6 research outputs found
Openly Accessible Microfluidic Liquid Handlers for Automated High-Throughput Nanoliter Cell Culture
Cell culture is typically performed in Petri dishes,
with a few million cells growing together, or in microwell plates
with thousands of cells in each compartment. When the throughput of
each experiment, especially of screening based assays, is increased,
even using microliter solution per well will cost a considerable amount
of cells and reagents. We took a rational approach to reduce the volume
of each cell culture chamber. We designed and fabricated a polyÂ(dimethylsiloxane)
based liquid pipet chip to deliver and transfer nanoliter (50–500
nL) samples and reagents with high accuracy and robustness. A few
tens to a few hundreds of cells can be successfully seeded, transferred,
passaged, transfected, and stimulated by drugs on a microwell chip
using this pipet chip automatically. We have used this system to test
the cell growth dynamically, observed the correlation between the
culture conditions and cell viabilities, and quantitatively evaluated
cell apoptosis induced by <i>cis</i>-diammineplatinumÂ(II)
dichloride (cisplatin). This system shows great potential to facilitate
large-scale screening and high-throughput cell-array based bioassays
with the volume of each individual cell colony at the nanoliter level
Openly Accessible Microfluidic Liquid Handlers for Automated High-Throughput Nanoliter Cell Culture
Cell culture is typically performed in Petri dishes,
with a few million cells growing together, or in microwell plates
with thousands of cells in each compartment. When the throughput of
each experiment, especially of screening based assays, is increased,
even using microliter solution per well will cost a considerable amount
of cells and reagents. We took a rational approach to reduce the volume
of each cell culture chamber. We designed and fabricated a polyÂ(dimethylsiloxane)
based liquid pipet chip to deliver and transfer nanoliter (50–500
nL) samples and reagents with high accuracy and robustness. A few
tens to a few hundreds of cells can be successfully seeded, transferred,
passaged, transfected, and stimulated by drugs on a microwell chip
using this pipet chip automatically. We have used this system to test
the cell growth dynamically, observed the correlation between the
culture conditions and cell viabilities, and quantitatively evaluated
cell apoptosis induced by <i>cis</i>-diammineplatinumÂ(II)
dichloride (cisplatin). This system shows great potential to facilitate
large-scale screening and high-throughput cell-array based bioassays
with the volume of each individual cell colony at the nanoliter level
Openly Accessible Microfluidic Liquid Handlers for Automated High-Throughput Nanoliter Cell Culture
Cell culture is typically performed in Petri dishes,
with a few million cells growing together, or in microwell plates
with thousands of cells in each compartment. When the throughput of
each experiment, especially of screening based assays, is increased,
even using microliter solution per well will cost a considerable amount
of cells and reagents. We took a rational approach to reduce the volume
of each cell culture chamber. We designed and fabricated a polyÂ(dimethylsiloxane)
based liquid pipet chip to deliver and transfer nanoliter (50–500
nL) samples and reagents with high accuracy and robustness. A few
tens to a few hundreds of cells can be successfully seeded, transferred,
passaged, transfected, and stimulated by drugs on a microwell chip
using this pipet chip automatically. We have used this system to test
the cell growth dynamically, observed the correlation between the
culture conditions and cell viabilities, and quantitatively evaluated
cell apoptosis induced by <i>cis</i>-diammineplatinumÂ(II)
dichloride (cisplatin). This system shows great potential to facilitate
large-scale screening and high-throughput cell-array based bioassays
with the volume of each individual cell colony at the nanoliter level
Microfluidic Device for Studying Controllable Hydrodynamic Flow Induced Cellular Responses
Hydrodynamic
flow is an essential stimulus in many cellular functions,
regulating many mechanical sensitive pathways and closely associating
with human health status and diseases. The flow pattern of blood in
vessels is the key factor in causing atherosclerosis. Hemodynamics
has great effect on endothelial cells’ gene expression and
biological functions. There are various tools that can be used for
studying flow-induced cellular responses but most of them are either
bulky or lack precise controllability. We develop an integrated microfluidic
device that can precisely generate different flow patterns to human
endothelial cells cultured on-chip. We monitored cell morphology and
used small-input RNA-seq technology to depict the transcriptome profiles
of human umbilical vein endothelial cells under uni- or bidirectional
flow. Such integrated and miniatured device has greatly facilitated
our understanding of endothelial functions with shear stimulus, not
only providing new data on the transcriptomic scale but also building
the connection between cell phenotypic changes and expression alternations
Single-Cell-Based Platform for Copy Number Variation Profiling through Digital Counting of Amplified Genomic DNA Fragments
We
develop a novel single-cell-based platform through digital counting
of amplified genomic DNA fragments, named multifraction amplification
(mfA), to detect the copy number variations (CNVs) in a single cell.
Amplification is required to acquire genomic information from a single
cell, while introducing unavoidable bias. Unlike prevalent methods
that directly infer CNV profiles from the pattern of sequencing depth,
our mfA platform denatures and separates the DNA molecules from a
single cell into multiple fractions of a reaction mix before amplification.
By examining the sequencing result of each fraction for a specific
fragment and applying a segment-merge maximum likelihood algorithm
to the calculation of copy number, we digitize the sequencing-depth-based
CNV identification and thus provide a method that is less sensitive
to the amplification bias. In this paper, we demonstrate a mfA platform
through multiple displacement amplification (MDA) chemistry. When
performing the mfA platform, the noise of MDA is reduced; therefore,
the resolution of single-cell CNV identification can be improved to
100 kb. We can also determine the genomic region free of allelic drop-out
with mfA platform, which is impossible for conventional single-cell
amplification methods
Tagmentation on Microbeads: Restore Long-Range DNA Sequence Information Using Next Generation Sequencing with Library Prepared by Surface-Immobilized Transposomes
The next generation
sequencing (NGS) technologies have been rapidly
evolved and applied to various research fields, but they often suffer
from losing long-range information due to short library size and read
length. Here, we develop a simple, cost-efficient, and versatile NGS
library preparation method, called tagmentation on microbeads (TOM).
This method is capable of recovering long-range information through
tagmentation mediated by microbead-immobilized transposomes. Using
transposomes with DNA barcodes to identically label adjacent sequences
during tagmentation, we can restore inter-read connection of each
fragment from original DNA molecule by fragment-barcode linkage after
sequencing. In our proof-of-principle experiment, more than 4.5% of
the reads are linked with their adjacent reads, and the longest linkage
is over 1112 bp. We demonstrate TOM with eight barcodes, but the number
of barcodes can be scaled up by an ultrahigh complexity construction.
We also show this method has low amplification bias and effectively
fits the applications to identify copy number variations