Cell Sorting Techniques

Cell Sorting Techniques
Approches
Applications
Techniques

FACS
MACS

1. “Cell Sorting Techniques” College of Health Sciences School of Medicine Department of Medical Physiology P.by: Habtemariam Mulugeta ID No. GSR/2895/14 1

2. “Cell Sorting Techniques” General Cell Biology 2 Habtemariam M.

3. Outline Objectives Definition Applications of Cell Sorting Preparation of Samples for Cell Separation Cell Separation Approaches General Categories of Cell Sorting Techniques Summary Acknowledgement References Habtemariam M. 3

4. Objectives After completing this session, students should be able to: Describe briefly about Cell Sorting Techniques Differentiate the different types of Cell Sorting Techniques Understand the functions of Cell Sorting Appreciate the differences between Cell Sorting Techniques Familiarize with Cell Sorting and its Techniques Habtemariam M. 4

5. Definition Cell sorting , aka cell isolation or cell separation , 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 . The resulting homologous populations of cells have important applications in research and as therapeutics . Habtemariam M. 5 Rosental et al. 2017

6. Cont. Cell separation can be performed on a variety of complex biological samples, including : Whole peripheral blood Leukapheresis products (e.g .  Leukopaks ) Peripheral blood mononuclear cells (PBMCs) Bone marrow Cord blood Spleen & lymph nodes Other tissues (e.g. skin, liver, lung, fat, brain, tumor , etc.) Habtemariam M. 6

7. Applications of Cell Sorting Capture of circulating tumor cells (CTCs) from blood Isolation of immune cells (T Cells, B Cells, etc.) from peripheral blood Isolation of WBC from tissue. Preparing a sample of blood separated from plasma Separation of pathogenic bacteria from food Conduct molecular analysis of a single cell type , including RNA expression and epigenetic analysis Habtemariam M. 7

8. Cont. Genetically modify and expand a particular cell type of interest for disease modelling or cell therapy research applications (e.g.  T cell therapy research ) Directly use purified cells for adoptive cell transfer experiments in various animal models Increase Sensitivity of Analytical methods (e.g .  cell isolation for HLA analysis ,  cell isolation for FISH analysis ) Study the in vitro effects of drug candidates on specific cell populations (e.g.  hematotoxicity testing ) Fuse enriched plasma cells with myeloma cells to  produce hybridomas Habtemariam M. 8

9. Preparation of Samples for Cell Separation Isolating cells from intact tissues: 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 . Ensure that the processing method does not affect cell surface epitopes , as this may negatively impact both cell isolation and downstream functional analyses . Habtemariam M. 9

10. Cont. There are several enzymes commonly used in tissue dissociation protocols : Collagenase  can hydrolyze collagen and is widely used for isolating cells from animal tissues . Hyaluronidase  is often used in combination with collagenase and catalyzes hydrolysis of 1,4-β-D-glycosidic linkages. DNase  is added to cell suspensions to minimize cell clumping due to DNA released by damaged cells . Elastase  is used to digest tissues containing high amounts of elastin . Trypsin  is a serine protease with a specificity for peptide bonds and is often combined with other enzymes (e.g. elastase and/or collagenase ) for tissue dissociation . Habtemariam M. 10

11. Buffer Suggestions Ca 2+ /Mg 2+ - free buffers: helps to reduce cell aggregation Use BSA (0.1 - 1%) or dialyzed FBS (1 - 5%) Use a minimal amount of BSA to decrease autofluorescence and to increase population resolution. Avoid non-dialyzed FBS , as it facilitates cell-cell adhesion by replacing Ca and Mg . Add EDTA (2 - 5 mM) → helps prevent cell adhesion. Add 10 – 25 mM of HEPES to improve pH stability . Add DNAse I ( 25 – 50 ug/mL ) and 5 mM of MgCl 2 → digests free DNA released by dead cells. Habtemariam M. 11 https://cancer.wisc.edu/research/resources/flow/

12. Single Cell Suspension Filter samples immediately before Sorting . Filters are available in the Flow Lab . When processing tissue samples , pass cells through a 25-gauge needle. Avoid keeping cells at unnecessarily high concentration . Keep cell suspension at 1 – 10 million/mL during processing, depending on cell type . Always check cells under the microscope as they are being prepared to ensure they are in a single cell suspension. Habtemariam M. 12 https://cancer.wisc.edu/research/resources/flow/

