Decellularized tissue-specific hydrogels support an engineered salivary gland within a microfluidic platform

Head and neck (HN) cancer is a broad category of tumor types arising from various anatomic structures including the craniofacial bones, soft tissues, salivary glands, skin, and mucosal membranes. Treatment often involves an intensive combination of surgery, radiotherapy and chemotherapy. Despite this, tumour recurrence rates remain high and survival rates are relatively poor. Here, we describe a novel high throughput drug screening platform combining the OrganoPlate, a microfluidic based 3D culture plate, and HN cancer-derived organoids. MIMETAS develops Organ-on-a-Chip-based models for evaluation of new medicines. Our unique microfluidic technology enables testing of compounds on miniaturized 3D organ models in high-throughput. These models are expected to show better predictivity as compared to laboratory animals and conventional 2D cell culture models, without compromising throughput or ease of use. Hereby we show the establishment of HN cancer-derived organoids in 2-lane OrganoPlate, and its usefulness for phenotypic drug screenings. The aim of the study is to evaluate the 2-lane OrganoPlate as a platform for growing HN cancer organoids and drug screening. Organoid lines (T2, T3 and T4) were embedded in the 2-lane OrganoPlate as single cells in an Extracellular Matrix gel. At day 3, cultures were treated with Cisplatin or Carboplatin for 120 hours. Drug response was evaluated by assessment of morphology (phase contrast), Cell Viability (Alamar blue) and proliferation (EdU incorporation). Organoids cultures grow well under perfusion in the 2-lane OrganoPlate and different sensitivity to cisplatin is captured by the used readouts. The high-throughput, microfluidic 2-lane OrganoPlate platform offers an attractive method for growing HN cancer-derived organoids, supporting development of individualized tumour models for phenotypic drug screenings. Citation Format: Karla Queiroz, Else Driehuis, Silvia Bonilla, Henriëtte Lanz, Hans Clevers, Jos Joore, Paul Vulto. A novel high throughput platform for head & neck cancer organoids drug screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1173.

ObjectiveSalivary gland (SG) hypofunction is a common clinical condition arising from radiotherapy to suppress head and neck cancers. The radiation often destroys the SG secretory acini, and glands are left with limited regenerative potential. Due to the complex architecture of SG acini and ducts, three-dimensional (3D) bioprinting platforms have emerged to spatially define these in vitro epithelial units and develop mini-organs or organoids for regeneration. Due to the limited body of evidence, this comprehensive review highlights the advantages and challenges of bioprinting platforms for SG regeneration.MethodsSG microtissue engineering strategies such as magnetic 3D bioassembly of cells and microfluidic coaxial 3D bioprinting of cell-laden microfibers and microtubes have been proposed to replace the damaged acinar units, avoid the use of xenogeneic matrices (like Matrigel), and restore salivary flow.ResultsReplacing the SG damaged organ is challenging due to its complex architecture, which combines a ductal network with acinar epithelial units to facilitate a unidirectional flow of saliva. Our research group was the first to develop 3D bioassembly SG epithelial functional organoids with innervation to respond to both cholinergic and adrenergic stimulation. More recently, microtissue engineering using coaxial 3D bioprinting of hydrogel microfibers and microtubes could also supported the formation of viable epithelial units. Both bioprinting approaches could overcome the need for Matrigel by facilitating the assembly of adult stem cells, such as human dental pulp stem cells, and primary SG cells into micro-sized 3D constructs able to produce their own matrix and self-organize into micro-modular tissue clusters with lumenized areas. Furthermore, extracellular vesicle (EV) therapies from organoid-derived secretome were also designed and validated ex vivo for SG regeneration after radiation damage.ConclusionMagnetic 3D bioassembly and microfluidic coaxial bioprinting platforms have the potential to create SG mini-organs for regenerative applications via organoid transplantation or organoid-derived EV therapies.

Exosomes are nanoscale lipid-bilayer vesicles that mediate intercellular communication by delivering bioactive molecules such as nucleic acids and proteins. Among them, exosomes derived from salivary gland epithelial stem cells (sgESC-Exos) exhibit antifibrotic effects in salivary gland models through the delivery of antifibrotic microRNAs, such as miR-1290 and miR-3162. However, their clinical translation is hindered by low production yield and particle heterogeneity. To address these challenges, exosome-mimetic nanoparticles (ENPs) are developed that mimic the lipid composition and therapeutic cargoes of sgESC-Exos using a microfluidic chip integrated with a reverse-Tesla structured micromixer. The microfluidic platform facilitates efficient mixing and self-assembly of lipid and aqueous phases, resulting in uniform ENPs with effective encapsulation of nucleic acids. The resulting ENPs exhibit physicochemical properties similar to exosomes, including comparable size distribution and structural features. Moreover, they demonstrate efficient encapsulation of exosomal miRNAs and more uniform physicochemical properties than sgESC-Exos, contributed to enhanced therapeutic efficacy, with ≈21-fold higher cellular uptake and two-fold faster wound closure rate. These findings highlight the potential of microfluidics-based bottom-up synthesis as a robust platform for exosome-inspired nanomedicine, addressing key limitations of exosomes and paving the way for clinical translation.

Head and neck cancers (HNCs) account for ~4% of all cancers in North America and encompass cancers affecting the oral cavity, pharynx, larynx, sinuses, nasal cavity, and salivary glands. The anatomical complexity of the head and neck region, characterized by highly perfused and innervated structures, presents challenges in the early diagnosis and treatment of these cancers. The utilization of sub-microliter volumes and the unique phenomenon associated with microscale fluid dynamics have facilitated the development of microfluidic platforms for studying complex biological systems. The advent of on-chip microfluidics has significantly impacted the diagnosis and treatment strategies of HNC. Sensor-based microfluidics and point-of-care devices have improved the detection and monitoring of cancer biomarkers using biological specimens like saliva, urine, blood, and serum. Additionally, tumor-on-a-chip platforms have allowed the creation of patient-specific cancer models on a chip, enabling the development of personalized treatments through high-throughput screening of drugs. In this review, we first focus on how microfluidics enable the development of an enhanced, functional drug screening process for targeted treatment in HNCs. We then discuss current advances in microfluidic platforms for biomarker sensing and early detection, followed by on-chip modeling of HNC to evaluate treatment response. Finally, we address the practical challenges that hinder the clinical translation of these microfluidic advances.