Tissue culture. Dr Saeb Aliwaini 1st lecture and lab

Transcription

1 Tissue culture 1st lecture and lab

2 Introduction The laboratory process of cell culture allows cells to be manipulated and investigated for a number of applications, including: - Studies of cell function, for example metabolism; - Testing of the effects of chemical compounds on specific cell types; - Cell engineering to generate artificial tissues; - Large-scale synthesis of biologicals such as therapeutic proteins and viruses

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5 1 st culture Harrison [1907] chose the frog as his source of tissue, why?? Whereas bacteria can double every 30 minutes or so, animal cells require around 24 hour. However, tissue culture became established as a routine laboratory method by the 1950s with the advent of defined culture media devised by Eagle and others. The discovery of antibiotics facilitated prolonged cell culture by reducing contamination issues.

6 It was shown in 1949 that poliovirus could be grown in cultures of human cells, and this became one of the first commercial largescale vaccine products of cultured mammalian cells , monoclonal antibodies were being commercially produced in multi-kilogram quantities. cultured epidermal keratinocyte autografts in vitro fertilization (IVF) Stem cell research is another cell culture application that holds huge promise for the future

7 The oldest lines L929

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9 Effects of Axin overexpression on the morphology of L929 cells.

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11 Trypsinization It was not until the 1950s that trypsin became more generally used for subculture, following procedures described by Dulbecco to obtain passaged monolayer cultures for viral plaque assays [Dulbecco, 1952]. Gey established the first continuous human cell line, HeLa [Gey et al., 1952 The 1950s were also the years of the development of defined media [Morgan et al., 1950; Parker et al., 1954; Eagle, 1955, 1959; Waymouth, 1959], which led ultimately to the development of serum-free media [Ham, 1963, 1965]

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15 Culture types The term organ culture implies a three-dimensional culture of un disaggregated tissue retaining some or all of the histological features of the tissue in vivo Cell culture refers to culture derived from dispersed cells taken from original tissue, from a primary culture, or from a cell line or cell strain by enzymatic, mechanical, or chemical disaggregation Histotypic culture cells have been re aggregated or grown to recreate a three-dimensional structure Organotypic implies the same procedures but recombining cells of different lineages, such as epidermal keratinocytes in combined culture with dermal fibroblasts, in an attempt to generate a tissue equivalent.

16 Cell line After the first subculture, the primary culture becomes known as a cell line or subclone. Cell lines derived from primary cultures have a limited life span (i.e., they are finite, and as they are passaged, cells with the highest growth capacity predominate, resulting in a degree of genotypic and phenotypic uniformity in the population.

17 Cell strain If a subpopulation of a cell line is positively selected from the culture by cloning or some other method, this cell line becomes a cell strain. A cell strain often acquires additional genetic changes subsequent to the initiation of the parent line. How to select?

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19 A - Control of the Environment Advantages Of Tissue Culture Physiochemical Environment(Ph, Temperature, Osmotic Pressure, And O2 And CO2Tension), Which Has To Be Controlled Very Precisely. physiological conditions, which have to be kept relatively constant serum, extracellular matrix (ECM) are important but not well defind B- Characterization and Homogeneity of Samples Because experimental replicates are virtually identical, the need for statistical analysis of variance is simplified C- Economy, Scale D- in vitro Modeling of In vivo Conditions

20 Limitations Expertise Quantity The cost of producing cells in culture is about 10 times that of using animal tissue Dedifferentiation and Selection (but, the development of serum-free selective media helps ) If differentiated properties are lost what do you need?? many cell lines have been misidentified due to crosscontamination or errors in stock control in culture or in the freezer Instability

21 What are the differences? 2 dimensional, When a cell line forms, it may represent only one or two cell types, and many heterotypic cell cell interactions are lost. The culture environment also lacks the several systemic components involved in homeostatic regulation in vivo The low oxygen tension due to the lack of oxygen transporter (hemoglobin) results in energy metabolism in vitro occurring largely by glycolysis; although the citric acid cycle is still functional, it plays a lesser role.

22 Types Of Tissue Culture 1- organ culture Organ culture implies that the architecture characteristic of the tissue in vivo is retained, at least in part. Cultured at the liquid gas interface (on a raft, grid, or gel), which favors the retention of a spherical or three-dimensional shape. organ cultures are usually placed at the interface between the liquid and gaseous phases, to facilitate gas exchange while retaining access to nutrients.

