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Clonal picking of stem cell colonies

Precise isolation of stem cell colonies and colony fractions for biomedical and pharmaceutical research

Stem cells have an outstanding role in the field of regenerative medicine as their high self-renewal and differentiation potential makes them particularly suitable for a wide range of biomedical and pharmaceutical research application.

The discovery that the reprogramming of differentiated cells back to pluripotent stem cells (so-called induced pluripotent stem cells or iPS cells) is possible has given an enormous impulse to stem cell research. The range of applications and research fields is tremendous and the expectations are high.

In research iPS cells are used for the development of cellular disease models and as test systems for the development of new drugs. In addition iPS cells are considered to have therapeutically relevant potential in regenerative medicine for the development of new therapies for cell and tissue degenerative diseases. With the use of iPS a wide variety of tissue types can now be cultivated which can either be used as replacement tissue or for research purposes.

For generation of iPSC the cells are subject to gene editing. Gene editing does not result in a homogeneous cell population however but rather in single cells with different phenotypes due to deviating gene integration.
It is therefore important to observe clonal outgrowth of the single cell pool. To get a clonal cell population single cells are grown into clones and then individual clones are isolated.
The whole process of generating clonal pools of iPS cells is very time-consuming. Using well-established methods only 1 in 100 tissue cells or less are converted into iPS cells.
Hence there is a high demand for automated solutions both for the identification and the targeted isolation of the desired stem cell colonies or clones. The requirements for automation are very high. The target objects must be clearly identified + isolated without any cross-contamination from neighboring clones. Furthermore the isolation + transfer must be as gentle as possible in order to avoid undesired changes or differentiation of the cells.

The ALS CellCelector with its specifically designed scrape process for picking adherent cells and cell colonies is extremely gentle, very specific and therefore ideally suited for the clonal passaging of stem cells, stem cell colonies as well as the isolation of specific parts of a stem cell colony.

Modules for picking of single stem cells, stem cell colonies or partial colonies

The Scrape module is the tool of choice for picking the entire colonies or large parts of it.
Different diameters allow the operator to use the most suitable ScrapeTip for each colony.


scrape tool for adherent stem cell colonies
For the precise isolation of specific colony parts e.g. undifferentiated areas within a stem cell colony as well as for the isolation of single stem cells the Single cell module can be used. This module utilizes a glass capillary which is available in different diameters from 20 µm of up to 220 µm.
single cell tool for picking of single stem cells
For isolation of 3D colonies, like embryoid bodies, spheroids, organoids or hematopoietic stem cell colonies the Semi-solid media module is the perfect tool. Depending on the colony size two different diameters are available.

picking tool for semi-solid media

Applications

  • Clonal picking of newly derived iPS colonies
  • Colony picking after genome editing (CRISPR)
  • Colony splitting with transfer to 2 or 3 destination plates (creation of replica plates)
  • Isolation of differentiated stem cell colonies
  • Scanning and isolation of hematopoietic stem cell colonies from methylcellulose
  • Isolation of single stem cells (single cell cloning or heterogeneity studies)
  • Cleaning up of stem cell cultures by removal of differentiated areas
Isolation of a stem cell colony: before and after picking

Automated isolation of induced pluripotent stem cell (iPS) and embryonic stem cell colonies with the ALS CellCelector

The transfer of stem cell colonies is often a stressful procedure resulting in a great number of dead cells influencing their living neighbors. Using Trypsin or similar enzymatic digestion methods to facilitate the transfer of colonies can have distinct effects on the phenotype of especially freshly reprogrammed stem cells and may result in unintentional differentiation.. Moreover it might lead to the cross-contamination of different colonies with clonality being lost. The incorporation of  a mechanical transfer using manual scraping with pipette tips or cell scrapers is laborious though and hardly fulfills the requirements for a clonal colony transfer. Therefore it is crucial to implement a gentlemechanical transfer method with high specificity maximizing both the viability as well as the clonality of picked colonies while maintaining their pluripotent characteristics.
Automated isolation of iPS colonies
Automated isolation of iPS colonies: the overview image shows the whole scan of the corresponding well (image on the left), before and after picking images provide a documentation of the picking process (middle and right image).

Gentle detachment of adherent cells for high viability while retaining pluripotency

For picking of adherent colonies the ALS CellCelector combines a very gentle, crosswise scrape movement with the simultaneous aspiration of the colony. This gently loosens the colony from the base of the culture plate or feeder cell layer.
scrape movement to detach adherent colonies
Propidium Iodide (PI) staining was used to assess cell survival of manually and automatically picked hESC colonies after the transfer into a new plate.
The images show a lower number of dead cells when isolated automatically compared to manual picking.
PI staining of dead cells
Phase contrast images of hESCs merged with the corresponding fluorescent images of PI staining (scale bar 50 µm).
Left: manually picked colonies, right: automatically picked colonies after transfer.
The morphology of a colony can also provide important information about its current condition. A phase contrast image of a representative colony was taken 3 days (left) and five days (right) after automated passage into a new culture dish. The colony showed normal growth. This shows that stem cell colonies picked automatically with the CellCelector™ show no significant differences to manually picked colonies of the same passage. The growth behavior is also comparable.
phase contrast image of a stem cell colony
Phase contrast image of a representative colony after automated passage into a new culture dish
Left: 3 days after passaging; right: 5 days after passaging
To test how stem cells react to the CellCelector picking process with regards to their state of pluripotency the hESC colonies were stained for the pluripotency associated markers Oct4, SSEA-1, Tra-1-60 and Nanog.

