Clonal Stem Cell Picking
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 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.
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.
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.
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.
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).
For picking of adherent colonies the 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.
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.
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 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.
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.
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 CellCelector enables an automatic transfer of stem cell colonies from feeder cells.
Human embryonic stem cells (hESC) on feeder cells.
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 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.
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.
Due to its powerful optics and imaging software as well as its high precision the 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 with the CellCelector.
Transfer of specific, undifferentiated parts of a human embryonic stem cell (hESC) colony using the single cell module of the CellCelector.
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.
Learn how to monitor, maintain and culture stem cells consistently and reliably while retaining high levels of cell health in this webinar.
Learn about an accelerated and streamlined workflow approach for the culture, monitoring and characterization of iPSCs.
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