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Development of a Microfluidic Culture Paradigm for Ex Vivo Maintenance of Human Glioblastoma Tissue: A New Glioblastoma Model?

ABSTRACT: BACKGROUND:One way to overcome the genetic and molecular variations within glioblastoma is to treat each tumour on an individual basis. To facilitate this, we have developed a microfluidic culture paradigm that maintains human glioblastoma tissue ex vivo. METHODS:The assembled device, fabricated using a photolithographic process, is composed of two layers of glass bonded together to contain a tissue chamber and a network of microchannels that allow continued tissue perfusion. RESULTS:A total of 128 tissue biopsies (from 33 patients) were maintained in microfluidic devices for an average of 72 hours. Tissue viability (measured with Annexin V and propidium iodide) was 61.1% in tissue maintained on chip compared with 68.9% for fresh tissue analysed at commencement of the experiments. Other biomarkers, including lactate dehydrogenase absorbance and trypan blue exclusion, supported the viability of the tissue maintained on chip. Histological appearances remained unchanged during the tissue maintenance period, and immunohistochemical analysis of Ki67 and caspase 3 showed no significant differences when compared with fresh tissues. A trend showed that tumours associated with poorer outcomes (recurrent tumours and Isocitrate Dehydrogenase - IDH wildtype) displayed higher viability on chip than tumours linked with improved outcomes (low-grade gliomas, IDH mutants and primary tumours). CONCLUSIONS:This work has demonstrated for the first time that human glioblastoma tissue can be successfully maintained within a microfluidic device and has the potential to be developed as a new platform for studying the biology of brain tumours, with the long-term aim of replacing current preclinical GBM models and facilitating personalised treatments.

SUBMITTER: Olubajo F

PROVIDER: S-EPMC6854064 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Development of a Microfluidic Culture Paradigm for Ex Vivo Maintenance of Human Glioblastoma Tissue: A New Glioblastoma Model?

Olubajo Farouk F Achawal Shailendra S Greenman John J

Translational oncology 20191111 1

<h4>Background</h4>One way to overcome the genetic and molecular variations within glioblastoma is to treat each tumour on an individual basis. To facilitate this, we have developed a microfluidic culture paradigm that maintains human glioblastoma tissue ex vivo.<h4>Methods</h4>The assembled device, fabricated using a photolithographic process, is composed of two layers of glass bonded together to contain a tissue chamber and a network of microchannels that allow continued tissue perfusion.<h4>R ...[more]

PMID: 31726354

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Project description:Background:In vitro maturation of immature testicular tissue (ITT) cryopreserved for fertility preservation is a promising fertility restoration strategy. Organotypic tissue culture proved successful in mice, leading to live births. In larger mammals, including humans, efficiently reproducing spermatogenesis ex vivo remains challenging. With advances in biomaterials technology, culture systems are becoming more complex to better mimic in vivo conditions. Along with improving culture media components, optimizing physical culture conditions (e.g., tissue perfusion, oxygen diffusion) also needs to be considered. Recent studies in mice showed that by using silicone-based hybrid culture systems, the efficiency of spermatogenesis can be improved. Such systems have not been reported for ITT of large mammals. Methods: Four different organotypic tissue culture systems were compared: static i.e., polytetrafluoroethylene membrane inserts (OT), agarose gel (AG) and agarose gel with polydimethylsiloxane chamber (AGPC), and dynamic i.e., microfluidic (MF). OT served as control. Porcine ITT fragments were cultured over a 30-day period using a single culture medium. Analyses were performed at days (d) 0, 5, 10, 20 and 30. Seminiferous tubule (ST) integrity, diameters, and tissue core integrity were evaluated on histology. Immunohistochemistry was used to identify germ cells (PGP9.5, VASA, SYCP3, CREM), somatic cells (SOX9, INSL3) and proliferating cells (Ki67), and to assess oxidative stress (MDA) and apoptosis (C-Caspase3). Testosterone was measured in supernatants using ELISA. Results: ITT fragments survived and grew in all systems. ST diameters, and Sertoli cell (SOX9) numbers increased, meiotic (SYCP3) and post-meiotic (CREM) germ cells were generated, and testosterone was secreted. When compared to control (OT), significantly larger STs (d10 through d30), better tissue core integrity (d5 through d20), higher numbers of undifferentiated spermatogonia (d30), meiotic and post-meiotic germ cells (SYCP3: d20 and 30, CREM: d20) were observed in the AGPC system. Apoptosis, lipid peroxidation (MDA), ST integrity, proliferating germ cell (Ki67/VASA) numbers, Leydig cell (INSL3) numbers and testosterone levels were not significantly different between systems. Conclusions: Using a modified culture system (AGPC), germ cell survival and the efficiency of porcine germ cell differentiation were moderately improved ex vivo. We assume that further optimization can be obtained with concomitant modifications in culture media components.

Dataset Information

Development of a Microfluidic Culture Paradigm for Ex Vivo Maintenance of Human Glioblastoma Tissue: A New Glioblastoma Model?

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Development of a Microfluidic Culture Paradigm for Ex Vivo Maintenance of Human Glioblastoma Tissue: A New Glioblastoma Model?

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