Oncomine Cancer Research Panel (OCP) | ESHG 2015 Poster PS12.131
CYTO2016_SPiS_final
1. Image Recognition Cell Dispensing System for Plating
of Single Cells and Monodisperse Spheroids
Immunotherapy is recently drawing many attentions in cancer therapy. This technique focuses on expression analysis of the cells, where
the activities of antibodies must be carefully monitored. For this purpose, there is a growing need for single-cell dispensing system in order to
prevent mixing in the expression profile analysis.
In cell-based drug assays, the assays incorporating spheroids are considered more suitable for evaluating drug efficacy than single cells,
because spheroid is a better representation of cell microenvironment in biological systems. For accurate evaluation in spheroid-based assays,
(1) preparation of monodisperse spheroid population and (2) plating of specific number of spheroids into reaction wells are necessary, due to
the spheroid size-dependent nature of the assays. However, there were not any instrument on the market that fulfilled such requirements.
For this reason, we have developed a system “On-chip SPiS” that allows for damage-less plating of single cells and size spheroids. This
instrument draws sample solution in a disposable transparent pipette tip for particle count and size image-recognition, then dispenses out
specified number of particles into multiple wells. On-chip SPiS can fit inside an biosafety cabinet. First, On-chip Sort, a cell sorter with
disposable microfluidic chip at its core, was used to sort a group of spheroids based on the size information from forward scattered light
intensity. On-chip Sort can recover spheroids of size up to 150µm in diameter. Then, the collected spheroids were plated one by one in
multititer plate. We anticipate that the size selection of spheroids by On-chip Sort and single cell dispensing by On-chip SPiS together will
become a useful tool for efficacy evaluation of drug candidates especially for anticancer drugs.
Introduction Spheroid Sorting and ATP Assay
Single Particle isolation System “On-chip SPiS”
On-chip SPiS is a tool for plating of single cells by image
recognition using our accumulated experience of cell
detection technology (Fig.1). To ensure only one cell is
dispensed at a time, the sample pipetted up in the
transparent polymeric pipette tip is photographed for
differentiation of cells from debris and scratches on the
pipette tip. The entire sample can be captured by taking
advantage of the depth of field and field of view of the
camera. Cells and spheroids of size ranging from 10µm up to
200µm can be plated without any damage at accuracy of 90%
and above. Although the pipette tips are custom made,
standard sample tubes and microtiter plates (both 96 wells
and 386 wells) can be used. This instrument can also
determine the size of the cells, and thus we can selectively
deposit the cells of particular size.
Single cell and Spheroid Dispensing
PC-9 cell line was plated in 96 well plate using On-chip SPiS. The cells
were initially stained with Hoechst, and they were evaluated under a
fluorescence microscope (Fig. 2(A)). The observation data showed 92%
of the wells contained 1 cells, and 8% of the wells had no cells (Fig. 2(B)).
Cell spheroids (cultured using NanoCulture Plate MS pattern, low
binding, 96 wells, NPC-LS96, SCIVAX Life Sciences, Ltd.) were also plated
in 384 well. Microscopy results confirmed that 95% of the wells
contained single spheroid, and 5% of the wells contained 2 or more
spheroids (Fig. 2(C)). These results show significantly greater single cell
dispensing accuracy than that of a conventional dispenser, which plates
single cells at a likelihood of 37% according to Poisson Distribution.
Single cell dispensing
results
Single spheroid
dispensing results
Spheroid Viability after Dispensing
Hoechst-stained
PC-9 cell in a well
100µm
Yuu Fujimura*, Tomiko Tanaka, Jin Akagi, Yohsuke Bansho, Ryoko Watanabe, Masayuki Ishige, Kazuo Takeda
On-chip Biotechnologies Co., Ltd.
Correspondence: Yuu Fujimura
y-fujimura@on-chip.co.jp
Viability of spheroids that have been dispensed using On-chip
SPiS inside a biosafety cabinet was evaluated. 7 spheroids were
plated individually, and they were cultured for 4 days. All 7
spheroids survived and became about 1.5 times larger than when
they were just plated (Fig. 3). This data indicates that On-chip SPiS
can dispense spheroids aseptically and without any damage.
