37 research outputs found
Cell-Based Dose Responses from Open-Well Microchambers
Cell-based assays play a critical
role in discovery of new drugs
and facilitating research in cancer, immunology, and stem cells. Conventionally,
they are performed in Petri dishes, tubes, or well plates, using milliliters
of reagents and thousands of cells to obtain one data point. Here,
we are introducing a new platform to realize cell-based assay capable
of increased throughput and greater sensitivity with a limited number
of cells. We integrated an array of open-well microchambers into a
gradient generation system. Consequently, cell-based dose responses
were examined with a single device. We measured IC<sub>50</sub> values
of three cytotoxic chemicals, Triton X-100, H<sub>2</sub>O<sub>2</sub>, and cadmium chloride, as model compounds. The present system is
highly suitable for the discovery of new drugs and studying the effect
of chemicals on cell viability or mortality with limited samples and
cells
Highly Stretchable Potentiometric pH Sensor Fabricated via Laser Carbonization and Machining of Carbon−Polyaniline Composite
The
development of stretchable sensors has recently attracted considerable
attention. These sensors have been used in wearable and robotics applications,
such as personalized health-monitoring, motion detection, and human–machine
interfaces. Herein, we report on a highly stretchable electrochemical
pH sensor for wearable point-of-care applications that consists of
a pH-sensitive working electrode and a liquid-junction-free reference
electrode, in which the stretchable conductive interconnections are
fabricated by laser carbonizing and micromachining of a polyimide
sheet bonded to an Ecoflex substrate. This method produces highly
porous carbonized 2D serpentine traces that are subsequently permeated
with polyaniline (PANI) as the conductive filler, binding material,
and pH-sensitive membrane. The experimental and simulation results
demonstrate that the stretchable serpentine PANI/C–PI interconnections
with an optimal trace width of 0.3 mm can withstand elongations of
up to 135% and are robust to more than 12 000 stretch-and-release
cycles at 20% strain without noticeable change in the resistance.
The pH sensor displays a linear sensitivity of −53 mV/pH (<i>r</i><sup>2</sup> = 0.976) with stable performance in the physiological
range of pH 4–10. The sensor shows excellent stability to applied
longitudinal and transverse strains up to 100% in different pH buffer
solutions with a minimal deviation of less than ±4 mV. The material
biocompatibility is confirmed with NIH 3T3 fibroblast cells via PrestoBlue
assays
Stretchable and Micropatterned Membrane for Osteogenic Differentation of Stem Cells
Stem cells have emerged as potentially
useful cells for regenerative
medicine applications. To fully harness this potential, it is important
to develop in vitro cell culture platforms with spatially regulated
mechanical, chemical, and biological cues to induce the differentiation
of stem cells. In this study, a cell culture platform was constructed
that used polydopamine (PDA)-coated parafilm. The modified parafilm
supports cell attachment and proliferation. In addition, because of
the superb plasticity and ductility of the parafilm, it can be easily
micropatterned to regulate the spatial arrangements of cells, and
can exert different mechanical tensions. Specifically, we constructed
a PDA-coated parafilm with grooved micropatterns to induce the osteogenic
differentiation of stem cells. Adipose-derived mesenchymal stem cells
that were cultured on the PDA-coated parafilm exhibited significantly
higher osteogenic commitment in response to mechanical and spatial
cues compared to the ones without stretch. Our findings may open new
opportunities for inducing osteogenesis of stem cells in vitro using
the platform that combines mechanical and spatial cues
Microfluidic Generation of Polydopamine Gradients on Hydrophobic Surfaces
Engineered surface-bound molecular
gradients are of great importance
for a range of biological applications. In this paper, we fabricated
a polydopamine gradient on a hydrophobic surface. A microfluidic device
was used to generate a covalently conjugated gradient of polydopamine
(PDA), which changed the wettabilty and the surface energy of the
substrate. The gradient was subsequently used to enable the spatial
deposition of adhesive proteins on the surface. When seeded with human
adipose mesenchymal stem cells, the PDA-graded surface induced a gradient
of cell adhesion and spreading. The PDA gradient developed in this
study is a promising tool for controlling cellular behavior and may
be useful in various biological applications
Spot Identification and Quality Control in Cell-Based Microarrays
Cell-based microarrays are being increasingly used as
a tool for
combinatorial and high throughput screening of cellular microenvironments.
