Microfluidic systems: A new toolbox for pluripotent stem cells

Conventional culture systems are often limited in their ability to regulate the growth and differentiation of pluripotent stem cells. Microfluidic systems can overcome some of these limitations by providing defined growth conditions with user‐controlled spatiotemporal cues. Microfluidic systems allow researchers to modulate pluripotent stem cell renewal and differentiation through biochemical and mechanical stimulation, as well as through microscale patterning and organization of cells and extracellular materials. Essentially, microfluidic tools are reducing the gap between in vitro cell culture environments and the complex and dynamic features of the in vivo stem cell niche. These microfluidic culture systems can also be integrated with microanalytical tools to assess the health and molecular status of pluripotent stem cells. The ability to control biochemical and mechanical input to cells, as well as rapidly and efficiently analyze the biological output from cells, will further our understanding of stem cells and help translate them into clinical use. This review provides a comprehensive insignt into the implications of microfluidics on pluripotent stem cell research. Conventional culture systems are often limited in their ability to regulate the growth and differentiation of pluripotent stem cells. In this review, the authors describe technologies that move small volumes of fluids (on microscales) and how they can be used with stem cells. These technologies can provide precise signals that control stem cells, causing them to self‐renew (produce more stem cells) or differentiate (become any of the cells in the body). They can also be used to investigate the biology of stem cells and test their quality for medical applications. These powerful tools could one day be used to combat degenerative diseases.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/96259/1/180_ftp.pd

Spheroids-on-a-chip: Recent advances and design considerations in microfluidic platforms for spheroid formation and culture

© 2018 Elsevier B.V. A cell spheroid is a three-dimensional (3D) aggregation of cells. Synthetic, in-vitro spheroids provide similar metabolism, proliferation, and species concentration gradients to those found in-vivo. For instance, cancer cell spheroids have been demonstrated to mimic in-vivo tumor microenvironments, and are thus suitable for in-vitro drug screening. The first part of this paper discusses the latest microfluidic designs for spheroid formation and culture, comparing their strategies and efficacy. The most recent microfluidic techniques for spheroid formation utilize emulsion, microwells, U-shaped microstructures, or digital microfluidics. The engineering aspects underpinning spheroid formation in these microfluidic devices are therefore considered. In the second part of this paper, design considerations for microfluidic spheroid formation chips and microfluidic spheroid culture chips (μSFCs and μSCCs) are evaluated with regard to key parameters affecting spheroid formation, including shear stress, spheroid diameter, culture medium delivery and flow rate. This review is intended to benefit the microfluidics community by contributing to improved design and engineering of microfluidic chips capable of forming and/or culturing three-dimensional cell spheroids

Filling-and-Dragging Technique for A Particle-Entrapment Using Triangular Microwells

Trapping particle such as a cell, cell spheroid or scaffold bead, in a large trapping spot, such as a microwell, is important in various biological aspects. To achieve high trapping efficacy, the management of two countering effects from hydrodynamic and gravitational force is a key requirement. To increase the possibility of controllable entrapment, this study proposed a new approach using the filling and dragging technique to trap particles. The investigation of the trapping efficacy in three different triangular microwells such as obtuse, equilateral and acute triangle was conducted. The extremely low flow rate was firstly introduced to fill the particles in the microwell, and the flow rate was subsequently increased to drag and rearrange the entrapped particles. High trapping rate of a single particle in an equilateral triangular microwell could reach 80% when trapping polystyrene beads. For biomaterial particle such as cell spheroid, the adhesiveness with the other and the microwell surface is the parameter that needs to be further investigated

Pulling and Pushing Stem Cells to Control Their Differentiation

Much has already been done to achieve precisely controlled and customised regenerative therapies. Thanks to recent advances made in several areas relevant to regenerative medicine including the use of stimuli-responsive materials, 4-dimensional biofabrication, inducible pluripotent stem cells, control of stem cell fate using chemical and physical factors, minimal access delivery, and information-communication technology. In this short perspective, recent advances are discussed with a focus on a recent report on the use of mechanical stretching of nanoparticle-laden stem cells by using external magnetic field to induce defined cardiac line differentiation. Although more and more tools are becoming available for engineering tissue models tissues and the range of potential applications is expanding, there is still much work to be done before it is proved to work with human cells, form tissues and ultimately achieve application in the clinic

