Multi-analyte biosensor interface for real-time monitoring of 3D microtissue spheroids in hanging-drop networks

Microfluidics is becoming a technology of growing interest for building microphysiological systems with integrated read-out functionalities.Here we present the integration of enzyme-based multi-analyte biosensors into a multi-tissue culture platform for‘body-on-a-chip’applications.The microfluidic platform is based on the technology of hanging-drop networks,which is designed for the formation,cultivation,and analysis of fluidically interconnected organotypic spherical three-dimensional(3D)microtissues of multiple cell types.The sensor modules were designed as small glass plug-ins featuring four platinum working electrodes,a platinum counter electrode,and an Ag/AgCl reference electrode.They were placed directly into the ceiling substrate from which the hanging drops that host the spheroid cultures are suspended.The electrodes were functionalized with oxidase enzymes to enable continuous monitoring of lactate and glucose through amperometry.The biosensors featured high sensitivities of 322±41 nA mM^(−1) mm^(−2) for glucose and 443±37 nA mM^(−1) mm^(−2) for lactate;the corresponding limits of detection were below 10μM.The proposed technology enabled tissue-size-dependent,real-time detection of lactate secretion from single human colon cancer microtissues cultured in the hanging drops.Furthermore,glucose consumption and lactate secretion were monitored in parallel,and the impact of different culture conditions on the metabolism of cancer microtissues was recorded in real-time.

Optimization and comparison of two different 3D culture methods to prepare cell aggregates as a bioink for organ printing

The ultimate goal of tissue engineering is to design and fabricate functional human tissues that are similar to natural cells and are capable of regeneration.Preparation of cell aggregates is one of the important steps in 3D tissue engineering technology,particularly in organ printing.Two simple methods,hanging drop(HD)and conical tube(CT)were utilized to prepare cell aggregates.The size and viability of the aggregates obtained at different initial cell densities and pre-culture duration were compared.The proliferative ability of the cell aggregates and their ability to spread in culture plates were also investigated.In both methods,the optimum average size of the aggregates was less than 500μm.CT aggregates were smaller than HD aggregates.5,000 cells per drop HD aggregates showed a marked ability to attach and spread on the culture surface.The proliferative ability reduced when the initial cell density was increased.Comparing these methods,we found that the HD method having better size controlling ability as well as enhanced ability to maintain higher rates of viability,spreading,and proliferation.In conclusion,smaller HD aggregates might be a suitable choice as building blocks for making bioink particles in bioprinting technique.

Thymic Nurse Cells Support CD4CD8^+ Thymocytes to Differentiate into CD4^+CD8^+ Cells

Thymic nurse cells （TNCs） represent a unique microenvironment in the thymus for T cell maturation. In order to investigate the role of thymic nurse cells during T cell differentiation, a TNC clone, RWTE-1, which formed a typical complex with fetal thymocytes in vitro was established from normal Wistar rat. Hanging drop culture method was applied to reveal the interaction between TNCs and thymocytes. Our result revealed that eighty percent of immature CD4^-CD8^＋ cells differentiated into CD4^＋CD8^＋ cells after a 12-hour hanging drop culture with RWTE-1. However, in a 12-hour culture of immature CD4^-CD8^＋ cells with or without RWTE-1 supernatant, only 30% of the cells differentiated into CD4^＋CD8^＋ cells spontaneously. This observation led to the conclusion that RWTE-1 cell has the capacity to facilitate immature CD4^-CD8^＋ thymocytes to differentiate into CD4^＋CD8^＋ T cells by direct interaction.