Cell culture

types of cell culture and technology

1. INTRODUCTION ABOUT CELL CULTURE in-vitro culture (maintain and/or proliferate) of cells ,tissue or organs Types of tissue culture: Organ culture Tissue culture Cell culture Histotypic Culture Organotypic Culture Primary culture Cell line 04/10/2018 1 KMCH COLLEGE OF PHARMACY,DEPT OF PHARMACOLOGY

2. TYPES OF CELL CULTURE PRIMARY CULTURE Adherent culture Suspension culture SECONDARY CULTURE CELL LINES Finite cell culture Continous cell culture 04/10/2018 KMCH COLLEGE OF PHARMACY,DEPT OF PHARMACOLOGY 2

3. ORGAN CULTURE The entire embryos or organs are excised from the body and culture Advantages Normal physiological functions are maintained Cells remain fully differentiated Disadvantages Scale up is not recommended Growth is alow 04/10/2018 3 KMCH COLLEGE OF PHARMACY,DEPT OF PHARMACOLOGY

4. TISSUE CULTURE Fragments of excised tissue are grown in culture media Advantages Some normal function may be maintained Better than organ culture for scale up but not ideal Disadvantages Original organization of tissue is lost 04/10/2018 4 KMCH COLLEGE OF PHARMACY,DEPT OF PHARMACOLOGY

5. CELL CULTURE Tissue from an explant is dispersed, mostly enzymatically , into a cell suspension which may then be cultured as a monolayer or suspension culture Advantages Development of a cell line over several generations Scale-up is possible Disadvantages Cells may lose some differentiated characteristics 04/10/2018 5 KMCH COLLEGE OF PHARMACY,DEPT OF PHARMACOLOGY

6. HISTOTYPIC CULTURE The culturing of the cells for their reaggregation to form a tissue-like structure represents histotypic culture 04/10/2018 6 KMCH COLLEGE OF PHARMACY,DEPT OF PHARMACOLOGY

7. ORGANO TYPIC CULTURE This culture techniques involves the recombination of different cell types to form a more defined tissue or an organ 04/10/2018 7 KMCH COLLEGE OF PHARMACY,DEPT OF PHARMACOLOGY

8. PRIMARY CULTURE The culture produced by the freshly isolated cells or tissue taken from an organism is the primary culture. These cells are heterogenous and slow gaining and represents the tissue of their origin with regard to their properties Isolation of tissues-Mechanical & Enzymatic Mechanical methods- sieving, syringing, vigorous pipetting Enzymatic methods- warm trypsin , cold trypsin & collagenase treatment 04/10/2018 8 KMCH COLLEGE OF PHARMACY,DEPT OF PHARMACOLOGY

9. TYPES OF ORGANS IN PRIMARY CELL CULTURE Mouse embryos Chick embryos Human biopsy materials Transplantable animal Tumour Chick embryo organ Rudiments (brain, heart, lungs, liver, gizzard, kidney, spinal cord, skin, muscle) 04/10/2018 9 KMCH COLLEGE OF PHARMACY,DEPT OF PHARMACOLOGY

10. ISOLATION OF TISSUES Must comply with local legislation and medical ethical rules. Sterilize the site with 70% alcohol. Remove tissue aseptically. Transfer to the laboratory in transport medium If delay in transporting to lab, keep at 4⁰C for up to 72 hour. 04/10/2018 10 KMCH COLLEGE OF PHARMACY,DEPT OF PHARMACOLOGY

11. PRIMARY CULTURE Is a stage of the culture after first isolation of the cells but before the first subculture. 4 stages: 1) acquisition of samples, 2) isolation of tissues, 3) disaggregation, 4) culture seeding into culture vessel. After isolation, a primary cell culture is obtained by allowing cells to migrate out from the fragment of tissue adhering to a suitable substrate by disaggregation. PRIMARY EXPLANT Suitable for small amount of tissues example skin biopsy. Attachment on the substrate by using plasma clots, or fibrinogen and trombin . Disaggregation by mechanical and enzymatic. 04/10/2018 11 KMCH COLLEGE OF PHARMACY,DEPT OF PHARMACOLOGY