Acoustic assembly of cell spheroids in disposable capillaries

Multicellular spheroids represent a promising approach to mimic 3D tissues in vivo for emerging applications in regenerative medicine, therapeutic screening, and drug discovery. Conventional spheroid fabrication methods, such as the hanging drop method, suffer from low-throughput, long time, complicated procedure, and high heterogeneity in spheroid size. In this work, we report a simple yet reliable acoustic method to rapidly assemble cell spheroids in capillaries in a replicable and scalable manner. Briefly, by introducing a coupled standing surface acoustic wave, we are able to generate a linear pressure node array with 300 trapping nodes simultaneously. This enables us to continuously fabricate spheroids in a high-throughput manner with minimal variability in spheroid size. In a proof of concept application, we fabricated cell spheroids of mouse embryonic carcinoma (P19) cells, which grew well and retained differentiation potential in vitro. Based on the advantages of the non-invasive, contactless and label-free acoustic cell manipulation, our method employs the coupling strategy to assemble cells in capillaries, and further advances 3D spheroid assembly technology in an easy, cost-efficient, consistent, and high-throughput manner. This method could further be adapted into a novel 3D biofabrication approach to replicate compilated tissues and organs for a wide set of biomedical applications

Development of Micro-actuators and Micro-sensors for the On-chip Interrogation of Cells and In Vitro Generated Tissues.

Microscale systems enable interrogation of biological mechanisms beyond the capacity of conventional macroscale techniques. The large surface-to-volume ratio of microscale platforms allows investigators to better control the spatial and temporal microenvironment presented to biological samples, manipulating samples at scales reminiscent of their native microenvironments. This research describes microscale technologies to advance the design, complexity, and control of tissue culture microenvironments in three areas – chemical stimulation, regulating cell culture dimensionality, and oxygen monitoring. These tools improve in vitro models to better emulate the native biological response. To regulate temporal patterns of biochemical stimulation I developed an autonomous microfluidic oscillator circuit that enables dynamic control of delivered fluids without external control signals. This work produced to (1) a practical system to modulate the duty cycle of an applied stimulus in a user-defined manner without requiring modification of the device itself; and (2) a method to couple multiple independent oscillators together to ensure uniformity of experimental parameters, such as frequency and duty cycle, across multiple devices. In other work, reproducibility of three-dimensional spheroid cultures was achieved by culture additives to generate increasingly complex, and robust microscale cultures. We also developed dispersible microsensors for tissue culture oxygen measurements. When recreating physiologic microenvironments, it is critical to monitor and quantify the presence of oxygen. The untethered biocompatible oxygen sensors can be embedded or dispersed within diverse culture conditions for the real-time/continuous detection of oxygen in vitro. Dispersible microsensors were used to visualize the oxygen environment within in vitro tumor models, which allow for the informed generation of tumor models to more accurately capitulate the necessary oxygen environments.PhDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/113506/1/sashacai_1.pd

3D Spheroid Culture Systems for Metastatic Prostate Cancer Dormancy Studies and Anti-Cancer Therapeutics Development.

Prostate cancer is the most common non-skin cancer in United States men. Despite recent advances, mortality still remains high due to the emergence of therapy-resistant cancer cells that metastasize. Recently it has been postulated that only cancer stem cells (CSCs) are able to establish metastases, and therefore are the essential targets to destroy. Unfortunately, current use of CSCs is limited by the small number of CSCs that can be isolated, and the difficulty of culturing the CSCs in vitro. Furthermore, current in vitro-based metastatic prostate cancer models do not faithfully recreate the complex multi-cellular, three-dimensional (3D) tumor microenvironment seen in vivo. It is therefore crucial to develop effective in vitro prostate cancer culture and testing systems that mimic the actual in vivo tumor niche microenvironment. Here we utilized novel microscale technologies to develop an accurate 3D metastatic tumor model for detailed study of metastatic prostate cancer dormancy as well as accurate anti-cancer therapeutics screening and testing in vitro. Guided by the observation that prostate cancer cells parasitize and stay quiescent in the hematopoietic stem cell niche that is rich in osteoblasts and endothelial cells in vivo, a microfluidic device was established to create 3D spheroid culture of prostate cancer cells supported by osteoblasts and endothelial cells. This 3D metastatic prostate cancer model recapitulates the physiologic, dormant growth behavior of prostate cancer cells in the hematopoietic stem cell niche. Furthermore, we developed a hanging drop-based high-throughput platform for general formation, stable long-term culture, and robust drug testing and screening of 3D spheroids. Using this platform, we found significant differences in drug sensitivities against cells cultured under conventional 2D conditions versus physiological 3D models. A variety of techniques and methods were also established to specifically pattern the spatial localization of different co-culture cell types within a spheroid in this platform for accurate engineering of the 3D metastatic prostate cancer niche microenvironment. Collectively, these biological findings and technological innovations have led to advances in the understanding of prostate cancer biology and progress towards development of novel tools and therapeutics to fight against tumorigenic cancer cells.Ph.D.Biomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/86364/1/ahsiao_1.pd