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.
Avatar Biotechnologies for Oral Health and Healthy Longevity Research Unit, Department of Research Affairs, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand.
SLAS Technol. 2023 Aug;28(4):278-291. doi: 10.1016/j.slast.2023.03.002. Epub 2023 Mar 24.
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.
表皮生长因子（EGF）是一种已知的信号通路，对于外分泌腺的发育和类器官生物制造尤其重要。本研究开发了一种体外 EGF 传递平台，使用 Nicotiana benthamiana 植物生产的 EGF（P-EGF）包封在透明质酸/藻酸盐（HA/Alg）水凝胶上，以提高短期培养系统中腺状类器官生物制造的有效性。将原代颌下腺上皮细胞用 5-20ng/mL 的 P-EGF 和市售的细菌衍生的 EGF（B-EGF）处理。通过 MTT 和基于荧光素酶的 ATP 测定来测量细胞增殖和代谢活性。P-EGF 和 B-EGF 在 6 天的培养中以相似的方式促进了腺上皮细胞的增殖，浓度为 5-20ng/mL。使用两种 EGF 传递系统（HA/Alg 包封和培养基补充）评估类器官形成效率、细胞活力、ATP 依赖性活性和扩增。磷酸盐缓冲盐水（PBS）用作对照载体。从 PBS、B-EGF 和 P-EGF 包封水凝胶制造的上皮类器官在基因型、表型和功能测定方面进行了表征。与 P-EGF 补充相比，P-EGF 包封水凝胶增强了类器官的形成效率、细胞活力和代谢。在培养第 3 天，从 P-EGF 包封的 HA/Alg 平台发育的上皮类器官包含具有特定腺上皮标志物表达的功能性细胞簇，例如外分泌前腺泡（AQP5、NKCC1、CHRM1、CHRM3、Mist1）、导管（K18、Krt19）和肌上皮（α-SMA、Acta2），并且具有高有丝分裂活性（38-62% Ki67 细胞）和大的上皮祖细胞群体（∼70% K14 细胞）。与其他组（B-EGF、PBS）相比，P-EGF 包封在整个培养过程中显著上调了前腺泡 AQP5 细胞的表达。因此，利用 Nicotiana benthamiana 进行分子农业可以生产适用于 HA/Alg 基体外平台包封的 EGF 生物制剂，该平台可以有效地、迅速地诱导外分泌腺类器官的生物制造。