23 TYPES OF TISSUE CULTURE

24 or by using a roller bottle or rotating tube rack Increased permeation of oxygen can also be achieved by using increasing O2concentrations up to pure oxygen

25 2- primary explant culture The Primary Explant Technique Was The Original Method Developed By Harrison [1907] Fragment of tissue was embedded in blood plasma or lymph, mixed with heterologous serum and embryo extract, and placed on a coverslip that was inverted over a concavity slide

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27 3- cell culture cell culture implies that the tissue, or outgrowth from the primary explant, is dispersed (mechanically or enzymatically) into a cell suspension, which may then be cultured as an adherent monolayer on a solidnsubstrate or as a suspension in the culture medium

28 Monolayer culture signifies that the cells are grown attached to the substrate. Anchorage dependence means that attachment to (and usually some degree of spreading onto) the substrate is a prerequisite for cell proliferation Suspension cultures are derived from cells that can survive and proliferate without attachment (anchorage independent)

29 This ability is restricted to hematopoietic cells, transformed cell a subculture or passage. The formation of a cell line from a primary culture inolves (1) an increase in the total number of cells over several generations (population doublings) (2) the ultimate predominance of cells or cell lineages with a high proliferative this will lead to (3) a degree of uniformity in the cell population

30 Histotypic culture : means the high-density, or tissue-like, culture of one cell type. Organotypic culture : the presence of more than one cell type interacting, as the cells might, in the organ of origin. Different interactions lead to integrated biology understanding.

31 Next lecture is Biology of Cultured Cells

32 The Cell Culture Laboratory Cell culture laboratory design Health and safety implications Services Water Pressurized gases Liquid nitrogen Microbiological safety cabinet

33 Microbiological safety cabinet

34 Incubators Controlled and reliably maintained: temperature, humidity and carbon dioxide concentration. 37 C (or to be safe, 36.8 C or 36.9 C, as cells are more tolerant to a low incubation temperature than a high one). with a relative humidity level of 95% and with CO2 concentration matched to the media in use (usually 5%) The atmosphere within the incubator is normally humidified by means of a tray of water placed in the bottom of the incubator, with a fan ensuring even distributionof humidity around the chamber

35 Microscopes, Centrifuges, A good inverted microscope is essential in any tissue culture laboratory to allow visualization of cultures in flasks and plates, and also for cell counting A low speed benchtop centrifuge capable of generating at least 200 g is needed for pelleting cells during various culture operations Refrigerators and freezers For storage of media and additives at least one refrigerator is needed Liquid nitrogen refrigerators for cell storage

36 Culture plasticware and associated small consumable items

37 Liquid handling and filtration volumetric pipettes,pasteur pipettes, centrifuge tubes, autoclavable micro-pipette tips, and filters for sterilizing tissue culture solutions. The pore size of sterilizing filters should normally be 0.2 µm, or even 0.1 µm in order to exclude mycoplasma as well as other micro-organisms.

38 Washing reusable tissue culture equipment Ultra-pure water Chloros (hypochlorite) solution for soaking Phosphate-free detergent

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40 General care and maintenance of the tissue culture laboratory it is useful to have a checklist of tasks that must be completed on a daily, weekly or monthly basis Daily checklist: Where appropriate, check room air handling pressure differential(s) before entry. Record incubator, refrigerator and freezer temperatures. Check incubator temperature and CO2 are at set points. Check CO2 cylinder pressures. Check conductivity of purified water. Weekly checklist: Wash floor and bench work surfaces (if not done daily). Clean underneath MSC work surface. Change water in water baths.

41 Change water in humidifier trays in incubators. Replenish stocks of routine reagents and plasticware. Empty aspirator jars as necessary (if not done daily). Top up LN in cell freezers (or twice weekly). Change floor sticky mat (or more often if required). Change used laboratory coats for clean ones.

42 Microbial contamination In media, flasks, serum, hood, incubator microscopic observation more difficult to detect such as viruses, mycoplasma or crosscontamination by other cell cultures Be honest???? As soon as possible, all users should be alerted to the fact that there is microbial contamination in the laboratory.

43 Identifying the source consumables raw materials (cells, media or media component) operator error external sources. Cell lines should only be obtained from reputable suppliers (preferably culture collections such as the American Type Culture Collection (ATCC) or the European Collection of Cell Cultures (ECACC).