High Density Cell Arraying Device• Membrane with nanowells for single cell trapping• Generate high density cell array• Single cell culture, staining, imaging and assay

20 μm nanowell

50 μm nanowell

Number of Nanowells 370,000（20 μm）

90,000（50 μm）

20 μm nanowell, MCF-7

The SIEVEWELL single cell arraying platform brings new insights to your research.

Cell Culture
Suspension cell culture
Spheroids generation

Cell of Interest
Molecular analysis

Cell Arraying
Rare Cell Detection
On-chip staining
Minimized cell-loss during staining

Assay
Cell-to-cell interaction
Immune Cell Killing AssayExpansion

Mechanism of Single Cell Capture
Arraying cells is an important step for single cell analysis. For example, cell overlap is unfavourable for single cell
imaging and isolation. Microcavity arrays are used to capture single cells. In general, the distribution of cells in acavity is by sedimentation and the single cell rate follows a Poisson distribution. Its single cell capture rate is relatively
low, especially when loading cells in higher quantities.
SIEVEWELL is designed to capture cells at a high single cell rate. Two pores are positioned at the bottom of thenanowell. After loading the cell suspension, a directional liquid flow can be created from the inner liquid chamber tothe side ports by aspirating liquid from the side ports with a standard pipette. The cells follow the liquid flow and are
captured in the nanowell. When a cell enters a nanowell, it blocks the pores, reducing the flow of liquid through thatnanowell. Other cells are then redirected to other empty nanowells. This mechanism allows a higher single cell rate
than a Poisson distribution.
Easy to use. Fluidic system or instrument is not required.
Add fluid to center chamber

Aspirate fluid from side port

Compatible with 8 channel pipette

Generation of directional flow by aspiration from side port
Micro-gap situated under
the nanowell structure

Trapped cell blocks the pores

Cells move toward empty
nanowells than cell trappednanowells

Flow
Cell Reagent

Cell
NanowellFlowSingle Cell Capture Rate
PC-3

HL60s

MCF-7

A549

K562

A549

Poisson distribution

20 μm nanowell

SIEVEWELL
Single cell rate

Single cell rate

SIEVEWELL

Poisson distribution

50 μm nanowell

Cell occupancy against total nanowell
Number of cells

37,00074,000111,000

148,000

185,000

9,000

18,00027,00036,00045,000

Arraying Cells at High-Density
Seeding cells at low density is one option to avoid cell overlapping for single cell imaging. This requires not only moreglass slide, microplate and reagents but also a time for taking images and analyzing to detect cell of interest.
SIEVEWELL has nanowells at high-density in 17 x 17 mm (1/3 of glass slide). This minimizes required number of glass
slide or microplate, resulting in reducing the required time for taking images.
Number of Nanowells
370,000（20 μm）
90,000（50 μm）

20 μm nanowell

50 μm nanowell

On-Chip Staining
Cell staining is standard procedure to visualize cells and cellular components under microscope. Conventional
methods require multiple transfer or wash steps during staining that risk causing loss of the rare cell population ofinterest, e.g. circulating tumor cells. The pore size of the SIEVEWELL is smaller than typical mammalian cells. Thanks
to this design, cell loss can be minimized during on-chip staining in the same way, e.g., fixation, permeabilization,
blocking, incubation and washing.
Cell loading

Cell distribution

Staining

Washing

DAPI

Alexa Fluor 488-CKPE-CD45Composit

Human PBMC spiked with MCF7 cell were loaded. Cells were fixed with 4% PFA, permeabilized with 0.2 % Triton X-100, blocked withProtein Block, stained with a cocktail of PE mouse anti-human CD45 antibody, Alexa Fluor 488 mouse anti-human CK antibody andDAPI, then washed.

