The interactions between fibers and cells in vitro have been studied over the past 30 years in an

Transcription

1 How to do it: in vitro cell studies. Armanda Pugnaloni

2 Cell cultures as in vitro research models in studies on cytotoxicity of Asbestos Mineral Fibers The interactions between fibers and cells in vitro have been studied over the past 30 years in an effort to understand the mechanisms of cell injury and lung disease. In vitro assays have elucidated physicochemical features of asbestos important in induction of toxicity, proliferation, and morphologic transformation of cells in vitro. The mechanisms of asbestos-induced cytotoxicity were first explored in red blood cells and later in cell cultures and tissues from the respiratory tract maintained in culture.

3 Cell lines in asbestos toxicity assays Priorization on body entrance/barrier organs: lung, intestin, skin

4 Schematic presentation of the structural organisation of the human respiratory immune system. The upper respiratory epithelium, lining the inner surface of the trachea, bronchi and bronchioles, is composed of a pseudostratified layer of ciliated cells, mucus-producing cells and secretory cells, and is responsible for the elimination of potential pathogens and foreign particles with the action of the mucociliary escalator. The distal regions of the lung epithelium, the alveolar septa, represent the site of the gas exchange. In both regions, macrophages are located at the apical side of the epithelial l layer and protect it from the inhaled antigen cells by phagocytosis. Dendritic cells will capture antigens, process and present antigen peptide to naive T cells, and trigger their differentiation into antigen- specific effector T cells. Fytianos, Drasler, Blank et al., Nanomedicine (Lond.) 2016

5 Asbestos-induced mesothelial carcinogenesis. Mesothelial l cells actively engulf asbestos st s fibers and the cells do not die thereafter. Shinya Toyokuni (2014) Iron overload as a major targetable pathogenesis of asbestos-induced mesothelial carcinogenesis, Redox Report, 19:1.

6 Human intestinal epithelial cell lines The human colon adenocarcinoma cell line Caco-2

7 To evaluate and compare the pathogenic potential of synthetic and naturally occurring fibers carefully mineralogically characterized preparations of fibers and particles target t cells of the lung: - alveolar epithelial cells, - tracheobronchial epithelial cells, - lung fibroblasts, - mesothelial cells, - macrophages

8 IN VITRO MODELS DISADVANTAGES ADVANTAGES Simplified f systems compared - quick, economy, reproducibility to an integrated body - control of experimental variables Conditions for exposure to - assessment of related reports substances other than in vivo -identification of critical The substances administered parameters for the adverse effects can interact with the culture -study of cellular and biochemical medium mechanisms based on biological i l effects -preliminary screening of new materials

9 Routine testing Reproducible, high-throughput opportunities.

10 Cell Culture Systems Cells may be loosely divided into two main types: 1- Suspension cell culture (Anchorage-independent) derived from cells which can divide and survive without being attached to a substrate, e.g.. cells of haemopoietic lineage magnetic stir plate. Can be maintained in culture vessels that are not tissue- culture treated, requires agitation for adequate gas exchange Easier to passage platform shaker

11 Cell Culture Systems 2- Adherent cell culture Anchorage-dependent must adhere to a surface to survive form monolayers cells derived from different tissue (breast, liver) growth is limited by surface area will cease proliferating once they become confluent (completely cover the surface of cell culture vessel) cells are dissociated enzymatically or mechanically from surface

12 Classes of Culture Cells Cultures of animal cells are usually 1. Primary cells 2. Cell lines

13 Cell Types & Culture Characteristics Primary Cultures Primary cultures are derived directly from excised, normal animal tissue and cultures either as an explant culture or following dissociation into a single cell suspension by enzyme digestion. ADVANTAGES The characteristics of In vivo cells DISADVANTAGES finished divisions number (cycles) senescenza Heterogeneous populations Complex isolation

14 Cell Lines Continuous Continuous Cultures Are comprised of a single cell type that can be serially yp propagated p in culture indefinitely. They undergo a transformation process that makes them "immortal (able to divide indefinitely) are called a cell line derived from tumors transformed spontaneously in culture by mutations. Viral infection with SV40 T

