Cell sorting .pptx

Cell sorting is the process through which a particular cell type is separated from others


It is based on its differences in
Size, morphological parameters, viability protein expression

Applications including research, diagnosis, and therapy

1. Addis Ababa University College of Health Sciences Department of Medical Biochemistry Cell s orting & Cell fractionation techniques By Ousman Mohammed (ID no. GSR/7263/15) 1

2. Outline Introduction to cell sorting techniques Functions of cell sorting Preparation of samples for cells sorting C ell sorting techniques Cell fractionation Summary References 2

3. Objectives After completing this session, learners should be able to: D escribe some common cell sorting techniques Explain the functions of cell sorting Differentiate the different types of cell sorting techniques Understand a dvantage and disadvantages of different cell sorting techniques E xplain about cell fractionation steps 3

4. Introduction Cell sorting  is the process through which a particular cell type is separated from others It is based on its differences in S ize , morphological parameters, viability protein expression A pplications including research, diagnosis, and therapy 4

5. Why Cell sorting? The cell is the basic functional unit of all organisms. Therefore, it is important to start with exactly the right cell for performing good cell-biology experiments 5

6. Cont… Capture of circulating tumor cells (CTCs) from blood Isolation of immune cells from peripheral blood Isolation of WBC from tissue Conduct molecular analysis of a single cell type, including RNA expression and epigenetic analysis 6

7. Preparation of samples for cells sorting A successful sort results in good purity and yield of the target population and is dependent on these things : Cell harvesting and preparation Cell staining and fluorochrome choice Proper controls Sample and sorting conditions Sample collection conditions 7

8. Preparation cont… Isolating cells from tissues or adherent cultured Disrupting the extracellular matrix holding the cells together using mechanical force and/or proteolytic enzymes Incomplete dissociation of cell clumps can lead to inefficient labeling of the target cells P rocessing method should not affect cell surface epitopes , as this may negatively impact both cell isolation and downstream functional analyses 8

9. Strategies for cell sorting Positive selection The cell type of interest is targeted by the removal mechanism and retained for downstream analysis T argeting a cell surface marker with a mAb or ligand and directly labeling desired cells for selection Isolated cells are bound by Ab or particles 9

10. Strategies cont… Negative selection Performed by removing several cell types to leave one cell type untouched Involve labeling unwanted cell types for removal with Ab targeting specific cell surface proteins Antibody cocktail target all unwanted cells but not desired cells Protocols are faster and easier E.g. Removing all cells except for T cells or the removal of all cells except for B cells from samples 10

11. Cont… 11

12. Techniques of cell sorting Major cell sorting techniques includes:   Fluorescence activated cell sorting (FACS) Magnetic-activated cell sorting (MACS ) Buoyancy-activated cell sorting (BACS) 12

13. Fluorescence activated cell sorting (FACS) One of the most commonly used methods in cell sorting This method relies on cell suspensions which contain a decent number of fluorescent target cells FACS utilizes flowcytometry to separate cells based on morphological parameters & protein expression Interestingly, these systems are able to isolate even rare and single cells 13

14. FACS cont… Three main components : Fluidics Optics Electronics This involves encapsulating cells into small liquid droplets which are selectively given electric charges and sorted by an external electric field 14

15. How Does FACS Work? Antibodies match with antigens on the surface of targeted cell Targeted cell is labeled with fluorescent marker and mixed into the cell sample One at a time, cells flow through a point of analysis where they are hit by a laser beam Cells are then emptied into containers based on their fluorescence 15

16. FACS cont… Cells are injected into a “flow cell” which is located in the optical path of a light source Cells are made to move (or focused) in single file using liquid pressure through a small (50-300 µm) orifice = hydrodynamic focusing 16

17. Fluorescence activated cell sorting (FACS) 17

18. FACS cont.. 18

19. Advantages and Disadvantages FACS Sort s ingle c ells Sort complex cell types with multiple markers at higher purity The ability to separate cells based on their surface markers as well as size and granularity allow for more in-depth isolations However, Bulky volume, expensive price Complicated operation Annoying sample preparation cell staining (fluorescence- labeling ) 19

20. Magnetic Activated Cell Sorting (MACS ) Based on the attachment of small, inert, supra-magnetic particles to mAbs specific for antigens on the target cell population The cells are separated by magnetic particles through an antibody interaction with surface markers of the targeted cells Cells labelled to these antibody-bead conjugates are then separated via a column containing a ferromagnetic matrix 20