Acknowledgement
Dead/Alive Determination of Spheroids using On-chip SpheroStain
We used a newly developed dye, On-chip SpheroStain, to observe the distribution of
dead cells present in spheroids. Fig. 6 shows the results of single cells stained using On-
chip SpheroStain, where live cells are not stained by the dye, while almost 100% of the
dead cells are stained. In the case of spheroid, spheroids of size 150µm and below
show almost no dead cells present inside (Fig. 7). However, the fraction of dead cells
present is higher for spheroids 200µm and above . The ordinary evaluation using ATP as
an index only provides information for the live cells, but On-chip SpheroStain has
proven it can provide information on the dead cells within spheroids. Consequently, we
conclude that use of spheroids 150µm and below is recommended for drug evaluation.
MG132: Cell permeable proteasome inhibitor. It inhibits activation of NF-κB, and leads to apoptosis
of various cancer cell lines.
ATP measurement was done using Infinite® 200 PRO, Tecan Group Ltd.
Fixed cell after staining for 10min
Almost all cells were stained.
Live cell after staining for 10min
Live cells are not stained.
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0 0.01 0.1 1 10
ATPConcentration(µM)
MG132 Concentration (µM)
No Sorting
After Sorting
Drug sensitivity evaluation using spheroids are commonly
undertaken using ATP as an index, but variations in the spheroid size
would hugely affect the experimental outcomes. This is considered
due to the difference in the extent of drug permeation to the core of
the cell spheroid. From this hypothesis, we collected a group of
spheroids of a similar size from a heterogeneous sample using our
microfluidic chip based cell sorter, On-chip Sort (On-chip
Biotechnologies Co., Ltd.). This instrument can recover particles of
size up to 150µm.
The spheroids were prepared using dedicated plate (NanoCulture
Plate MS pattern, low binding, 96 wells, NPC-LS96, SCIVAX Life
Sciences, Ltd.). Fig. 4(B) shows (in blue) the distribution of spheroids
after 3 days of culture. The initial sample contained mostly singular
cells (over 1000 cells indicated in light blue), while the spheroid size
varied from 30µm to 240µm. From this sample, On-chip Sort was
used to collect spheroids of size around 100µm based on the size
information from forward scattered light intensity (Fig. 4(C)). The
distribution of the sorted spheroids are plotted in red in Fig. 4(B),
which suggest that the size distribution have narrowed down
significantly (between 80-140µm).
These sorted spheroids and unsorted spheroid sample were used
for single spheroid dispensing using On-chip SPiS. After plating,
MG132 was added to each well at various concentration (0, 0.01,
0.1, 1, and 10µM). Then, these spheroids were cultured over 4 days
and the concentration of ATP was measured. Fig. 5 shows the
average concentration of ATP present in each spheroid, where the
blue bars indicate the unsorted spheroid sample, whereas the
orange bars represent the sorted spheroids. As the concentration of
MG132 increases, the ATP concentration decreases due to the dying
spheroid cells. Note that the concentration of ATP varies significantly
more for the unsorted spheroid samples than for the sorted
spheroids, shown by the error bars. Therefore, preparation for
monodisperse spheroids are crucial for an accurate drug evaluation.
Fig. 1 On-chip SPiS and its dispensing method
Number of cells in each
well
Percentage of wells (%)
0 8
1 92
2 0
Number of spheroids in
each well
Percentage of wells (%)
0 0
1 95
2 5
Fig. 2 Results of single cell and single spheroid plating
(A) (B) (C)
An image of the pipette
tip by built-in camera
Spheroid shortly after dispensing
100µm
Spheroid 4 days after dispensing
100µm
Fig. 3 Spheroid growth over 4 days of culture
200µm
Before Sorting
Sorting
Sorted Population
200µm
After Sorting
(A)
(B) (C)
Fig. 4 Spheroid sorting
0
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80
10 20 30 40 50 60 70 80 90 100110120130140150160170180190200210220230240250260
NumberofSpheroids
Spheroid Size (µm)
Uneven Spheroids
Sorted Spheroids
Fig. 5 ATP concentration vs MG132 concentration for sorted and unsorted spheroid samples
Fig. 6 Single cells stained by On-chip SpheroStain
Many of the data and results presented are provided by the courtesy of SCIVAS Life Sciences. Ltd. as part of collaborative work.
On-chip Sort
Fig. 7 Distribution of dead cells within spheroids