=—— GEB 425 Fisheries Biotechnology Lecture 9 Fish Cell Technology A PDM GEB, SUST Lecture Contents 1.Background of Fish Cell Culture IL. Cells Types ML Introdaction to Cell Culture Lab 1V. Culture Condition V. Culture Techniques ‘VE Examples in Fish Cell Technology Major Developments in Cell Culture Technology First development was the use of antibiotics which inhibits the growth of contaminants. ‘Second was the use of trypsin to remove edherent cells to subculture further from the culture vessel. ‘Third was the use of chemically defined culture medium. anero2 | Scanned with CamScannerCell Culture which prokaryoti, eukaryotic or plant cells are grown under refers to the culturing of cells derived from animal cells. ison in 1907. Cell culture is the process bY! controlled conditions Pratie Cell culture was fist successfully undertaken by Ross Harr Rous in 1888 for the first time maintained embryonic chick cells in a cell culture, se I. Fish Cell and Tissue Culture ‘+ Fish Cell Cultue: Fisb/shrimp/aquatic animal cells removed from tissues, will continue to ‘grow if supplied with the appropriate nutrients and conditions. ‘+ Tissue culture: In vitro cultivation of organs, tissues & cells at defined temperature using an incubator & supplemented with a medium containing cell nutrients & growth factors. + Different types of cells grown in culture includes connective tissue elements such as fibroblasts, skeletal tissue, cardiac, epithelial tissue (liver, breast, skin, Kidney) and many different types of tumor cells, Why is cell culture used for? Aras her eletue eatoy in urety pying a major ale + Model etm for studying basic ell ley, ntratons between disease causing agents and Call eet of drugs onal sce ae tere mrarions between disease ceasing + Ty esting Say eects of nw des. 1 Cancer reser: Sia a of Various chemical, virus & radiation to convert norma Cae sue Saye fen of iin cenies,vrs & radiation to conver normal + Virology: Coltvation of vrs fr vacin produstion, lo used to study there infectious eel. * Genetic Ensinesing: Production of commercial protein, large scale production of vrwes fr us in vain prediction eg. pli, bits, chicken pox, hepatitis B& measles * Gene trary: Celi having a fnctioel gene can be replaced to cls whic ae bang so | il Scanned with CamScannerLe ——— ate/202, Ii. Types of cells Morphological (hape & appearance) or functional characteristics Epithelial like-attached toa substrate and appears flattened and polygoatl in shape Lymphoblast like- cells do not attach remain in suspension witha spherical shape Fibroblast like- cells attached to an substrate spears elongated and bipola® | Ce cultures may contain he following thre types of els: 1. Stem cells- Stem cells are undifferentiated cells which can differentiate under correct inducing conditions into one of several kinds of eels. . 2, Precursor cells- Precursor cells are derived fom stem cells, are commited to ferentiation, liferentiated; these cells retain the capacity for proliferation cells ites cts sully do aot ave the eapaciy to did but are not yet IIL Cell Culture Lab (Equipment) + COpthermostats + Solutions + Dishes + Freezers + Liquid nitrogen + Centrifuges + Autoclave + Vacuum ovens + Cryotubes + Microscopes + ELISA-readers Scanned with CamScannerAer dopends on the type ‘of cell being | "Sea ageacetany Suess smn Som (FCOADS) lumen Med tepleenod ih antics Vi Poe sake cd streptomycin etc. =o ‘Prepared media is| filtered and incubatec * Environment; ets ~ High humidity < Split Trypsia-EDTA a ere «Safety aspect in cel culture ». Basi aseptic conditions + Possibly keep cultres fv of antibiotics in order + If working on te bench us «Bunsen lime to heat” tg beable to recognize te contamination ‘their surounding the Bunsen + Never use the same media botl for different cell * Swab ll bottle tops & aks wth 70% ethanol Tia tape are dropped or bots touched + Flame all botde necks & pipette bypassing very unconditionally