13. Dead Cell Discrimination Strongly recommended to use a Dead Cell Exclusion Dye with any cell sorting experiment . It will greatly reduce autofluorescence and lower non-specific baselines , which will result in increased population resolution. Even with careful optimization, tissue preparation protocols can result in some cell death . To ensure that apoptotic cells do not interfere with the experiments , they can be depleted from the sample prior to performing further cell isolation or downstream assays . (e.g. with the  EasySep ™ Dead Cell Removal (Annexin V) Kit ) Habtemariam M. 13 https://fccf.mskcc.org

14. Physical Manipulations Centrifugation: Use minimal speed to sediment cells. A good starting point for most preparations is 300g for 10 minutes . Vortexing : avoid vigorous vortexing Pelleting : do not generate a dry pellet at any time during processing. Air Bubbles: avoid introducing air bubbles . Surface tension forces can kill cells . Habtemariam M. 14 https://fccf.mskcc.org

15. Cont. Temperature: Keep samples on ice , unless otherwise required by a specific protocol. Slowing intracellular metabolism helps cells survive longer outside the incubator. Habtemariam M. 15 https://fccf.mskcc.org

16. Cell Separation Approaches Positive selection, Depletion, and Negative selection. Habtemariam M. 16

17. Positive Selection The cell type of interest is targeted by the removal mechanism and retained for downstream analysis Performed by targeting a cell surface marker (CD4, CD8, etc.) with a monoclonal antibody or ligand and directly labeling desired cells for selection . Antibody cocktail targets a unique surface marker on the target cells. Isolated cells are highly purified. Isolated cells are usually bound by antibodies. Habtemariam M. 17

18. Depletion A single cell type is targeted and removed from a biological sample. For example, the removal of red blood cells from peripheral blood mononuclear cells (PBMCs) is completed via depletion . Habtemariam M. 18

19. Negative Selection Similar to depletion , the negative cell separation approach is when several cell types are removed to leave one cell type untouched . Involve labeling unwanted cell types for removal with antibodies or ligands targeting specific cell surface proteins Antibody cocktail target all unwanted cells and do not target desired cells. Protocols are faster and easier with minimal sample manipulation Isolated cells are not bound by Antibodies or magnetic particles example is the depletion of all cells except for T cells or the removal of all cells except for B cells from samples like whole blood or bone marrow . Habtemariam M. 19

20. Cont. Habtemariam M. 20

21. General Categories of Cell Sorting Techniques Bulk cell sorting Single cell sorting Habtemariam M. 21

22. 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. Example: Filtration ,  Centrifugation , and  Magnetic   Cell   Sorting Habtemariam M. 22

23. Single cell sorting Based on Intracellular and Extracellular Properties . A series of statistical methods designed by data scientists used. 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 obtained via bulk sorting methods . The main Example : Flow Cytometry   or  Fluorescence Activated Cell Sorting Habtemariam M. 23

24. Filtration Filtration requires a membrane with a consistent pore size. The pore is smaller than the target cell , but larger than the unwanted debris . Membranes with a variety of pore sizes are available to select from . Used for rough separation of cells. Habtemariam M. 24

25. Centrifugation Centrifugation is commonly used for initial separation of Blood into Plasma , WBC , and RBCs . Density gradient centrifugation improves the separation process by adding a material with a density between the WBC fraction and the RBC ; this allows for a clean separation between the two . Heavier , or more dense cells fall to the bottom during centrifugation , while the less dense plasma remains at the top . Habtemariam M. 25

26. Cont. Common Applications include: Fractionation of PBMC , Exclusion of Dead Cells from a cell culture , and Separation of plasma from blood cells . Density gradient centrifugation is an inexpensive cell separation technique but has Limited Specificity , Low Purity , and Low Throughput . In addition, even though it is a common laboratory technique , density gradient centrifugation can be a slow and laborious process that is difficult to master . Habtemariam M. 26

27. Cont. There are several types of density gradient media , each with unique properties that render them ideal for different purposes . The following are examples of the most well-known types : Lymphoprep ™ , Lympholyte ® , and Ficoll-Paque ®   are similar media: consist of saccharides and sodium diatrizoate ; have a density of 1.077 g/ mL . commonly used to isolate mononuclear cells from peripheral or cord blood , and bone marrow.  Habtemariam M. 27

28. Cont. Percoll ®   density : 1.131 g/mL consists of colloidal silica particles coated with polyvinylpyrrolidone (PVP) widely used to separate cells , organelles , viruses , and other subcellular particles. OptiPrep ™   is a medium consisting of iodixanol in water used to isolate viruses , organelles , macromolecules , and cells . Habtemariam M. 28

29. Cont. Habtemariam M. 29 These techniques are useful for rough separation of cells ; more specialized techniques are needed for enriching specific cell types based on cell surface markers.

