Joao N Ferreira⁸

Affiliations

¹ Avatar Biotechnologies for Oral Health and Healthy Longevity Research Unit, Department of Research Affairs, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand; International Graduate Program in Oral Biology, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand.
² Avatar Biotechnologies for Oral Health and Healthy Longevity Research Unit, Department of Research Affairs, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand.
³ Medical Sciences Program, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand; Research Affairs, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.
⁴ Monitoring and Surveillance Center for Zoonotic Diseases in Wildlife and Exotic Animals, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom, Thailand.
⁵ Avatar Biotechnologies for Oral Health and Healthy Longevity Research Unit, Department of Research Affairs, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand; Department of Oral Pathology, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand.
⁶ Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand.
⁷ Avatar Biotechnologies for Oral Health and Healthy Longevity Research Unit, Department of Research Affairs, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand; Research Affairs, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.
⁸ Avatar Biotechnologies for Oral Health and Healthy Longevity Research Unit, Department of Research Affairs, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand. Electronic address: Joao.F@chula.ac.th.

PMID: 36966988
DOI: 10.1016/j.slast.2023.03.002

Abstract

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.

Keywords: Drug delivery; Epidermal growth factor; Exocrine gland; Hydrogel; Organoids; Plant molecular farming.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Adenosine Triphosphate
Epidermal Growth Factor* / pharmacology
Hyaluronic Acid / pharmacology
Hydrogels*
Molecular Farming
Organoids

Substances

Epidermal Growth Factor
Hydrogels
Hyaluronic Acid
Adenosine Triphosphate