SC assays Flashcards
What is a colony formation assay?
You grow cells at clonal density and see if you can grow anything. Colonies with stem cells present are expected to grow the most
What cells are used for colony formation assays?
probably primary cells
What parameters are assesed in colony formation assays?
- parameters assesed:
- number of colonies per input cells
- size of colonies
- difeferentiation
What is the benefit of colony formation assays?
relatively simple and fast . Can take place immediately after tissue dissociation
What can be a downside of colony formation assays?
it can be hard to obtain colonies from stem cells
What is a potential usecase for a colony formation assay?
A clonogenic assay is the most direct quantitative method of measuring human hematopoietic progenitor cellsin vitro Hematopoietic colonies are essentially clones of cells produced by a single progenitor cell. The aim of colony-forming unit (CFU) assays is to define the potential of hematopoietic stem and progenitor cell populations for proliferation and lineage differentiation.
What is a sphere formation assay?
primary cells from certain tissues can be grown in the absence of feeder cells in three dimensional sphere culture
What can you use sphere formation assays for?
- it is believed that only stem cells cam give rise to serial spheres
- sphere formation is an assay that can be performed to obtain an indication of the presence of stem cells in a poorly characterised tissue
What might be the downside of sphere formation assays?
it is impossible to avoid differentiation within the sphere and to control/study molecular signals exchanged between cells within the spheres
What are organoids?
Organoids are tiny, self-organized three-dimensional tissue cultures that are derived from stem cells. Such cultures can be crafted to replicate much of the complexity of an organ, or to express selected aspects of it like producing only certain types of cells.
Why can organoids be particularly appealing?
Most of what we know about embryonic development has been learned by extrapolating to human biology what is observed in mice and other animal models. Now, thanks to organoids, researchers have the possibility of culturing tiny versions of each tissue using human cells.
What does Carla Kim study and what can it be helpful with?
Researchers like Carla Kim, PhD are using organoids to determine the role of stem cells in tissue regeneration, maintenance, and function and to understand how these cells talk to each other. Kim and her group were the first scientists to grow lung organoids that mimic two distinct parts of the lung: the airways and the alveolar sacs where gas exchange occurs. They did it by using a special culture setup that allowed the cells to be in contact with both air and liquid, mimicking the lung environment. Their culture also included helper cells derived from blood vessels to stimulate stem cell growth.
Why would you induce in vitro differentiation assays on stem progenitor cells?
- for some stem progenitor cells it is possible to induce differentiation in vitro in order to:
- evaluate the differentiation potential of distinct cell populations
- evaluate the role of genes/compounds in regulating differentiation along a specific lineage
What do in vitro differentiation assays allow?
- allow retrospective identification of stem/progenitor cells (you mnake them do something and then you can tell what cell it actually was)
- used to see what the tissue you have can actually do and then you can take it to further experiments
- allow detailed analysis of proliferation and differentiation potential
What are some benefits and issues with in vitro differentiation assays?
- very highly controlled
- do not necessarily reflect what happens in vivo