15 Culture Morphology HeLa epithelial The form taken by a cell line reflects the tissue from which h it was derived d BAE-1 endothelial e.g.. cell lines derived from blood (leukaemia leukaemia, lymphoma) tend to grow in suspension whereas cells derived d from solid tissue (lungs, kidney) tend to grow as monolayers. Attached cell lines can be classified as endothelial such as BAE-1, MRC-5 fibroblast epithelial such as HeLa, neuronal such as SH-SY5Y, SY5Y, or fibroblasts such as MRC-5 and their morphology reflects the area within the tissue of origin. SH-SY5Y neuronal

16 Phases of Cell Growth A typical growth curve for cultured cells displays a sigmoid pattern of proliferation. The growth phases associated with normal cells are defined as: 1. Lag Phase at this stage the cells do not divide. During this period the cells adapt to the culture conditions and the length of this phase will depend upon the growth phase of the cell line at the time of subculture and also the seeding density. 2. Logarithmic (Log) Growth Phase cells actively proliferate and an exponential increase in cell density Note: Different cell lines have different timescales for each arises. The cell population is considered to be the phase, this graph is provided as a general example of a typical most viable at this phase. Each cell line will show growth curve. different cell proliferation kinetics during the log 3. Plateau (or Stationary) Phase cellular proliferation phase and it is therefore the optimal phase for slows down due to the cell population becoming determining i the population doubling time. confluent. It is at this stage the number of cells in the Cells are also generally passaged at late log phase. active cell cycle drops to 0-10% and the cells are most Passaging cells too late, can lead to overcrowding, susceptible to injury. apoptosis and senescence. 4. Decline Phase cell death predominates in this phase and there is a reduction in the number of viable cells. Cell death is not due to the reduction in nutrient supplements but the natural path of the cellular cycle.

17 In Vitro Age of a Cell Culture Two terms are predominantly used to define the age of a cell culture: (i) passage number - indicates the number of times the cell line has been subcultured. (ii) the population doubling (pd pd) number - indicates the number of cell generations the cell line has undergone i.e. the number of times the cell population has doubled.

18

19 Basic Requirements For Successful Cell Culture 1. The first necessity is a well-established established and properly equipped cell culture facility. 2. All facilities should be equipped with the following: A certified biological safety cabinet protects both the cells in culture and the worker from biological l contaminants A centrifuge, preferably capable of refrigeration A microscope for examination of cell cultures and for counting cells A humidified incubator set at 37 C with 5% CO 2 in air A 37 C water bath filled with water containing inhibitors of bacterial and fungal growth can also be useful if warming of media prior to use is desired

20 Aseptic Technique Work with cells in a biological safety cabinet laminar flow hood prevent airborne organisms from entering your cultures always use ETOH to clean hood before and after use UV lights can be turned on after the work is finished to sterilize surfaces.

21 Cabinets may be ducted to atmosphere or re-circulated through a second HEPA filter before passing to atmosphere. Microbiological Safety Cabinets A microbiological safety cabinet is probably the most important piece of equipment for cell culture since, when operated correctly, it will provide a clean working environment for the product, whilst protecting the operator from aerosols. In the laminar flow hood operator and/or product protection is provided through the use of HEPA (high efficiency particulate air) filters. The level of containment provided varies according to the class of cabinet used.

22 Incubators Cell cultures require a strictly controlled environment in which to grow. Specialist incubators are used routinely to provide the correct growth conditions, such as temperature, degree of humidity and CO 2 levels in a controlled and stable manner. Generally, they can be set to run at temperatures in the range of 37 o C and set to provide CO 2 at the required level 5% Flasks and capsules with media and cultured cells CO 2 incubator Some incubators also have the facility to control the O 2 levels. Copper-coated incubators are also now available. These are reported to reduce the risk of microbial contamination within the incubator due to the microbial inhibitory activity of copper. The inclusion of a bactericidal agent in the incubator water trays will also reduce the risk of bacterial and fungal growth. However, there is no substitute for regular cleaning.