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14. Enzymatic disaggregation Warm trypsin , 37˚C for 30 mins , cell damaged if too long exposure. Cold pre exposure, soak at 4C overnight and 37C for less 30 mins . Advantage: higher yield of viable cells, preserve more cell types Other enzyme - collagenase benefit for connective tissues and muscle (fibrous tissue) dipase,DNase,hyaluronidase Mechanical disaggregation (prevent proteolytic damage) Scrapping Spillage Sieving Syringes Trituration by pipette 04/10/2018 14 KMCH COLLEGE OF PHARMACY,DEPT OF PHARMACOLOGY

15. SECONDARY CULTURE Derived from a primary cell culture. Isolated by selection or cloning. Becoming a more homogeneous cell population. Finite life span in vitro. Retain differentiated phenotype. Mainly anchorage dependant. Exhibit contact inhibition. 04/10/2018 KMCH COLLEGE OF PHARMACY,DEPT OF PHARMACOLOGY 15

16. CONTINUOUS CULTURES Derived from a primary or secondary culture Immortalised : Spontaneously (e.g.: spontaneous genetic mutation) By transformation vectors (e.g.: viruses &/or plasmids) Serially propagated in culture showing an increased growth rate Homogeneous cell population Loss of anchorage dependency and contact inhibition Infinite life span in vitro 04/10/2018 KMCH COLLEGE OF PHARMACY,DEPT OF PHARMACOLOGY 16

17. 04/10/2018 KMCH COLLEGE OF PHARMACY,DEPT OF PHARMACOLOGY 17 Genetically unstable Characteristics of continous cell lines -smaller, more rounded, less adherent with a higher nucleus /cytoplasm ratio -Fast growth and have an euploid chromosome number -reduced serum and anchorage dependence and grow more in suspension conditions -ability to grow up to higher cell density -different in phenotypes from donor tissue -stop expressing tissue specific genes

18. CELL LINE The sub culturing of the primary culture gives rise to cell lines. the term continuous cell lines implies the indefinite growth of the cells in the subsequent subculturing . Finite cell lines represent the death of cells after several subcultures. 04/10/2018 18 KMCH COLLEGE OF PHARMACY,DEPT OF PHARMACOLOGY

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20. ESTABLISHED CELL LINE CULTURE 04/10/2018 20 KMCH COLLEGE OF PHARMACY,DEPT OF PHARMACOLOGY

21. Subculturing Subculturing Subculturing or "splitting cells," is required to periodically provide fresh nutrients and growing space for continuously growing cell lines. The frequency of subculture and the split ratio, or density of cells plated depend on the characteristics of each cell line being carried. Subculturing - Adherent Cells Suspension culture. 04/10/2018 KMCH COLLEGE OF PHARMACY,DEPT OF PHARMACOLOGY 21

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24. Physical Environment for Culture: Physical Environment for Culture the aim is to provide an environment that mimics, to the greatest extent possible, the in vivo environment of that specific cell type. The cell culture incubator, the culture dish or apparatus, and the medium together create this environment in vitro. Provides an appropriate temperature, pH, oxygen, and CO 2 supply, surface for cell attachment, nutrient and vitamin supply, protection from toxic agents, the hormones and growth factors that control the cell's state of growth and differentiation. 04/10/2018 24 KMCH COLLEGE OF PHARMACY,DEPT OF PHARMACOLOGY

25. CELL CULTURE MEDIA Cell culture media generally comprise an appropriate source of energy and compounds which regulate the cell cycle A typical culture medium is composed of; Amino acids Vitamins Inorganic salts Glucose and serum 04/10/2018 25 KMCH COLLEGE OF PHARMACY,DEPT OF PHARMACOLOGY

26. MEDIA Media Support survival and growth Natural Media Clots -Plasma clots (male fowl) Biological Fluids- Amniotic, Ascitic fluid, serum Tissue extracts- Chicken and bovine embryo extract Artificial Media Defined & Complex media Immediate Survival Prolonged Survival Indefinite growth Specialized function Classes Serum containing media Serum free media Chemically defined Media Protein free media 04/10/2018 26 KMCH COLLEGE OF PHARMACY,DEPT OF PHARMACOLOGY