30. Sedimentation works on the basis that gravity will cause larger and denser components to sediment faster than materials that are smaller and less dense . The largest and densest components in a sample can be pelleted through an initial low-force centrifugation due to their high rate of sedimentation. The supernatant can then be spun again . Through successive centrifugations , components with an increasingly lower rate of sedimentation can be isolated. Leukocytes are commonly separated from erythrocytes through dextran sedimentation.  HetaSep ™  is an example of an erythrocyte aggregation agent that is used to separate nucleated cells from RBCs in whole blood. Sedimentation is inexpensive but generally results in lower purity than other methods. Habtemariam M. 30

31. Adhesion The unique adhesion profiles of different cell types can be used to separate target cells from heterogeneous populations . By choosing suitable growth factors and cell culture plates to selectively favor or inhibit adhesion , adherent cells can be separated from cells in suspension. Macrophages are inherently adherent and often isolated from peripheral blood and bone marrow by adhesion. Habtemariam M. 31

32. Cont. Mononuclear cells can be cultured with serum and a differentiation cocktail , promoting the formation of an adherent monolayer of macrophages . After removing the supernatant containing unwanted cells , the macrophages can be isolated . Alternatively, cells that naturally grow in suspension or have lost anchorage dependency can be isolated by culturing the heterogeneous cell population in plates designed for ultra-low attachment . Without a surface to adhere to , adherent cells will fail to survive and the target cells will remain in suspension. Habtemariam M. 32

33. Microfluidic Cell Separation Microfluidics is an umbrella category of cell separation methods .  Designed to manipulate fluids on a microscopic level to facilitate single-cell isolation. M icrofluidic technologies are frequently built onto microchips and are commonly known as " lab-on-a-chip " devices. These devices have several advantages , including The smaller volumes of samples and reagents required for use . They are also portable , making them particularly useful as field-based diagnostic tools . Habtemariam M. 33

34. Cont. Microfluidic methods can be divided into active and passive systems. Active microfluidic systems involve external forces . passive microfluidics make use of the cell’s density and mass in combination with gravity. These methods can also be classified by the presence or absence of cell labeling ; Some Methods involve labeling cells with antibodies , Most Methods are known for being label-free . Habtemariam M. 34

35. Cont. There are several different microfluidic methods used for cell isolation, including: Acoustophoresis : a gentle , non-contact , continuous , and label-free separation method for cells and particles. The ability to separate cells with acoustophoresis depends primarily on differences in the biophysical characteristics of cells/particles Aqueous two phase systems : a liquid–liquid fractionation method. is based on incompatibility of two aqueous solutions such as a polymer/salt system [e.g. polyethylene glycol (PEG) and potassium phosphate], a polymer/polymer system (e.g. PEG/dextran) Habtemariam M. 35

36. Cont. Biomimetic microfluidics Cell affinity chromatography Deterministic lateral displacement Electrophoretic sorting Field flow fractionation Gravity and sedimentation Magnetophoresis Microfiltration Optical sorting: Researchers are using light and new image processing tools for label-free cell characterization Habtemariam M. 36

37. Magnetic Cell sorting techniques MACS (Magnetic Activated Cell Sorting) Superparamagnetic nanoparticles  are made of a core of iron oxide , typically magnetite (Fe3O4), which is not innately magnetic , but becomes magnetized by an applied magnetic field . These particles or beads are coated with silica or a polymer surface to prevent clumping , and a well-chosen coating also provides a rich surface for the covalent attachment of functional groups and antibodies . Provide high degree of specificity to a cell enrichment protocol. Habtemariam M. 37

38. Cont. The attachment of antibodies provides the superparamagnetic particles with specificity . The functionalized particles are incubated with the target cell solution , and the cells with surface antigens complementary to the antibodies will bind to form a cell-bead conjugate . The conjugates are enriched by magnetic cell separation . is a good choice when specificity is desired . is rapid and efficient . Habtemariam M. 38

39. Cont. Advanced Bio-Magnetic Separation Systems can increase sort accuracy by providing standard curves and optical monitoring of the sorting process. Habtemariam M. 39 Advanced Separation Systems are engineered to provide gentle and consistent magnetic forces throughout the working volume to increase cell viability. Specific , Rapid , and Efficient when care is taken to develop and finely tune a sorting strategy .

