Stem Cell and Organoid Platform

The IGH 3D culture platform hosts the Montpellier organoid platform (POM) (website). It provides the scientific community with a sorter for fragile cells or tissues called Cellenone from the brand Cellenion (see device).

The CellenONE allows to automate the cloning steps with a single-cell dispenser based on acoustic manipulation. It allows the sorting of cells in suspension, the separation of two cell populations or the cloning by sorting on multi-well plates. It dispenses only single-cell drops into the microplate. Any remaining drops will be dispensed into a collection tube, so no sample is wasted and the cells can be re-sorted by another criterion.

This device will be installed in culture room 30 on the ground floor of the Institute of Human Genetics in March 2022. An engineer from the IGH will be in charge of training and accompanying the users in the realization of their projects. She will guarantee continuity of service through regular maintenance of this equipment and will ensure its development. A reservation site will be set up; this service will have to be paid for. It will be open to academic, clinical and private sector users. This equipment will be accessible after a training to its use given by the engineer in charge of the organoid platform;

We also invite you to contact this engineer for any 3D culture project: Spheroids, organoids, tumoroids of human, animal or plant origin (isabelle.peiffer@igh.cnrs.fr).

For information, price offers for the following companies: Promega, Sigma, Peprotech, Thermoscientific /fisher ; Stemcell technology ; Eurobio, Biotehne (R&D, Tocris), Miltenyi, Corning, startsted, Euromedex, Ozyme, and cliniscience will be available for POM members.

Procedure

Preparation of cell samples

10 min*

Place the sample in the holder

Aspirate the sample

Select culture media (DMEM, RPMI, Ham s F12) or buffer (PBS)
Prepare a minimum volume of 2 μL, ideally around 10 μL
Optimal cell concentration less than 200 cells/μL

Cell mapping

5 min*

System setup takes only a few minutes

Users can walk away during the isolation process

Automated cell tracking
Automated report generation
Choose the isolation parameters
Select target type (96 384 1536 wp)

A mapping report is automatically generated, it contains:

Evaluation of the boundary ejection area Cell detected, diameter, elongation and circularity, average concentration distribution Cell

Automated Cloning

4 min*

Up to 2 microplates per cycle 96 single cells in ~ 4 min (or 384 in ~ 16 min)
Automated
Temperature control to avoid RNA degradation
Sample will be processed autonomously until all programmed positions have filled with a single cell.

* Time per 96-well plate at optimal cell concentration

Technical information

Dispensing technology: piezo acoustic
Dispensed volume: 50-800 pL per drop
Resolution: 1 μm
Accuracy (Absolute Position): < 10 μm
Accuracy (Repeat Position): < 3 μm
Maximum speed: 100 cm/sec
Tracking area(mm): x=180; y=120 (2 plates)
Dimensions: LxWxH (mm): 740 x 750 x 1580
-> including arm L = 1260 mm
-> with open door H = 1970 mm
Weight: 205 kg
Voltage: 110 V; 220 V

It is equipped with a module that allows the visualization of fluorescent particles. It contains 4 light sources and filters for the visualization of the following fluorophores: DAPI (ex.375nm; em. 432nm); FITC (ex.490nm; em.515nm); Cy3 PE (ex.565nm; em.580nm); Cy5 (ex. 625nm; em.670nm)

Glass capillary with piezoelectric ceramic
Generation of acoustic droplets
50 to 800 pL per drop
Typical operating frequency: 500 Hz
Viscosity up to ~20 mPa. s (eq. at ~50% glycol)
CV 0.2 %.

Technology

(1) The cell suspension is loaded into a glass capillary
(2) The CellenONE allows a perfect control of the generation of drops in the air.
(3) Thanks to the automated imaging inside the glass capillary, the CellenONE determines, whether the next drop will contain a single cell or not
(4) The single cell contained in the drop is dispensed into a well of the plate. Drops without cells or with more than one cell are dispensed into a recycling tube.

The CellenONE is an automated single cell dispensing system based on a patented piezo acoustic technology: It allows precise cell deposition on a wide range of microplates (96, 384, 1536) and microwell substrates. Most dispensing and microfluidic technologies follow Poisson distribution, which leads to multiple cells per position and low efficiency.
The CellenONE uses visual feedback built into the software to ensure that only single cells are dispensed. With the CellenONE, the cell sample is divided into droplets of equal volume.

Each drop can contain :

(A) No cells

(B) 1 cell

The samples are analyzed live and the images are recorded.

The CellenONE allows sorting of cells in suspension, separation of two cell populations or cloning by sorting in multi-well plates. It dispenses only single cell drops into the microplate. Any remaining drops will be dispensed into a collection tube, resulting in no sample waste and the cells can be re-sorted according to another criterion.

Utilisation

The CellenONE allows low volume, high viability and precision single cell isolation. It is mainly used for "sensitive and fragile" cell line development and sequencing.

Isolation of single live cells (live/dead) (Remove dead cells from scRNA-seq libraries)
Isolation of very rare single cells (Isolate only cells of interest)
Cloning of the best expressers (Stimulate clonal recovery and expresser levels)