SUSPENSION CULTURE

1. SUSPENSION CULTURE Dr.K.Geetha, Assistant Professor, Nanotechnology Division/Dept Of ECE, PMIST

2. CONTENTS • What? • Principle? • Types • How? • Pros & Cons • Recents in it

3. WHAT? • Suspension culture is a type of culture in which single cells or small aggregates of cells mul­tiply while suspended in agitated liquid medium. • It is also referred to as cell culture or cell suspen­sion culture. • SIMPLY-Initially by transferring pieces of undifferentiated and friable callus to a liquid medium agitated by a suitable device.

4. ? Friability-improved by-manipulating culture components by repeated cell culture/ by culturing on semi-solid medium.

5. SPINNING CULTURE APPARATUS-specifically designed for suspension cell culture allow for superior gas exchange and permit higher volumes of cells to be cultured.

6. WHY AGITATION? • Facilitates gaseous exchange. • maintains uniform distribution of cell and cell clumps in the medium. • Eliminates nutrient gradients (change in temperature, pressure , concentration) in the nutrient medium and at the surface of the cells. • Eliminates polarity of all cells due to gravity. • Polarity-spatial diff in shape, structure, size, organization of cellular components.

7. PRINCIPLE • Callus proliferates unconditionally. • Difficult to go after its metabolism during growth and development. • To overcome this difficulties, suspension culture (single cell / small aggregates grow under condition) was developed to study about its biochemical pathways & physiology during its growth and development. • IDEAL SUSPENSION CULTURE-Contains only single cells with uniform physiology and biochemical property. • To attain ideal culture-friable callus is used • Ideal condition can be obtained – synchronize the cell division, cell enlargement and differentiation within cell population. • Callus- unorganized mass of parenchymal cells. • 2types-compact/ (Friable callus-Callus without organ regeneration.) • Parenchymal cell-Alive at maturity stage-storage-photosynthesis-found below epidermal tissue.

8. Continued…. • To know the time period for sub-culturing of a particular species the growth measurement in suspension culture is very much needed. • growth measurement- done by counting the cell number under simple microscope-after staining and macerating the small aggregates. • The growth can also be measured by collecting the cell mass and by taking the fresh weight or dry weight of the cell mass i.e., the packed cell volume (PCV) measurement. • TWO METHODS • Mechanical methods=> Sterile explant-grinded by glass homogenizer-homogenate containing intact cells or small masses of cells-used to initiate the culture. • Enzymatic method=> Used for isolation of single cells-uses-pectinases to digest the pectin wall-shaker-initiates the process.

10. GROWTH PHASES OF SUSPENSION CULTURE • Lag phase-cells are ready to divide. • Log / Exponential phase-High rate of cell division. • Linear phase- lesser the cell division , higher the cell elongation. • Deceleration phase- lesser the rate of both cell division and elongation. • Stationary phase-No. of cells & its size remains constant.

11. TYPES OF SUSPENSION CULTURE • Batch culture-biomass growth in batch culture follows the fixed pattern. • Slowly rotating culture-nipple flasks are used-8 nipple like projections-capacity-250ml-flat disc rotates-speed of 1-2 rpm. • Shake culture-simple and effective method-conical flask are used-square plate moves by a circular motion at 60-180 rpm. • Spinning culture-used for large volume up to 10L-rotated in a culture spinner at 120 rpm at an angle of 45degree. • Stirred culture-large scale batch culture-culture vessel not rotated-culture inside vessel continuously dispersed by bubbling sterile air-internal magnetic stirrer revolves at 200-600 rpm-convenient to agitate.

12. Continuous culture-old liquid medium is continuously replaced by the fresh liquid medium to stabilize the physiological stage of the growing-separate port to remove the old medium and inlet for fresh medium. • Chemo stats-cylindrical or circular in shape and posses inlet and outlet pores for aeration and for introduction of and removal of cells and medium cells-stirred by a magnetic stirrer. • 2. Turbid stats-automatic monitoring unit is connected with the culture vessel and such unit adjusts the medium flow in such a way as to maintain the optical density or PH at chosen, present level. {turbidity should be in control- turbidity of a suspension culture medium changes rapidly when cells increase in number due to their steady state growth. The changes in turbidity of the culture medium can be measured by the changes of optical density of the medium}

13. Conical flak-autoclaved nutrient medium. • Transfer 3-4 pieces of callus-spatula-flask. • Sterile flask mouth by heating-cotton plug/aluminium foil-Shaker-80 to 120rpm. • After 7 days-pour-sterilized sieve pore diameter -60µ- 100µ-filtrate has only free cells / small aggregates. • Centrifuge-500 to 1,000 rpm-re suspend the residue in several flask in same amount of fresh medium. • For subculture-repeat the previous step-one-fifth of the residual cells as the inoculum-dispense equally in flasks-shaker. • Haemocytometer-definite number of cells measured accurately. SUSPENSION CULTURE AND PLANT REGENERATION THROUGH EMBRYOGENESIS.

16. RECENTS….. 1.Circulating Tumor Cell-Derived Pre-Clinical Models for Personalized Medicine Main cause of death-development of metastases-inability of current therapies to cure patients at metastatic stages-so Circulating Tumor Cells (CTCs)-good candidates for generating preclinical models, making it possible to follow up the spatial and temporal heterogeneity of tumor tissues-to develop pre-clinical models they have used suspension culture. 2.Biolistic Transformation of Cotton Embryogenic Cell Suspension Cultures Particle bombardment-(gold/tungsten particles)-using helium-driven biolistic device (Bio-Rad PDS1000/He)-used to bombard gold particles coated with plasmid into embryogenic cells-Regeneration of fertile transgenic plants from embryogenic cells is 2months-Advantage of suspension culture-susceptible for cryopreservation and long-term storage.

17. A Refined Culture System for Human Induced Pluripotent Stem Cell-Derived Intestinal Epithelial Organoids

18. NOTE: Each supplemented with 25% WRN CM and requisite ingredients for human organoid culture. MATRIGEL- Gelatinous protein mixture-derived from mouse tumor cells-help to retain stem cells in undifferentiated state. HAPPY CELL ASM- Revolutionizing 3D cell culture- unique, low viscosity & density, reagent that suspends the cells permanently. iPSC-derived from skin or blood cells-reprogrammed back into an embryonic-like pluripotent state-development of an unlimited source of any type of human cell needed for therapeutic purposes. Day 3 Day 6

19. Laser Ablation of the Recipient Area With Platelet-Rich Plasma–Enriched Epidermal Suspension Transplant in Vitiligo Surgery Keratinocyte/melanocyte

20. REFERENCES • Protocol for sub culturing the suspension cell by Culture Collections, Public Health England, Porton Down, Salisbury, SP4 0JG, UK. • https://www.phe-culturecollections.org.uk/media/126320/m236_cell- culture-protocol-subculture-of-suspension-cell-lines.pdf • Cell suspension culture technique-https://nptel.ac.in/courses/102103016/3 • A Refined Culture System for Human Induced Pluripotent Stem Cell-Derived Intestinal Epithelial Organoids-https://www.researchgate.net/figure/Growth-of-Human-iPSC-Derived-Organoids-by-Suspension-Culture-Using-Happy-Cell-ASM-A_fig4_321658270 • Laser Ablation of the Recipient Area With Platelet-Rich Plasma–Enriched Epidermal Suspension Transplant in Vitiligo Surgery- https://www.ncbi.nlm.nih.gov/pubmed/30188329

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