Immunocytochemical expression analysis of typical markers is a reliable method to elucidate the effects of the cell transfer. Particular proteins playing a pivotal role in the maintenance of pluripotency or determination of cell fate can be visualized by fluorescence-labelled antibodies and are easily detected by the imaging software of the CellCelector.

The pluripotency of hESC colonies could be confirmed and the expression levels are comparable to those of conventionally propagated hESCs.
pluripotency of hESC colonies
Immunocytochemical staining for the pluripotency-associated surface marker Tra-1-60 (B), the pluripotency factors Oct4 (C) and Nanog (D) and  SSEA-1, a surface marker for differentiated human cells (E). Primary antibodies were visualized with Alexa-555-coupled secondary antibodies. Cell nuclei were counterstained with Hoechst or DAPI.
(F) Quantification of cells expressing the pluripotency-associated marker Tra-1-60 and the differentiation marker SSEA-1 was done by FACS analysis.

Isolation of undifferentiated stem cell colonies from feeder cell layer

It is a commonly used method to co-cultivate undifferentiated stem cells with feeder cells (mostly fibroblasts) in order to provide an environment that keeps stem cells stable and viable.
However the isolation of stem cells without transferring feeder cells requires refined skills. The ALS CellCelector enables an automatic transfer of stem cell colonies from feeder cells.
hESC on feeder cell: before and after picking
Human embryonic stem cells (hESC) on feeder cells

Clonal picking after gene editing

Genetically modified model organisms are of great importance for the study of biological relationships and the investigation of diseases, as they can be used to examine the function and regulation of genes in the physiological environment. With their help new insights can be gained in basic research, which can be reflected in new forms of therapy.

The first step of the classical method for the generation of transgenic organisms consists of the transfection of embryonic stem cells and the subsequent selection of positive clones. The ALS CellCelector is ideally suited for clonal picking of individual cell colonies by combining sophisticated image documentation from single cell to clone with precise and gentle isolation + transfer of grown clones.

Generation of replica plates

If the size of the colony is big enough a single colony can be automatically picked into two or more replica plates. One destination plate can then be used for cultivation while the other is used for quality control PCR analysis.
generation of replica plates: first pick
generation of replica plates: second pick
generation of replica plates

Isolation of specific parts from stem cell colonies

Due to its powerful optics and imaging software as well as its high precision the ALS CellCelector can also isolate specific areas out of stem cell colonies. Based on the morphological properties and differences of the stem cell colonies the CellCelector can distinguish for example between differentiated and undifferentiated parts of a colony and selectively pick them out.
Isolation of already differentiated parts from an hESC colony: before picking
Isolation of already differentiated parts from an hESC colony: after picking
Isolation of already differentiated parts from an hESC colony with the ALS CellCelector™.
Transfer of specific, undifferentiated parts of a human embryonic stem cell (hESC) colony: before picking
Transfer of specific, undifferentiated parts of a human embryonic stem cell (hESC) colony: after picking
Transfer of specific, undifferentiated parts of a human embryonic stem cell (hESC) colony using the single cell module of the ALS CellCelector™.

Isolation of embryoid bodies (EB) from semi-solid media

Stem cell colonies can be cultivated in 3D using highly viscose media like Methylcellulose or Matrigel to form organoids or embryoid bodies which are interesting in pharmaceutical research and for organ formation studies. The CellCelector with its dedicated picking module for semi-solid media is perfectly suited for the isolation of these structures.
Isolation of embryoid bodies: before picking
Isolation of embryoid bodies: after picking
RELATED PUBLICATIONS




  • Zoldan, K. et al. Automated harvest of induced pluripotent stem cell colonies and colony fractions using the cell separation robot CellCelectorTM  nature methods application notes (2010)  

  • Zoldan, K. et al. Automated isolation of semi-adherent  macrophage-like cells from a fibroblast-contaminated culture using the cell separation robot CellcelectorTM nature methods application notes (2010)  

  • Haupt, S. et al. Automated selection and harvesting of pluripotent stem cell colonies using the CellCelector  Nature Methods (2009)  



  • Planes, E. et al. Life Cell Imaging for Quality Control of Pluripotent Stem Cell Culture Poster ISSCR 2013 Boston
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