ว ชา Advanced Biochemical Techniques Animal Cell Culture

Transcription

1 ว ชา Advanced Biochemical Techniques Animal Cell Culture Assist. Prof. Thanaset Senawong, Ph.D. Department of Biochemistry, Faculty of Science, Khon Kaen University หน งส ออ างอ ง 1. Davis JM Basic Cell Culture: A Practical Approach. Second Edition. Oxford University Press Inc., New York. 1

2 Content Laboratory for Cell Culture Culture media Animal Cell Culture Basic cell culture technique and the maintenance of cell lines Primary cell culture and the establishment of cell lines Objectives To understand the basic animal cell culture technique Be able to specify the key ingredients of basal medium Be able to tell the difference between primary cultured cells and cell line 2

3 1.The Laboratory Self-contained tissue culture laboratory 3

4 1.1 Major items of equipment and instrumentation Laminar flow hoods -Class II Microbiological Safety Cabinet -HEPA filter (0.3 µm) Incubators -temp. (37 C) -gas phase (5% CO 2 ) -humidity (95%) (water tray) air-techniek-bv/laminar-flow-hoods html hp?companyid=9&cms_id=80 4

5 1.1 Major items of equipment and instrumentation (continued..) Microscope -inverted microscope with phase contrast optics and a photographic facility. Centrifuge 5

6 1.1 Major items of equipment and instrumentation (continued..) Fridges and freezer Miscellaneous small items of equipment -water-bath -balance -ph meter -magnetic stirrer -aspirator jar with a pump 6

7 1.2 Culture plasticware and associated small consumable items Tissue culture plasticware -for growing cells -for handling solutions and cell suspensions -for storage Filters for sterilizing tissue culture solutions -0.2 µm pore size (standard) -0.1 µm pore size (mycoplasma) Pipettes Pipetting aids Miscellaneous small items -pipette cans, hemocytometers, tube racks, ect. 7

8 1.3 General care and maintenance of the tissue culture laboratory Daily check list: -record incubator temperatures -check CO 2 cylinder pressures -top up water-baths -empty pipette soak cylinders -check glassware soak tanks for wash load -draw chloros through aspirator lines -replenish stocks of sterile glassware, solutions,etc. Weekly check list: -wash down all work surfaces (if not done daily) -check under laminar flow cabinet work surfaces for media spills -change water in water-baths -top up humidifier trays in incubators -aspirate condensation from incubator bases -check stocks of routine reagents, plasticware, etc. -empty aspirator jars as necessary (if not done daily) -top up liquid nitrogen in cell freezers (or twice weekly) 8

9 1.3 General care and maintenance of the tissue culture laboratory (continued..) Monthly check list: -check equipment services/safety test due -defrost freezers (as necessary) -calibrate instruments and monitors -strip down and cleanse/sterilize insides of incubators Good practice in the lab: -Clearing away promptly after finishing sterile work -Dealing promptly with used glassware etc. to be soaked -Labelling opened sterile equipment and solutions, for re-use only by the same named individual -Keeping good communal records, e.g. of shared cell or reagent stocks -Checking incubated cells regularly for early signs of contamination -Checking incubators regularly for unused cell cultures and discarding surplus (or contaminated) stocks according to approved 9 safety procedures

10 2.Culture media A complete cell culture medium is composed of two distinct parts: 1) A basal medium that satisfies cellular requirements for nutrients, salts, and ph control. :- A basal medium is composed of simple low molecular weight components. 2) A set of supplements that satisfy other cellular requirements and permit growth of cells in the basal medium. 10

11 2.1 Basal media -have been developed to cover the main types of cells in common use Types of basal media: 1) Eagle s medium and derivatives ----> BME (basal medium Eagle s) ----> EMEM (minimum essential medium with Earl s salts) ----> AMEM (minimum essential medium with alpha modification) ----> DMEM (Dulbecco s modified minimum essential medium) ----> GMEM (minimum essential medium with Glasgow modification) ----> JMEM (minimum essential medium with Joklik s modification) 2) Roswell Park Memorial Institute (RPMI) designed media ----> RPMI 1629, RPMI 1630, RPMI ) Basal media designed for use with serum supplementation ----> Fischer s, Liebovitz, Trowell, and Williams 4) Basal media designed for serum-free formulations ----> CMRL 1060, Ham s F10 and derivatives, TC199 and derivatives, MCDB and derivatives, NCTC, and Waymouth 11