Circulating tumor cell from patient blood
SIEVEWELL
1.5 mL tube

Cell retention rate [%]1201008060402020 μm nanowell0Fixation
Permeabilization-+-++

Direct comparison of staining procedure.
A549 cells were stained using 1.5 mL tube or SIEVEWELL 20 μm.
Case 1: no fixation, no permeabilization
Case 2: fixation with 4 % PFA, no permeabilization
Case 3: fixation with 4 % PFA, permeabilization with 0.2 % Triton X-100

Data provided by Professor Hans Neubauer
Department of Obstetrics and Gynaecology, University Hospital and Medical
Faculty of the Heinrich Heine University Düsseldorf, Düsseldorf, Germany

Alexa Fluor is a registered trademark of Molecular Probes Inc, a Thermo Fisher Scientific Company, in the United States and other countries.

Monitoring of Cell Growth from Single Cells
Suspension Cell Culture from Single Cells
For cell growth monitoring from single cell, limiting dilution or sorting with cell sorter is well known method togenerate single cell state in microwell plate. However, many microplates and medium is required to analyze single
cell growth, and it is laborious to check if each well contains single cell or not. Also it takes time for imaging of many
microplates. Suspension cells are floating in the culture medium and roaming freely, so it is difficult to track growthfrom single cell.
SIEVEWELL, high density cell arraying device, is suitable for growth monitoring from single cell. Suspension cells arecaptured in each nanowell during proliferation.
Day 0Day 2CompositeDay 2Day 0K562 cells (stained with CellBrite Green) were cultured in SIEVEWELL Slide 50 μm. Images were taken every 2 hours with IncuCyte S3 (10x objective lens). Images of day 0 and day 2 were ovelayed using ImageJ.

Monitoring of cell growth from a single cell clarify that even cell line is a mixture of different growth rate of cells. K562cells are a mixture of cells with different growth rates, 18 hours, 26 hours and 38 hours. Cells in the nanowell wereretrieved using micromanipulator and transferred into 96 well microwell plates for further expansion, and RNA
sequencing was conducted.
Day 0Day 2
Doubling time
38 hours

26 hours
18 hours

50 μm nanowell, K562 cells

Cell Culture

Cell RetrivalExpansion

mRNA expression profiles in three cell types

18 hours
26 hours
38 hours

RNA Sequencing

Spheroid Generation from Single Cells
The ability of a single cell to form a spheroid is considered a potential self-renewal ability. SIEVEWELL 50 μm makes
it possible to monitor the formation of spheroids from a single cell. Over 50,000 spheroids could be generated on asingle chip.

Day 0Day 3

A549 cells were cultured in SIEVEWELL Slide 50 μm. Images were taken every 1 hour with CM30 Incubation Monitoring System, Evident.

Monitoring of Drug Response
Culture in SIEVEWELL allows the behaviour of cells exposed to drugs to be monitored from individual cells. Highdensity cell arrays could capture many individual cell images simultaneously. For example, over 1,000 cells could betracked in a single image. This will be useful to identify rare drug-resistant cells and provide clues to elucidate drug
resistance mechanisms and develop better drugs.
Effect of Imanitib on K562 Cells
Without Imatinib

Effect of Etoposide on A549 Cells
Without Etoposide

0.5 μM Imatinib

StartStart

70 hours

90 hours

Cells were cultured in SIEVEWELL Slide 50 μm. Images were taken every 1 hour with CM30 Incubation Monitoring System, Evident.

500 μM EtoposideCell-to-Cell Interaction Assay
SIEVEWELL 50 μm is suitable for capturing pairs of two different cells to study cell-to-cell interactions. Combinationof flow based capture and gravitational sedimentation enables to create a large amount of 1:1 cell-to-cell pairs in asingle device.

1st Cell Type Loading
Flow based procedure

2nd Cell Type Loading
Sedimentation based procedure

Immune Cell Killing Assay
Cytotoxic immune cells can recognize and kill target cancer cells. Immune
cell killing assays are a valuable tool for immuno-oncology research
projects for in vitro assessment of these cells. With SIEVEWELL, thedynamic interactions of immune and cancer cells can be visualized.

