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The Design of a Low-Cost, Stage-Top Cell Incubator for Long-Term Cell Imaging
Early imaging chambers designed in the 1970s involved the sandwiching of two
coverslips, separated by rubber O-rings, into a metal-plate holder. This design remained common for several years, but failed to allow for easy addition of culture medium or other drugs and did not actively maintain optimal cell-conditions. When the need for stage warmers or stage incubators was more widely realized, simple microscope slide warming plates were used. However, the heating plate did not reach the area on the slide with the specimen [2]. Over the years, stage incubators evolved into the current systems, which exist in two main types: (1) a small, stage-top incubator; and (2) a large, microscope-encasing box. The stage-top incubator is generally cheaper than the large enclosures (but still expensive), but fails to maintain steady temperatures and CO2 levels due to the large heatsink into the microscope stage and the high levels of fluctuations. Additionally, these incubators are typically only compatible with 10-cm petri dishes. This precludes applications for commonly-used rectangular well-plates. Conversely, the large enclosure has relatively small deviations, but is very expensive and takes several hours to reach equilibrium. There is a need for a low-cost cell incubator for long-term microscopy and
maintains steady parameters. We are developing a microscope stage-top incubator that is more affordable than existing systems, and that controls the internal temperature, humidity, and CO 2 levels to maintain cell viability for at least 24 hours. We are designing the control parameters (temperature, humidity, CO 2 ) to be modular, based on user-input, but the default settings will be 37 o C and 5% CO 2, Additionally, the new incubator will fit over a universal mounting frame to allow for varied experimental applications, and reach equilibrium parameters faster than the existing large enclosure. The development of an affordable, accurate stage-top incubator will allow more researchers to conduct long-term cell imaging (> 2 days).
Low Cost, Compact Cell Stretcher
In vitro cell culturing techniques are critical tools for cellular investigations for both basic science and translational applications. Growing cells on petri dishes, flasks, and well plates have long been considered the gold standard for cell culturing as the platforms allow for confluent cell growth. Environmental factors can be meticulously modulated by changing nutrient content in cell media or incubator conditions. Unfortunately, these in vitro cell cultures are not sufficient to model all biologically relevant structures. Culturing cells on rigid substrates fails to uniaxially align cells in favor of a non-ordered distribution. Furthermore, these existing methods fail to replicate the in vivo mechanical motion associated with certain cell types, such as fibroblasts, myocytes, epithelial cells (Camelliti, Gallagher, Kohl, & McCulloch, 2006). To this end, numerous cell stretchers have been developed to better mimic these mechanical forces and explore cellular behaviors in response to controlled external stretch. The Computer-controlled Cell Deforming System (Cell Stretcher CS10-series, Electron Microscopy Sciences Company) is a device prepared for uniaxial mechanical straining or compression with simultaneous live-cell imaging and motion compensation. Cells are cultivated on elastic membranes and the entire membrane is deformed, which strains the attached cells. This approach is useful but can create complications when imaging. Cells imaged during or after membrane deformation are displaced relative to the optical axis of the microscope, and microscopic monitoring is virtually impossible. To address this issue, an upgraded version of this device moves the entire membrane in the x-direction during the stretch to counteract the displacement. As a result, the region of interest is still precisely positioned above the objective of the microscope during (and after) the stretch. The benefit is cells can be imaged during the stretch. In addition, since the stretchable cell chamber is made of curved PDMS membrane with no vessel wall, and thus the relative displacement between PDMS membrane bottom and vessel wall does not exist, so the large strain can be achieved (Electron Microscopy Sciences, n.d.). Another widely used commercial cell stretcher is Automated Cell Stretching System. (STB-1400, STREX Inc.) This device is capable of stretching cells in CO2 incubator and simultaneously stretching cells in multiple chambers to enable comparison between samples. This cell stretcher system is also equipped with the detachable stretch chamber mounting unit, which can be transferred to a clean bench, enabling aseptic operations (“High Throughput & Long Term Cell Stretching Systems | Strex,” n.d.). In order to better study and understand cellular mechanisms involved in mechanical strain and stress from different directions, Cell Stretching Systems (NST-190-XY, Nepa Gene Co., Ltd.) has the capability of exerting biaxial stretch and compression on biological samples. Furthermore, this cell stretching system also includes cell chambers of different culture areas (4 cm2 and 10 cm2), allowing the incubation and stretching of different types and volumes of cells (“Cell Stretching Systems,” n.d.). However, the major limitation of commercial cell stretchers is the high price of the device and cell chamber. The price of the standard dcCS10 cell stretcher is $25,000 and the price of the upgraded dcCS10 with x-axis compensation is $39,000. While the cost of each cell chamber is small, they are single-use devices so the costs will add up over time. The bulkiness of currently available cell stretchers also restricts its compatibility with other devices and adds issues with transportation. Finally, since these systems are commercialized, users have limited access to the software controlling the device, reducing experimental customizability. We propose a small, open-source cell stretcher than is fabricated using low-cost electronics and hardware that can be mounted on a light microscope for easy imaging. Cells are seeded onto a custom-molded PDMS vessel that is uniaxially stretched on a linear stage actuator with micrometer precision. Linear movement is automated by a servo motor controlled by an Arduino Uno. The system is highly adaptable with a simple user interface that allows for toggling of experimental length, strain, strain rate, and frequency. Critically, this hardware fits on a universal microscope stage and allows for in-focus imaging during stretching to monitor cellular response.
An Economic, Modular, and Portable Skin Viscoelasticity Measurement Device for in situ Longitudinal Studies
In anticipation of studying skin mechanics with tissue engineering approaches, we designed a non-invasive, modular, and portable device at a low cost (<100 USD) for the in situ assessment of viscoelastic properties of the skin, as well as those of the fabricated tissues for in vitro studies. Our device is coined “indentation-based mechanical analyzer (IMA)”. Its design takes advantage of the recent maker movement, which incorporates the multitude of inexpensive mechanical and electronic parts that are commercially available for consumers. IMA implements an indenter to make prescribed, small deformations in the sample with a linear actuator, and records the reaction force from the indented tissue over time using a force sensor. The actuator and the sensor are controlled by Arduino, a popular electronic board for input/output signals. Because the probe, which consists of the indenter and sensor, is designed to be interchangeable, IMA can measure samples of peculiar geometries by switching between probes of different sizes and shapes. Because IMA is inexpensive, easy to build, and simple to calibrate and operate, we envision that it can be widely adopted by the community of tissue mechanics, and that the values of mechanical properties measured in different laboratories can be directly compared using IMA as a common measurement platform.
How to Build and Operate the Economic Bioprinter
This video shows a home-bulit 3D bioprinter and how to print microtissues with specific geometry.
Multiplexing Imaging-Ready Microgravity Simulator
The 3rd-generation device allows imaging cells growing in simulated microgravity without dismounting the cell culture cassettes. The parameters of microgravity can be adjusted. The video explains how.
Magnetic Tweezers: An Effective and Economic Method for a Five Micron Level Tolerance Tip Realignment Alignment system
The goal of this project is to address the issue with current magnetic tweezers system that has inconsistency in location of the tweezers tip. To resolve the problem, a mechanical fixture is designed and employed to decouple the movement of the magnetic tweezers and the slide fixture. In addition, a software based and sensor based calibration of the exact position of the tweezers tip applied to the system.
Cell Stretcher 2.0 Tutorial
This video demonstrates how to assemble the universal cell stretcher (Generation 2.0) for real-time dynamic imaging of cells under variable strains.