different cell line use in absorption study

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2. oocell culture is the complex process by which cells are grown under controlled conditions, generally outside of their natural environment. By this process prokaryotic,eukaryotic or plant cells are grown under controlled condition but in practice it refers to the culturing of cells derived from animal cells.2 3. o o oooCell culture was first successfully undertaken by Ross harrison in 1907 In 1885 Roux first time maintained embryonic chick cells in cell culture. 1911: Lewis and Lewis made the first liquid media consisted of sea water, serum, embryo extract, salts and peptones. They observed limited monolayer growth 1913: Carrel introduced strict aseptic techniques so that cells could be cultured for long periods. 1952: Gey established a continuous cell line from a human cervical carcinoma known as HeLa (Helen Lane) cells. 3 4. oo ooo1927: Carrel and Rivera produced the first viral vaccine - Vaccinia. 1933: Gey developed the roller tube technique. 1982: Human insulin became the first recombinant protein to be licensed as a therapeutic agent. 1985: Human growth hormone produced from recombinant bacteria was accepted for therapeutic use. 1987: Tissue-type plasminogen activator (tPA) from recombinant animal cells became commercially available4 5. oooFirst development was use of antibiotics which inhibits the growth of contaminants. Second was the use of trypsin to remove adherent cells to subculture further from the culture vessel. Third was the use of chemically defined culture media.5 6. oooMODEL SYSTEM: for studying basic cell biology, interaction between disease causing agent and cells, effect of drugs on cells, process and triggering of aging & nutritional studies. TOXICITY TESTING: in study of effect of new drugs. CANCER RESEARCH: study function of various chemical, virus & radiation to convert normal culture cell to cancerous cells. 6 7. oooVIROLOGY: cultivation of virus for vaccine production. GENETIC ENGGINERING: production of commercial protein, large scale production of viruses for use in vaccine production. GENE THERAPY: cells having a functional gene Can be replaced to cell which are having nonfunctional gene.7 8. o o3 Types of cell cultures 1)Primary cell culture: This is the maintenance of growth of cells dissociated from the parental tissue (such as kidney or liver) using mechanical or enzymatic methods, in culture medium using suitable glass or plastic containers. The primary cell culture could be of two types depending upon the kind of cells in culture. Adherent cells - Cells shown to require attachment for growth are said to be anchorage dependent cells. The adherent cells are usually derived from tissues of organs such as kidney where they are immobile and embedded in connective tissue. 8 9. ooSuspension cells - Cells which do not require attachment for growth or do not attach to the surface of the culture vessels are anchorage independent cells/suspension cells. All suspension cultures are derived from cells of the blood system because these cells are also suspended in plasma in vitro e.g. lymphocytes. 2)Secondary cell cultures: When a primary culture is subcultured, it becomes known as secondary culture or cell line. Subculture (or passage) refers to the transfer of cells from one culture vessel to another culture vessel. This is periodically required to provide fresh nutrients and growing space for continuously growing cell lines. The process involves removing the growth media and disassociating the adhered cells (usually enzymatically). Such cultures may be called secondary cultures.3)Cell Line: A cell line or cell strain may be finite or continuous depending upon whether it has limited culture life span or it is immortal in culture. On the basis of the life span of culture, the cell lines are categorized into two types: 9 10. Finite cell lines - The cell lines which have a limited life span and go through a limited number of cell generations (usually 20-80 population doublings) are known as finite cell lines. These cell lines exhibit the property of contact inhibition, density limitation and anchorage dependence. The growth rate is slow and doubling time is around 24-96 hours. Continuous cell lines - Cell lines transformed under laboratory conditions or in vitro culture conditions give rise to continuous cell lines. These cell lines show the property of ploidy (aneupliody or heteroploidy), absence of contact inhibition and anchorage dependence. They grow either in a monolayer or in suspension (see below). The growth rate is rapid and doubling time can be 12-24 hours. Monolayer cultures - When the bottom of the culture vessel is covered with a continuous layer of cells, usually one cell in thickness, they are referred to as monolayer cultures.10 11. Suspension cultures - Majority of continuous cell lines grow as monolayers. Some of the cells which are