40. Cont. MACS has several advantages , including: High purity Fast protocols Ease of use Low equipment cost Many cells can be isolated at once Potential for automation High cell viability Habtemariam M. 40

41. Flow Cytometry (Fluorescence-based Cell Sorting) Fluorescence-activated cell sorting (FACS) analyzes each cell individually. Extremely Powerful Technique that can provide a large amount of information at once . Ideal Quantification method for multiplex immunoassays . Uses Flow Cytometry and Fluorescent Probes to sort heterogeneous mixtures of cells . The cells are incubated with fluorophore-labeled antibodie s before the sort . The antibodies are specific to surface antigens on target cells . Each antibody has a different emission wavelength and is uniquely identifiable . One method of precisely labeling antibodies with fluorophores is on-bead labeling with Protein A. Habtemariam M. 41

42. Cont. After incubation with the labeled antibodies , the cell solution is sent through the flow cytometer. This machine guides the solution through a micron-sized nozzle one cell at a time . Each cell moves through a laser excitation area , where the laser excites the fluorophores bound to the cell surface. The fluorescent emission is recorded , and the cell is directed either into a collection container or a discard container according to user-defined parameters . Habtemariam M. 42

43. Cont. Multiple cell types can be enriched in a single run , and Quantitative Information  about cell numbers and percent of total population are simultaneously recorded. Highly Informative Method , and is the most precise cell sorting technique , but it is also Very Expensive . The machine itself is often prohibitively expensive , and requires a trained operator . For this reason many laboratories are turning back toward magnetic sorting techniques . Habtemariam M. 43

44. Cont. FACS has several Advantages over MACS including the ability to : Sort Single Cells Isolate cells based on Intracellular Markers (e.g. GFP) Isolate cells based on Surface Marker Expression Levels Sort complex cell types with multiple markers at higher purity. Habtemariam M. 44

45. Other Cell Separation Techniques less commonly used, cell separation methods Aptamer Technology Aptamers are single-stranded RNA or DNA oligonucleotides that form structures that can bind to highly specific targets. Through Systematic Evolution of Ligands By Exponential Enrichment (SELEX) Technology , aptamers can be screened and synthesized to target any cell type . These aptamers have high affinity and specificity toward their targets , and can be labeled with fluorochromes or magnetic particles to facilitate cell separation. The main advantage of aptamers is that they lack immunogenicity . Fluorophore-labeled aptamers have been used to sort mesenchymal stem cells  from bone marrow RNA aptamers have been used to isolate mouse embryonic stem cells Habtemariam M. 45

46. Buoyancy-Activated Cell Sorting Utilizes glass 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 . Habtemariam M. 46

47. Complement Depletion The complement depletion method takes advantage of the proteolytic cascade initiated by the complement system of the immune system . The complement system consists of plasma proteins that can be activated by pathogens or antibodies. Once activated , the plasma proteins induce the formation of a membrane-attack complex on a cell , resulting in cell lysis . With specific monoclonal antibodies , any cell population can be targeted and lysed through the complement cascade . Habtemariam M. 47

48. Laser Capture Microdissection (LCM) LCM is a technique that uses a narrow laser beam to cleave target cells or areas from mostly solid tissue samples . Through microscopic visualization , LCM can isolate cell populations from heterogeneous mixtures using: Cell Morphology or Specific Histological & Immunological Staining . LCM is particularly useful when working with small sample sizes . Habtemariam M. 48

49. Immunoguided Laser Capture Microdissection Immunoguided LCM combines Immunostaining with laser capture microdissection . This allows Immunophenotypes to be used, in addition to morphology and tissue location , to identify and isolate target cells from the tissue sample . This technique employs Immunohistochemistry or Immunofluorescence to guide the dissection process for isolating cells expressing a specific molecular marker . It is particularly useful when histological stains do not recognize certain cell populations . Habtemariam M. 49

50. Limiting Dilution Limiting dilution involves isolating single cells through the dilution of a cell suspension. This technique can be carried out with standard pipetting tools and is commonly used to produce monoclonal cell cultures and single cell cultures for single-cell analysis. Habtemariam M. 50