23 The Cell Environment cultured cells require a sterile environment and a supply of nutrients for growth. Growth media Cells require a medium on which to grow. This medium will contain the nutrients that will provide the cell with the energy and building materials they need to grow and divide.

24 The culture environment should be stable in terms of ph and temperature. Over the last 30 years various defined basal media types have been developed, modified and enriched with amino acids, vitamins, fatty acids and lipids. Basic Constituents of Media Inorganic salts Carbohydrates Amino Acids Vitamins Fatty acids and lipids Proteins and peptides Serum Trace Elements Roswell Park Memorial Institute Media are supplemented with: 10% foetal bovine serum, 1% non-essential amino acids, 2mML-glutamine and a mixture of standard antibiotics: 100U/ml penicillin and 100U/ml streptomycin

25 Serum Serum is a complex mix of albumins, growth factors and growth inhibitors is probably one of the most important components of cell culture medium. The most commonly used serum is foetal bovine serum (FBS). Other types of serum are available including newborn calf serum and horse serum. The quality, type and concentration of serum can all affect the growth of cells.

26 Buffering in Cell Culture Most cells require ph conditions in the range and close control of ph is essential for optimum culture conditions. Most commercial culture media include phenol red as a ph indicator so that the ph status of the medium is constantly indicated by the colour. Usually the culture medium should be changed/replenished if the colour turns yellow (acid) or purple (alkali). Generally in the cell culture medium ph indicator, commonly phenol red is used to analyze the ph of environment in which cells are growing Phenol red is: yellow in acidic medium (ph 68) 6.8), ph 8.0 tomato red at neutral ph (7.0), red at an alkaline ph (7.4) and finally purple at high h ph ph 7.2 The presence of an infectious agent sometimes can be detected t d by turbidity and a sharp change in the ph of the medium (usually indicated by a change in the color of the medium), and/or cell culture death. ph 6.5

27 Culture Vessels Culture vessels provide a contamination ti barrier to protect t the cultures from the external environment while maintaining n ng the proper internal environment For anchorage-dependent cells, the vessels provide a suitable and consistent surface for cell attachment Other characteristics of vessels include easy access to the cultures and optically clear viewing surfaces

28 Sterility Plasticware and Consumables Almost every type of cell culture vessel, together with support consumables such as tubes and pipettes, pp are commercially available as single use, sterile packs. The use of such plasticware is more cost effective than recycling glassware, enables a higher level of quality assurance and removes the need for validation of cleaning and sterilisation procedures. Plastic tissue culture flasks are usually treated to provide a hydrophilic h surface to facilitate attachment of anchorage dependent cells.

29 Culture Vessels Flasks Plastic flasks are available with a range of growing g areas, a variety of shapes, with several different neck designs Flasks surfaces are specially treated t for growing anchorage-dependent cells Flasks are commonly used to carry and expand cells. Either vented or non-vented tops.

30 Adherent Cells Multiwell plates offer significant savings in space, media, and reagents Multi-well plates 6, 12, 24, 96, 384 wells Allow for multiple replicates of experiments effectively Different Growth Areas for each size

31 Adherent Cells

32 Cultures of cells growing in adhesion Adhering cells grow to occupy the entire available surface: at this stage they are confluent. At confluence, growth stops and cells are detached and transferred to new plates. A) Transfer involves the use of EDTA (Ca 2 + and Mg 2 +, which are essential for adhesion) and / or trypsin (degrades the protein of the matrix). B) After detachment the EDTA and trypsin action is neutralized by the addition of fresh culture medium containing excess divalent cations and trypsin inhibitors. C) The cells are then counted and seeded in new plates. The time needed to duplicate cells is about 20 to 24 hours depending on the cell type.

33 Cell cultures are observed by inverted phase-contrast light microscope fitted with a digital camera device.