21. How Does MACS work? Magnetic beads coated with Ab or enzymes associated with surface markers of the targeted cells Magnetic beads label the cells with recognizable surface markers The targeted cells attach to the magnetic beads and are magnetized to the column walls while non-targeted cells flow through the sample column Cells isolate between targeted and non-targeted 21

22. Advantages and Disadvantages MACS is 4-6 times faster than  FACS This allows researchers to run a higher quantity of samples in a shorter time On the other hand, MACS can also be extremely harsh on fragile cells The magnetic nature of the process can cause damage to the target cell membrane 22

23. Buoyancy-activated cell sorting (BACS) Is a cell selection process that involves sorting cells with buoyant microbubbles Microbubbles labeled with antibodies specific to the target cells When mixed into the sample, the microbubbles bind to the target cells Due to the augmented buoyancy force , the microbubbles float to the surface , separating the target cells 23

24. BACS cont… The microbubbles are first mixed with the cell sample, and bind to the target cells After binding with the target cells, the microbubbles float to the top. The target cells float with them, leaving behind the non-target cells In the final step, the microbubbles and target cells are removed from the top of the sample 24

25. BACS cont… 25

26. BACS cont… A quick alternative to more traditional cell sorting technology It has proven ability to reduce cell sorting time and is easy to use T akes an average of 10-30 minutes for cell selection Relatively inexpensive 26

27. Single-cell sorting Based on intracellular and extracellular properties Every cell is individually analyzed Enable understanding of cellular properties that may be obscured or non-evident Results in highly enriched cell populations that are more homogeneous than those o btained via bulk sorting methods Microfluidic device with micro-channel valves to trap a single cell in a chamber E.g. FACS 27

28. Bulk cell sorting Highly rely on cell characteristics like size and density Results enriched cell populations that are less homogeneous than those obtained via single cell sorting methods All of the target cells are collected in one sweep E.g. Filtration , centrifugation , and magnetic c ell s orting 28

29. Cell fractionation I s the process used to separate cellular components while preserving individual functions of each component This is a method that was originally used to demonstrate the cellular location of various biochemical processes Other uses of subcellular fractionation is to provide an enriched source of a protein for further purification, and facilitate the diagnosis of various disease states Cell fractionation involves 3 steps: Extraction, Homogenization and Centrifugation 29

30. Cont… Extraction The first step toward isolating sub-cellular structure Suspension should be keep in an appropriate medium C ells or tissues suspend in a solution with an adequate pH and salt content, typically an isotonic solution with a pH of 7.0 30

31. Cell fractionation cont… Homogenization Tissue is typically homogenized in a buffer solution Mechanisms for homogenization include: G rinding , mincing, chopping, pressure changes, osmotic shock, freeze-thawing The samples are then kept cold to prevent enzymatic damage It involves grinding of cells in a suitable medium in the presence of certain enzymes with correct pH, ionic composition, and temperature 31

32. Cell fractionation cont… Purification I s achieved by differential centrifugation results in the sequential separation of organelles according to their density 32

33. Summary Cell sorting is a process to isolate one or more specific cell populations from a heterogeneous mixture of cells They are separated majorly based on differences in cell size , morphology & surface protein expression They have important applications in research and as therapeutics Common methods of cell sorting techniques include FACS , MACS Cell fractionation is the process used to separate cellular components 33

34. References Steven A Soper , Malgorzata A Witek (2020 ). "Cell Separations and Sorting". Anal Chem. 92 (1): 105–131 Zhu B and Murthy SK (2013). Stem cell separation technologies. Curr Opin Chem Eng 2, 3–7. Dirican , Enver Kerem (2012), "Magnetic-Activated Cell Sorting of Human Spermatozoa", Practical Manual of in Vitro Fertilization, Springer New York, pp. 265–272 Chen YW et al. (2017). A three-dimensional model of human lung development and disease from pluripotent stem cells. Nat Cell Biol 19, 542–549. Ma , Z et al.( 2017). "Fluorescence activated cell sorting via a focused traveling surface acoustic beam". Lab on a Chip. 17 (18): 3176–3185. 34

35. THANK YOU! 35