51. Micromanipulation Micromanipulation , a form of manual cell picking . It is a cell isolation technique involving the use of an inverted microscope and ultra-thin glass capillaries connected to an aspiration and release unit . The system moves through motorized mechanical stages , allowing the operator to carefully select a specific cell and apply suction via micropipette to aspirate and isolate the cell . Habtemariam M. 51

52. How do you choose the best cell separation method for your research? Depending on the intended downstream application for the isolated cells , scientists consider: Performance (i.e. purity and recovery) of the cell separation method Efficiency of the cell separation method, Viability and Function of the isolated cells Habtemariam M. 52

53. Performance The key measures of performance for cell separation methods are typically purity and recovery Purity   refers to the proportion of desired cells in the final isolated cell fraction , and is generally expressed as a percentage of total live cells . It is most commonly measured using flow cytometry . It indicates whether the final isolated cell population can sufficiently represent the characteristics of that particular cell type without the interfering effects of other cell types . Recovery   answers the question : O ut of all the desired cells you can possibly obtain from your sample, how many are you actually able to isolate? How many of your desired cells have you lost through your cell separation method ? Habtemariam M. 53

54. Viability and Function When researchers need live, purified cells for downstream cell culture and other applications Viability  can be expressed as the percentage of total cells in the isolated sample that are live . Function   of the cells you isolate should be preserved throughout the cell separation process to ensure that your downstream assays accurately represent the physiological function of your cell type of interest . Habtemariam M. 54

55. Efficiency All Important Variables to consider for maximizing the efficiency of your cell isolation. Throughput, Speed, Ease-of-use Automation Habtemariam M. 55

56. Cont. Throughput  refers to the rate at which cell separations can be completed in terms of sample volume, number of cells, or number of samples . If you’re working with large sample volumes or multiple samples at a time, you will want to consider which cell separation technology can support your desired throughput. Speed  refers to the amount of time it takes to complete the cell isolation procedure. Faster cell separation protocols are desirable if you need to increase your throughput and accomplish more with your time in the lab. Some of the fastest cell isolation kits can  isolate highly purified cells in as little as 8 minutes . ( EasySep ™ ) Habtemariam M. 56

57. Cont. Ease-of-use  contributes to the reliability and reproducibility of a cell separation method. Simple protocols are key to reducing user-caused errors and variability . Automation  can reduce variability , limit the amount of hands-on-time required , and allow you to do more with your time in the lab.  Automated cell separation instruments  also reduce the risk of exposure to dangerous pathogens when working with potentially  infectious samples . Habtemariam M. 57

58. 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. Cell Separation Approaches: Positive , Depilation , Negative . General Categories of Cell Sorting Techniques : Single and Bulk . Centrifuges, FACS, MACS Habtemariam M. 58

59. Acknowledgement Firstly, I would like thanks Our Lord and Savior Jesus Christ Son of the true Living God, Son of Theotokos. Next m y deepest gratitude goes to my instructor Dr. Sisay who gave me this chance to prepare and present on “Cell Sorting Techniques.” Finally , I would like to thank my classmates for their constructive comments. Habtemariam M. 59

60. References Rosental , Benyamin; Kozhekbaeva , Zhanna ; Fernhoff , Nathaniel; Tsai, Jonathan M.; Traylor-Knowles, Nikki (Dec 2017). "Coral cell separation and isolation by fluorescence-activated cell sorting (FACS)" (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5575905). BMC Cell Biology. 18 (1): 30. doi:10.1186/s12860-017-0146-8 (https://doi.org/10.1186%2Fs12860-017-0146-8). ISSN 1471-2121 (https://www.worldcat.org/issn/1471-2121). PMC 5575905 (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5575905). PMID 28851289 ( https://pubmed.ncbi.nlm.nih.gov/28851289 ). Chaffey N. Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K. and Walter, P. Molecular biology of the cell. 4th edn . Colter DC et al. (2000)  Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow.  Proc Natl Acad Sci U S A 97(7):3213-8. Dalili A et al. (2018)  A review of sorting, separation and isolation of cells and microbeads for biomedical applications: microfluidic approaches.  Analyst 144(1):87-113. Guo KT et al. (2009)  A new technique for the isolation and surface immobilization of mesenchymal stem cells from whole bone marrow using high‐specific DNA aptamers.  Stem Cells 24(10):2220-31. Gross A et al. (2015)  Technologies for single cell isolation.  Int J Mol Sci 16(8): 16897-919. https://cancer.wisc.edu/research/resources/flow/ https://fccf.mskcc.org Habtemariam M. 60

61. Thank You Habtemariam M. 61