12 2.1.2 Constituents of basal media: 1) Balanced salt solution ----> is composed of a combination of inorganic salts ----> inorganic salts maintain osmotic pressure, buffer the medium at physiological ph, maintain membrane potential and acts as cofactors in enzyme reactions and in cell attachment. ----> Na +, K +, Mg 2+, Ca 2+, Cl -, SO 2-4, PO 3-4 and HCO - 3 2) Buffering systems ----> media need to be buffered to compensate for evolution of CO 2 and the production of lactic acid from the metabolism of carbohydrates ----> media are frequently buffered with bicarbonate which forms a buffering system with dissolved CO 2 produced in the medium by growing cells. ----> cells must be grown in an atmosphere of 5-10% CO 2 to maintain the required optimal ph 12

13 Chemical reactions of the bicarbonate buffering system 13

14 3) ph indicator ----> to facilitate inspection and control of cultures ----> the most widely used indicator is phenol red :cherry red ---- ~ph :yellow ---- ph is too acid :purplish ---- ph is too high 4) Energy sources ----> carbohydrates (mostly glucose) ---- lactate (glycolysis) : galactose, fructose ---- less lactate ----> glutamine ---- oxidation of glutamine yields ATP 5) Amino acids ----> essential amino acids : arginine, cystine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, (cysteine 14 and tyrosine)

15 6) Vitamins ----> necessary for cell growth and multiplication ----> Folic acid, Niacinamide, Riboflavin etc. 7) Hormones and growth factors ----> affect the survival and proliferation of cells. 8) Proteins and peptides ----> supplements 9) Fatty acids and lipids 10) Accessory factors ----> trace elements; iron, zinc, copper, and selenium 11) Antibiotics ----> bacteria ;mixture of penicillin (100 IU/ml) and streptomycin (50 µg/ml) ----> fungi ; Amphotericin B (2.5 µg/ml), Nystatin (25 µg/ml) 15

16 2.2 Serum Enhances the growth of cells ----> contains a cocktail of most of the growth factors. ----> buffers the cell culture system against ph change, presence of heavy metals, proteolytic activity, and endotoxin. Types of serum ----> Calf serum, Bovine serum, Horse serum, Human serum ----> Fetal bovine serum (FBS) is the most widely used serum in the culturing of eukaryotic cells. Constituents of serum ----> growth factors, albumin, transferrin, anti-proteases, attachment factors

17 3. Basic cell culture technique and the maintenance of cell lines 3.1 Sterile technique and contamination control Successful cell culture is absolutely dependent on fastidious aseptic technique. Working within the laminar flow hood. Mouth pipetting is never permissible. Avoid wetting the mouth or lips of bottles, tubes, and flasks. Never pour from a culture dish or flask. Do not overfill culture flasks and dishes. Prevent cellular cross-contamination by: - eliminate aerosols. - never work in the hood with more than one cell line at a time. - use separate bottles of medium for different cell lines. 17

18 3.2 Maintenance of stock cultures Antibiotic-free stock cultures Characterize and verify the cell line identity (yearly) 3.3 Routine culture substrates glass (in vitro means on/in glass) Tissue culture plastic -polystyrene -growth surface is treated by gas-plasma discharge technique :oxidation----> increase negative charge (more hydrophilic) 18

19 3.4 Cryopreservation of cells Cells that are properly frozen (and properly stored) can be kept for extended periods (many years, and perhaps indefinitely). Cryoprotective agents:glycerol or dimethyl sulfoxide (DMSO) added to the resuspension medium (5-10%, v/v) acts: 1) to permeabilize the plasma membrane and allows water to flow out of the cell as cooling occurs 2) to decrease the freezing point so that ice crystals begin to form at about -5 C. For permanent storage, cells must be kept below -130 C. - liquid nitrogen: -196 C (immersed) : -140 C to -180 C (vapour phase) 19

20 4. Primary culture and the establishment of cell lines 4.1 Primary cultures Explant cultures Enzymatic dissociation Explant culture 20

21 4.2 The establishment of cell lines 21