Analysis of microarrays requires several steps, including microarray
imaging, identification of cell spots, quality control, and data exploration.
While high content image analysis, cell counting, and cell pattern
recognition methods are established, there is a need for new postprocessing
and quality control methods for cell-based microarrays used to investigate
combinatorial microenvironments. Previously, microarrayed cell spot
identification and quality control were performed manually, leading
to excessive processing time and potentially resulting in human bias.
This work introduces an automated approach to identify cell-based
microarray spots and spot quality control. The approach was used to
analyze the adhesion of murine cardiac side population cells on combinatorial
arrays of extracellular matrix proteins. Microarrays were imaged by
automated fluorescence microscopy and cells were identified using
open-source image analysis software (CellProfiler). From these images,
clusters of cells making up single cell spots were reliably identified
by analyzing the distances between cells using a density-based clustering
algorithm (OPTICS). Naïve Bayesian classifiers trained on manually
scored training sets identified good and poor quality spots using
spot size, number of cells per spot, and cell location as quality
control criteria. Combined, the approach identified 78% of high quality
spots and 87% of poor quality spots. Full factorial analysis of the
resulting microarray data revealed that collagen IV exhibited the
highest positive effect on cell attachment. This data processing approach
allows for fast and unbiased analysis of cell-based microarray data
Expressions of integrin CD11b and CD31.
<p>(A) Fluorescent expression intensity and area of CD11b in hMSCs and hASCS. (B) Green fluorescent expression intensity and area of CD31 in hMSCs and hASCS. Star (*) indicates comparison of statistical difference of stimulation to control in the same cell type. *<i>p</i><0.05. It also indicates statistical difference between stimulated hMSCs and hASCs.</p
Osteogenesis related transcript levels and β1 integrin expression.
<p>(A) Bone sialoprotein (BSP), (B) Osteopontin (OP), (C) Runt-related transcription factor 2 (Runx2), (D) β1 integrin (*<i>p</i><0.05)</p
Microchip and experimental setup for evaluating stem cells towards osteogenesis under mechanical stimulation.
<p>(A) The microchip is comprised of a cover, an air chamber, looped microvalves, and twelve cell culture chambers. These paired cell chambers share the inlet/outlet channel. The cells (hMSCs and hASCs) are loaded into half of the chip, individually. Scale bar = 1 cm. (B) Schematic diagram of top view (I) and simplified cross-sectional view (II) of the device. The device was designed to culture two different stem cells simultaneously and to apply mechanical stimulation using cyclic pneumatic force. (C) The experimental setup for mechanical stimulation, including a controlled nitrogen gas pressurized air chamber. The frequency of pneumatic pressure is controlled with a switching solenoid valve derived by a control circuit. During mechanical stimulation, microvalves are closed with higher pressure (P2>P1) to prevent undesired shear stress in the cell chambers.</p
Immunocytochemical staining of hMSCs and hACSs.
<p>(A) The expression of osteogenic markers after 1 and 7 days. Bone sialoprotein (BSP), Osteopotin (OP), and Collagen type I (Col I) were stained with GFP and strongly expressed in the stimulated BMSCs. Blue = DAPI Nucleic Acid Stain. (Scale bars: 100 (m) Green fluruorecent expression intensity (B) and area (C) of ECMs in hMSCs and hASCS. Data presented in the line graph represent mean value with SD (n = 12). *p<0.05. Star (*) indicates comparison of statistical difference of stimulation to control and statistical difference between stimulated hMSCs and hASCs.</p
Osteogenesis characterizations of hMSCs and hASCs after 7 days.
<p>hASCs and hMSCs cultured in the microchip with osteogenic medium for 7 days were stained with ALP and Alrizarin red. The stimulated group of BMSCs resulted in significantly enhanced ALP activity and calcium deposits. (Scale bars: ALP staining 100 (m, Alrizarin red staining 200 (m).</p