single cells on demand

single cells on demand

single-cell isolationproven | viable | pure

a single cell printer that isolatessingle, living cells automatically

from a biological sample

Clonal cell line development is acrucial step in various applicationsincluding generating biopharma-ceuticals (eg. monoclonal anti-bodies). Current workflows in cellline development have major draw-backs such as missing proof of clonality, inefficient single-cellisolation and reduced cell viability.

The single-cell printer technologyoffers documented proof of clonality and provides efficient and fast single-cell seeding combined with excellent cell viability and zero risk of cross-contamination.

The single-cell printer supportsSLAS/SBS format 96-well and 384-well plates. A great variety of typical cell lines used in cellline development such as CHO-K1,HEK 293, L929 can be processed.

Discover the single-cell printertechnology for your clonal cell line application.

cell line developmentap

plications

day 7day 6day 5day 4day 3day 0

4

5

The isolation of single cells remainsa challenging task in single-cell genomics. Current methods lack the evidence that only a single-cellhas been isolated in the analysisvessel. It is important that the integrity of the cells is maintainedprior to their lysis in order to preserve their DNA.

Furthermore, cells should undergo aslittle stress as possible prior to theirlysis in order to preserve their RNAand its expression level. The single-cell printer deposits single cells in a very gentle manner, guaranteeinghigh purity and high viability.

This provides optimal basis fordownstream single-cell genomicanalysis.

single-cell genomics

workflow

applications

6

single-cell isolation

lysis amplification sequencing

whole genome amplification mutational analysis by sequencing

Single cells of the osteosarcoma cellline U2OS were printed in wells of a384-well microtiter plate, preloadedwith 1 µl PBS. Additional cells wereprinted in dry wells resulting in atotal single-cell printing efficiencyof 98%.

Whole genome amplification (WGA)was performed on the cells andcomparable DNA yields were achie-ved for dry and PBS wells.The WGA DNA was evaluated by amultiplex PCR on repetitive LINE1transposons, which revealed positiveresults in all WGA samples.

In addition, U2OS-specific muta-tions in SLC34A2 (c.1538G>T), andin TET2 (c.1394C>T) were detectedin representative WGA samples ofsingle cells printed in PBS.

TET2c.1394C>TENST00000380013

TET2c.1538G>TENST00000382051

cell bulk

AATC CATC T AATC TATC T ATC C TCATG ATC C TCATG

single cell B3

90 60 80 80

cell bulk single cell B1

0

1

2

3

4

5

prefilled PBS dry NTC PC

WGA

DNA

-yield (µg

)

single cells7

The patented single-cell printingtechnology, developed by cytena,enables fully automated isolation ofsingle cells into standard microwellplate formats. The instrument usesan inkjet-like principle featuringa disposable, one-way printing cartridge.

The cell sample is pipetted into thecartridge and an external actuator is used to eject droplets out of it. The integrated optical sensor allowsfor determination of cell number ineach droplet. A fast shutter mechanismsorts the droplets containing exactlyone single-cell into the substrate.Unwanted droplets are deflectedinto waste.

the single-cell printer: proven | viable | pure

single-cell printer

cytena’s single-cellprinter is a benchtopsize, automated labora-tory instrument.Thanks to its intuitiveand user-friendly soft-ware, it can be easilyoperated by each labmember without requi-ring special skills. Theinstrument setup timeis less than 10 minutes.Filling e.g. a 96-wellplate with single cellstakes a few minutesonly.

45 cm

51 cm

55 cm8

tech

nology

An image sequence for each singlecell can be used as proof of clona-lity. The images are assigned to thewell of the specific cell and storedon a hard-drive.

proven

Cells are processed with an excellentviability (as gentle as pipetting). No cell labelling is required.

viable

The use of a disposable cartridge ensures the absence of any cross-contamination. This cartridge is the only part that comes into contact with cells before they are dispensed into the well.

pure

cell

nozzle

printing cartridge

cytena's disposable, one-way cartridge takes up to 80 µl of cell sample. The single-packed, sterilized cartridges can simply be loaded by hand-pipetting. Its silicon micro-fluidic chip generates free-flying picoliter droplets acting as transport vessels for thecells. One cartridge can fill many well-plates with singlecells. After use, the cartridge is disposed. This inno-vative approach prevents cross-contamination andsaves extensive cleaning steps.

disposable cartridge

9

2

5

3

schematic1 cartridge 2 cell suspension 3 pneumatic shutter 4 free-flying droplet 5 micro-well plate

1

resolution optics and software algorithm detect a single cell in the nozzle

2

5

344

1

droplet with a single cell isejected while pneumatic shutter is closed

2

5

3

1

droplet with a single cell landsin the dedicated well

working principle

droplet with one cell

10

tech

nology

2

5

3

1

resolution optics and software algorithm detect more than onecell in the nozzle

2

5

34

1

droplet with more than one cell isejected and immediately deflectedinto waste by pneumatic shutter

2

5

3

1

resolution optics and software algorithm detect no cell in thenozzle

2

5

34

1

empty droplet is ejected and immediately deflected into wasteby pneumatic shutter

droplet with more than one cell

droplet without a cell

11

perfor

man

ce data

single-cell printing efficiency

• printing efficiency* > 90 %• confirmed and proven by single-cell images• tested on many common cell lines • throughput 5-10 min per 96-well plate

(one cell per well)

single-cell viability (clonal recovery rate)

• excellent cell viability• high clonal recovery rates*• no pressure, no lasers, no electric fields• as gentle as hand-pipetting

sing

le-cells per w

ell-plate

0 %10 %20 %30 %40 %50 %60 %70 %80 %90 %

100 %

cell type

CHO HEK L929

viab

le clona

l colon

ies

per well-plate

0 %10 %20 %30 %40 %50 %60 %70 %80 %90 %

100 %

cell type

CHO HEK L929

12

*clonal recovery rate = number of viable colonies derivedfrom a confirmed single-cell

*printing efficiency = number of wells containing a single-cell per total number of wells addressed.

A

proof of clonality

before cell printing tracking a cells' way towards nozzle

B Ctargeted single-cell

Dtargeted single-cell withcontour overlay

Eafter cell printing, confirmation that cellleft nozzle

Proof of cell line clonality. Images taken during the selectionand isolation process of single-cells.

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*These cell types have been successfully printed and post-printing viability is confirmed by successful proliferation

human cancer HeLa cell lines CaSki

SiHa C33a U2OS

human B-cells cell lines Jurkat

Raji HEK 293U2OS

human fibroblastsprimary cells keratinocytes

animal CHO cell lines RBL

3T3-FIBL929

sample type suspension

prior filtering 40 µm(mesh size)

cell type eukaryotic

min. cell 5 µmdiameter

max. cell 35 µmdiameter

print medium DPBS

min. 104 cells/mlconcentration

optimal 105-106

concentration cells/ml

max. 107 cells/mlconcentration

chip silicon-glass

reservoir polymer

min. sample 10 µlvolume

max. sample 80 µlvolume

dead volume < 1 µl

microfluidic 40 x 40 µmoutlet (nozzle)

droplet volume 150 pl

dispensation non contactmode

perfor

man

ce data

14

approved cell types* sample requirements

cartridgecharacteristics

founders team

Benjamin Steimle | CFO Jonas Schöndube | CEOAndre Gross | CTODr. Peter Koltay | BD

cytena.comsingle cells on demand

cytena GmbHGeorges-Koehler-Allee 103 | 79110 Freiburg | Germany

+49 (0) 761 / 203 732 19info@cytena.com

cytena.com

single cells on demandget in touch!