Organotypic Spheroid Culture to Mimic Radiation-Induced Salivary Hypofunction

Abstract

Radiation treatment often leads to irreversible damage to normal salivary glands (SGs) because of their proximity to head and neck cancers. Optimization of the in vitro model of irradiation (IR)-induced SG damage is warranted to investigate pathophysiology and monitor treatment outcome. Here, we present an organotypic spheroid culture model to investigate the impact of IR on SGs and the mechanisms underlying IR-induced structural and functional changes. Human parotid epithelial cells were obtained from human parotid glands and plated on either plastic plates or Matrigel. A number of 3-dimensional (3D) spheroids were assembled on Matrigel. After IR at 10 and 20 Gy, morphologic changes in cells in 2D monolayers and 3D spheroids were observed. As the structural integrity of the 3D spheroids was destroyed by IR, the expression levels of salivary epithelial and structural proteins and genes decreased proportionally with radiation dosage. Furthermore, the spheroid culture allowed better measurement of functional alterations following IR relative to the monolayer culture, in which IR-inflicted spheroids exhibited a loss of acinar-specific cellular functions that enable Ca influx or secretion of α-amylase in response to cholinergic or β-adrenergic agonists. p53-mediated apoptotic cell death was observed under both culture conditions, and its downstream signals increased, such as p53 upregulated modulator of apoptosis (PUMA), Bax, cytochrome c, caspase 9, and caspase 3. These results suggest that the organotypic spheroid culture could provide a useful alternative model for exploration of radiobiology and mode of action of new therapies for prevention of radiation-induced salivary hypofunction.

Radiation-associated changes in salivation of patients with cancer of maxillofacial region

“…Исследование молекулярного состава СЖ методом ионной хроматографии до начала и после лучевой терапии выявило значительное увеличение в РЖ по сравнению с исходным уровнем хлоридов, сульфатов, лактатов с сопутствующим снижением уровня нитратов и тиоцианатов вне зависимости от возраста и пола пациентов, подвергшихся воздействию ионизирующего излучения [28,29]. Снижение же в слюнной жидкости количества нитратов, выполняющих роль антимикробных агентов, приводит к изменению защитной функции организма в целом [17].…”

“…В первые 2-3 нед после начала лучевой терапии отмечают повышение уровня IgA [31] и α-амилазы в слюнной жидкости пациентов со ЗНО ЧЛО, что временно обеспечивает некоторую защиту от радиоиндуцированных инфекций [18]. В более поздние сроки наблюдают снижение концентрации и активности α-амилазы и муцина, что может привести к увеличению восприимчивости к местным инфекциям [23], поскольку муцин относят к защитным белкам, придающим вязкость слюне и связывающим большое количество воды [16,28].…”

Cellular Therapies for Treatment of Radiation Injury: Report from a NIH/NIAID and IRSN Workshop

“…However, success has been seen growing cells in substances such as matrigel. For example, salivary gland stem cells grown in matrigel will form organoids, which is similar to their in vivo growth (156). …”

A magnetic three‐dimensional levitated primary cell culture system for the development of secretory salivary gland‐like organoids

“…In an effort to recreate a new salivary secretory compartment in the SG from dry mouth patients, researchers have been developing in vitro three‐dimensional (3D) SG‐like organotypic cultures, also referred as organoids or spheroids (J. N. Ferreira, Rungarunlert, Urkasemsin, Adine, & Souza, ; Leigh, Nelson, Mellas, McCall, & Baker, ; Pringle et al, ; Srinivasan et al, ). Several approaches have been recently utilized to culture these SG primary or stem/progenitor cells into organoids including the ones using suspension/levitation methods in combination with different spheroid assembly systems, 3D matrices, or nanoparticles to tag cells (J. R. Ferreira et al, ; Pringle et al, ; Shin, An, Choi, Kim, & Lim, ; Srinivasan et al, ; Tung et al, ). The rising of 3D cell culture systems addresses the limitations of two‐dimensional cell monolayers where rigid substrates/surfaces are present.…”

“…Conversely, 3D cell cultures can mimic in vivo cell–cell and cell‐matrix interactions allowing the creation of organoids with complex networks of different cellular compartments (Maria, Liu, El‐Hakim, Zeitouni, & Tran, ; Pringle et al, ; Srinivasan et al, ). These organoids can also recapitulate in vivo morphological, physiologic, and pathological conditions in SG secretory organs (Shin et al, ; Su et al, ).…”