non-adhesive e.g. cells of leukemia or certain cells which can be mechanically kept in suspension, can be propagated in suspension.11 12. HUMAN CELL LINES: DU145 (prostate cancer) THP-1 (acute myeloid leukemia) MCF-7 (breast cancer) Saos-2 cells (bone cancer) PC3 (prostate cancer) o PLANT CELL LINES: Tobacco BY-2 cell o Rat tumor cell lines: GH3 (pituitary tumor) PC12 (pheochromocytoma) o Mouse cell lines: MC3T3 (embryonic calvarium) o12 13. ooooChoice of media depends on the type of cell being cultured Commonly used Medium are GMEM, EMEM,DMEM etc. Media is supplemented with antibiotics viz. penicillin, streptomycin etc. Prepared media is filtered and incubated at 4 C.13 14. o ooo oLaminar cabinet-Vertical are preferable Incubation facilities- Temperature of 25-30 C for insect & 37 C for mammalian cells, co2 25% & 95% air at 99% relative humidity. To prevent cell death incubators set to cut out at approx. 38.5 C Refrigerators- Liquid media kept at 4 C, enzymes (e.g. trypsin) & media components (e.g. glutamine & serum) at -20 C Microscope- An inverted microscope with 10x to 100x magnification Tissue culture ware- Culture plastic ware treated by polystyrene 14 15. ooo o oPossibly keep cultures free of antibiotics in order to be able to recognize the contamination Never use the same media bottle for different cell lines. If caps are dropped or bottles touched unconditionally touched, replace them with new ones Necks of glass bottles prefer heat at least for 60 secs at a temperature of 200 C Switch on the laminar flow cabinet 20 mts prior to start working Cell cultures which are frequently used should be subcultered & stored as duplicate strains. 15 16. oo oooUse actively growing cells that are in their log phase of growth, which are 80-90% viable Keep exposure to trypsin at a minimum Handle the cells gently. Do not centrifuge cells at high speed or roughly re-suspend the cells Feeding & sub culturing the cells at more frequent intervals then used with serum containing conditions may be necessary A lower concentration of 104cells/ml to initiate subculture of rapidly growing cells & a higher concentration of 105cells/mlfor slowing growing cells 16 17. oo oo o o oSeveral cultured epithelial cell lines of human or animal origin can be used as in vitro drug absorption models to predict the absorption of compounds across intestinal tissue in vivo. The Caco-2 human colonic cell line is perhaps the most widely used cell line for this purpose. . Caco-2 cells adopt a polarized morphology and express many intestinal transport proteins and other enzymes characteristic of epithelial cells of the small intestine. Absorption Systems offers: 1)In vitro assays for absorption across human or animal intestinal strips 2)Oral bioavailability studies in rodents, dogs, pigs and non-human primates 3)Intestinal loop perfusion studies in rats17 18. oooThe Caco-2 cell line is an immortalized line of heterogeneous human epithelial colorectal adenocarcinoma cells. Caco-2 cell monolayers spontaneously differentiate to express morphological and functional characteristics of mature smallintestinal enterocytes. The differentiated monolayers are polarized, with microvilli on the apical side. it is developed by the Sloan-Kettering Institute for Cancer Research18 19. oooSpontaneously differentiate to express morphological (polarized columnar cells) and functional characteristics of mature small-intestinal enterocytes. Four times higher in transepithelial resistance compared to HT 29-cell monolayer. It expresses various drug metabolizing enzymes like aminopeptidase, esterase, and sulfatase.19 20. oo o o o o oTissue in the villus contains more than one cell type Dose not produce the mucus and unstirred water Observed in the intestine No P-450 drug metabolizing enzyme activity has been reported Expensive method Time consuming as 21 days required for full cell differentiation The necessity of LC / MS or HPLC for quantitation Influence of P-gp is difficult to estimate. 20 21. 21 22. 22 23. 3 DAYS21 DAYS 23 24. ooooA. Trans Epithelial Electrical Resistance (TEER) measurements: Measurement of the integrity of the caco-2 cell monolayer After washing the cell monolayer with 37C tempered D-PBS (with Ca2+, Mg2+) (Dulbecco's Phosphate Buffered Saline), 1600 l transport medium was added into the apical and 2800 l transport medium was added into the basal compartment.Allow for equilibrium for 60 min in the cell culture incubator. 24 25. oo o o o4. The measurement chamber was tempered to 37C with transport medium before the measurement. For the post-experimental TEER measurement, the withdrawn volume in the apical compartment was replaced with transport medium before TEER was measured. 5.Caco-2 monolayer with TEER values exceeding 250 cm2 were used for transport experiments. 6. Background TEER may be recorded in wells without cell monolayers, and can be subtracted from the raw TEER values with cells.25 26. oRinse the monol