Apoptosis induced by NK cell

50 μm nanowell, K562; green, KHYG-1; magenta

The erythroleukemia K562 cell is known as a NK-cell sensitive target. Calcein AM-stained K562 cells were loaded into
SIEVEWELL 50 μm, then KHYG-1 cells, NK leukemia cell line, were loaded by sedimentation. Time-lapse images weretaken every 3 minutes.
Target: K562

Effector: KHYG-1

Composite

Composite

Killing assay
Killing assay videoSupport for Cell Isolation
SIEVEWELL is top-open chamber for accessing glass capillary from top side. SIEVEWELL and glass capillary based cell
pick up tool is an ideal combination for cell isolation, not only for single cell cloning, but also for picking up spheroids
generated from single cells and cells of interest after assay.

Molecular analysis

Single cell culture
Cell pick up

Rare cell detection
Expansion

Assay

Examples of Surface Modification
Surface of nanowell is coated with polymer to prevent cell attachment. However, for adherent cell culture, protein
binding surface type is available for coating with ECM e.g. laminin, collagen by request.

On-Chip Neural Differentiation of PC12 Cells

20 μm nanowell

SIEVEWELL 20 μm was coated with Cellmatrix Type IV (Collagen Type IV, Nitta Gelatin Inc). Rat pheochromocytoma cell line PC12 cells wereloaded into SIEVEWELL 20 μm. Cells were cultured with RPMI 1640/10% horse serum/5% fetal bovine serum containing 10 ng/μL NGF. After
7 days, cells were fixed with PFA, permeabilized with 0.05% Tween 20/PBS, blocked with 1％BSA/PBS, stained with mouse anti-rat Tubulin
β3 (TUBB3) antibody (Clone, TUJ1) followed by staining with Alexa Fluor Plus 488 labelled anti-mouse IgG antibody and DAPI. Images weretaken with THUNDER Imaging Systems (Leica Microsystems).

Specifications

SIEVEWELL Slide

Product code

SWS 2001-5

SWS 5001-5

Nanowell size

Width 20 μm、Depth 25 μm

Width 50 μm、Depth 50 μm

Number of nanowell370,000

90,000

50 μm

25 μm

50 μm

20 μm

*10 μm cell

Dimension
Chamber size
Working volume
MaterialPackage

25 mm x 75 mm x 12 mm
17 mm x 17 mm, Cell repellent surface
0.3 - 2 mL
PS, PC, Biocompatible polymer
5 slides per box （sterilized）

References
Therapeutic application of human type 2 innate lymphoid cells via induction of granzyme B-mediated tumor cell deathCell. 2024 Feb 1;187(3):624-641.e23.
doi: https://doi.org/10.1016/j.cell.2023.12.015
Quantitative single cell mass spectrometry reveals the dynamics of plant natural product biosynthesis
bioRxiv. 2024.04.23.590720.
doi: https://doi.org/10.1101/2024.04.23.590720
Single-cell multi-omics enabled discovery of alkaloid biosynthetic pathway genes in the medical plant Catharanthus roseus.
Nature Chem Biol. 2023 Aug;19(8):1031-1041.
doi: https://doi.org/10.1038/s41589-023-01327-0.
Implementing microwell slides for detection and isolation of single circulating tumor cells from complex cell suspensions
Cytometry A. 2022 Dec;101(12):1057-1067.
doi: https://doi.org/10.1002/cyto.a.24660.
Validation of Cell-Free RNA and Circulating Tumor Cells for Molecular Marker Analysis in Metastatic Prostate Cancer
Biomedicines. 2021 Aug; 9(8): 1004.
doi: https://doi.org/10.3390/biomedicines9081004.
Improvement of single circulating tumor cells isolation with sievewell slidesGeburtshilfe Frauenheilkd 2020; 80(10): e212
doi: https://doi.org/10.1055/s-0040-1718200.

Contact

contact@sievewell.com

Please visit our website for further information.

Tokyo Ohka Kogyo Co., Ltd.
New Business Development Division
1590 Tabata, Samukawa, Koza, Kanagawa
253-0114, JapanFor research use only. Not for use in diagnostic procedures.
All specifications are subject to change without notice.

SIEVEWELL and SIEVEWELL logo are trademarks or registered trademarks of Tokyo Ohka Kogyo Co., Ltd. All trademarks, logos and brand names are the property of theirrespective owners. All company, product and service names used in this brochureare for identification purposes only. Use of these names, logos, and brands does not implyendorsement.