27. Serum Containing Media: Serum Containing Media MEM, DMEM, M199, F12, DMEM/F12,etc EMEM with 5-20% serum Provide plasma protein, peptides, lipids carbohydrates, minerals and enzyme Hormones (cortisone, insulin and testosterone and prostaglandin) Growth factors (PDGF, TGF-p, epidermal growth factors etc) Supply protein ( fibronectin ) spreading factor Binding factors (albumin, transferrin ) Increase viscosity of medium Protease inhibitor Buffer Minerals (Na, K, Fe, Zn, and Cu etc) 04/10/2018 27 KMCH COLLEGE OF PHARMACY,DEPT OF PHARMACOLOGY

28. Disadvantages of using serum media: Serum may inhibit growth of some cell types, e.g., epidermal keratinocytes . Serum may contain some cytotoxic or potentially cytotoxic constituents . For example, foetal calf serum contains the enzyme polyamine oxidase which converts polyamines like spermidine and sperrnine (secreted by fast growing cells) into cytotoxic polyaminoaldehydes There is a large variation in serum quality from one batch to another this requires costly and time consuming testing every time a new batch has to be used . Some growth factors may be inadequate for specific cell types and may need supplementation . It interferes with downstream processing when cell cultures are used for production of biochemicals . The supply of serum is always lower than its demand. 04/10/2018 28 KMCH COLLEGE OF PHARMACY,DEPT OF PHARMACOLOGY

29. Serum Free Media: Analytical approach Synthetic approach Limiting factor approach Defined Media EMEM, DME, Ham,s F12, CMRL 1066, RPMI 1640, Iscove,s modified Dulbecco,s medium 04/10/2018 29 KMCH COLLEGE OF PHARMACY,DEPT OF PHARMACOLOGY

30. Serum-Free Media -advantages: Improved reproducibility of results from different laboratories and over time since variation due to batch change of serum is avoided. Easier downstream processing of products from cultured cells. Toxic effects of serum are avoided. Bioassays are free from interference due to serum proteins. There is no danger of degradation of sensitive protein by serum proteases. They permit selective culture of differentiated and producing cell types from the heterogenous cultures. 04/10/2018 30 KMCH COLLEGE OF PHARMACY,DEPT OF PHARMACOLOGY

31. Serum-Free Media - disadvantages: 1. Most serum-free media are specific to one cell type . Therefore, different media may be required for different cell lines. 2. Reliable serum-free preparations, for most of the media formulations are not available commercially. This necessitates time consuming task of preparing the desired formulations in the laboratory. 3. A greater control of pH , temperature etc. is necessary as compared to that with serum containing media. 4. Growth rate and the maximum cell density attained are lower than those with serum containing media. 5. Cells tend to become fragile during prolonged agitated cultures unless biopolymers or synthetic polymers are added. 04/10/2018 31 KMCH COLLEGE OF PHARMACY,DEPT OF PHARMACOLOGY

32. Preparation and Sterilization of Medium The various media constituents and other reagents used in cell cultures must be carefully sterilized either by autoclaving or by filtration. Heat stable constituents tike water, salts, supplements like peptone or tryptose etc. are autoclaved at 121°C for 20 min. heat labile constituents like serum, trypsin , proteins, growth factors etc. must be sterilized by filtration through a 0.2 mm porosity membrane filter. Each filtrate should be tested for sterility to avoid failure due to contamination. In case of soda glass, caps should be left slack to avoid breaking during autoclaving. Autoclaving is preferred to filtration since it is cheaper, needs less labour and is uniformly effective. Therefore wherever possible, autoclaving should be resorted to and autoclavable versions of the media should be used. Most of the media, however, now available commercially are usually pre sterilized. . 04/10/2018 32 KMCH COLLEGE OF PHARMACY,DEPT OF PHARMACOLOGY

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35. References: Text book of biotechnology, Dr.U.Satyanarayana , pg.no 415-437 https://www.slideshare.net/nadiamohdkp/types-of-cell-culture www.biotechnology4u.com/animal_biotechnology_types_cell_cultures.html https://microbeonline.com  04/10/2018 35 KMCH COLLEGE OF PHARMACY,DEPT OF PHARMACOLOGY

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