In vivo cells express their characteristics in three-dimensional (3D) microenvironments via cell-cell interactions through autocrine, contact-dependent, paracrine, and synaptic signaling, often between heterologous cell types. Various in vitro 3D microwell-based culture methods have been proposed to further identify cellular characteristics by recreating cellular environments, typically in the form of spheroids and organoids, thereby realizing contact-based cell-cell interactions. However, in vivo cells generally exhibit multiple cellular interaction modes that have not been completely evaluated using existing microwell-based methods. This has led to a demand for more advanced and comprehensive methods. This study introduces a novel apparatus, the membrane-bottomed microwell (MBM) for non-contact co-cultures and 3D cell cultures. The MBM is a combination of a Transwell and a microwell array; these have previously been utilized to facilitate heterologous cell co-culturing and spheroid 3D cell culturing, respectively. In the Transwell insert, the lower part of the MBM is immersed in the culture media in which the cells are being two-dimensionally (2D) cultured, and the spheroids of the MBM are affected by the 2D cultured cells via the membrane at the bottom of the microwell. Here, we describe the methods for manufacturing the MBM in detail and elucidate the results of simulations of diffusion through the bottom of the membrane. We validate the proposed MBM for the spheroid culture of spermatogonial stem cells (SSCs), which had previously been 2D co-cultured with Sandos inbred mouse (SIM)-derived 6-thioguanine-and ouabain-resistant (STO; a mouse embryonic feeder cell line) feeder cells. The proposed system is shown to facilitate successful SSC spheroid culturing with paracrine signaling of STOs through an apparatus that simplifies both the loading and the evaluation processes; therefore, we believe that our findings will enable a more comprehensive understanding of SSCs and associated phenomena and that our system can be applied to various in vitro cell and tissue experiments.
Immunotherapy of bladder cancer is known to have favorable effects, although it is difficult to determine which patients will show a good response because of the different tumor microenvironments (TME). Here, we developed a bladder cancer-on-a-chip (BCOC) to mimic the TME using three-dimensional (3D) bioprinting and microfluidic technology. We fabricated a T24 and a 5637-cell line-based BCOC that also incorporated MRC-5, HUVEC, and THP-1 cells. We evaluated the effects of TME and assessed the immunologic reactions in response to different concentrations of Bacillus Calmette–Guérin (BCG) via live/dead assay and THP-1 monocytic migration, and concentrations of growth factors and cytokines. The results show that cell viability was maintained at 15% filling density in circle-shaped cell constructs at 20 μL/min microfluidic flow rate. A 3D co-culture increased the proliferation of BCOCs. We found that the appropriate time to evaluate the viability of BCOC, concentration of cytokines, and migration of monocytes was 6 h, 24 h, and three days after BGC treatment. Lastly, the immunotherapeutic effects of BCOC increased according to BCG dosage. To predict effects of immunotherapeutic agent in bladder cancer, we constructed a 3D bioprinted BCOC model. The BCOC was validated with BCG, which has been proven to be effective in the immunotherapy of bladder cancer.
Microfluidic devices have emerged as powerful tools for cell-based experiments, offering a controlled microenvironment that mimic the conditions within the body. Numerous cell experiment studies have successfully utilized microfluidic channels to achieve various new scientific discoveries. However, it has been often overlooked that undesired and unnoticed propagation of cellular molecules in such bio-microfluidic channel systems can have a negative impact on the experimental results. Thus, more careful designing is required to minimize such unwanted issues through deeper understanding and careful control of chemically and physically predominant factors at the microscopic scale. In this paper, we introduce a new approach to improve microfluidic channel design, specifically targeting the mitigation of the aforementioned challenges. To minimize the occurrence of undesired cell positioning upstream from the main test section where a concentration gradient field locates, an additional narrow port structure was devised between the microfluidic upstream channel and each inlet reservoir. This port also functioned as a passive lock that hold the flow at rest via fluid-air surface tension, which facilitated manual movement of the device even when cell attachment was not achieved completely. To demonstrate the practicability of the system, we conducted experiments and diffusion simulations on the effect of endocrine disruptors on germ cells. To this end, a bisphenol-A (BPA) concentration gradient was generated in the main channel of the system at BPA concentrations ranging from 120.8 μM to 79.3 μM, and the proliferation of GC-1 cells in the BPA gradient environment was quantitatively evaluated. The features and concepts of the introduced design is to minimize unexpected and ignored error sources, which will be one of the issues to be considered in the development of microfluidic systems to explore extremely delicate cellular phenomena.
Over the past few decades, research on life in space has increased. Owing to the expensive nature of and the challenges associated with conducting experiments in real space, clinostats, which continuously randomize the gravity vector by using motors, have been used to generate simulated microgravity (SMG) on Earth. Herein, by using a 3D printing method, we develop a customized small-sized clinostat (CS clinostat) that is easy to manufacture, inexpensive, and robust. Moreover, we develop and fabricate a gas-permeable polydimethylsiloxane culture dish that fits inside the CS clinostat. To validate SMG generation, ovarian cancer cells (OV- 90, TOV-21G, and Caov-3) were applied to demonstrate a significant reduction in caveolin-1 expression, a biomarker of SMG, indicating SMG generation. The proposed CS clinostat system has good accessibility for SMG research, which makes it useful as a tool for biologists, who are unfamiliar with conventional clinostat equipment, to conduct preliminary studies in the space environment.
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