touched, replace them with now ‘uickly though the hotest part of the fae a . ee ee Ness fs oes peters teat fr £0 ia rk either | Fight or vice versa, so that al bl recec Mirkeither eft iaktorviee eso the) Sich on the laminar flow cabinet 20 mts rior o + Clean up spills immediately & always leave the work Start working Place nat & ly + Calleaars which are Bequenty wed should be ‘settee & stored copie eran V. Cell Culture Techniques + Metabolic activity (MTT) + Detection of Apoptosis and Necrosis + Westem blot fom cell ‘Transfestion Gene deletions (Demonstration) (Clinieal Application of ultured Human Stem Cells Flow Cytometric Methods _ Fistiprobes DNA Array Rules for working with cell culture [Never use contaminated material within a sterile arca Use the correct sequence when working with more than one cel ines, + Diploid cells (Primary cultures, lines forthe production of vaccines etc,) Diploid cells (Laboratory lines) Continous, slow growing line CContitous, rapidly growing lines Lines which may be contaminated Virus producing lines | ‘Scanned with CamScanner3ner202 issociareo C&L ‘CULTURE, ‘CULTURE once tenon Seaside mere ceed well of edu, seen in section in oterged dtl in section i ‘esa ett ‘Seear ronan 1 an organ culture on filter disk on triangular saints the lower diagram, 2, explant cultures in afisk, with scton below 2 the lowest diagram, showing the explant and radial ou enzymatic disaggregation generating a cll supeasion 5 well showing an array of eels, seen in setion in the lower os tel id vet i 1d with an grow under the arrows: in the lower diagram. 49, a filter ode a nna ea ‘matrix and stromal cells. diagram, combined with ‘Types of Cell and Tissue Culture + CL CHORE TERRA OUTORE arene «Tissue from an explant is dispersal. | + Isthe growth ‘entire embryos oF organs are me fom an ara fs |” itouch eafmite_] HH ee sy ands enon wh gene] on es Shes nomine |. Tsiypal ned vaweot | Nema) phyblogl edn “cores | Remy se cin me aaalonc och bah res Shite iy irene seppewepnent of ell Soe oer|Admantgst > — _severa) generations ‘+ Some normal functions may be ‘Scab on recommer + Sealeupispositie anne — + Absolute control of feat | + Better than sre for seal ‘Fresh explantation is required for enim steal) « Beer remeron experiment i Disiratoss | Homogencity of sample + Less. compound needed than in nil dels Disadvantages + Cells may Tose some ditferntised characterises, + Handtomalnaio or goo amount of dss at + Dediterntiaion + Tesabily,aneuplokdy : + Original organization of secs tst | Scanned with CamScannerWat + Primary cultures + cell ines + Secondary eaates sanoet - ispension = ional + cals om ATCC and BTCC gpontanenus Toso” * Transfection ‘i 2 eeennje cel Fusion (Hybridomas, Hybrids) cls taton by tnehaneal act echnical action mir Pri Q— @~ confluent cultare unre onene | seatee scondry sbciture—> conan bie ae pi po oatinues apto @-@ @-@~ 2 canes secondary faite avs cells cated fom 0, callie a — o”h—m™ét™™C—~—OW cavaivison : pe —» continues up @-@-@- colnet tivslons 1. Primery Culture + Cells when surgically or enzymatically removed from an organism and placed in suitable ‘culture ‘vironment wil tach and grow are called es primary culture. ‘+ Tissue preparation ffom young fish or isolation of cells from blood, intraperitoncal fluid, ete, + Tissue dissociation = Dissection then Homogenization with Knife or Blender = Enzymatic digestion (collagenase, papain, trypsine)cleaving of DNA of damaged cell with DNAse = Dissociation of cells inmedium and selection of organic cel types. + Primary cells have a finite life span. 2 primary eulture contains a very heterogeneous population of cells. ‘Sub culturing of primary cells leads to the generation of cell lines. Call lines have limited life span, they passage several times before they become senescent. {Cells such as macrophages and neurons donot divide in vitro so can be used as primary cultures. {Lineage of ells originating from the primary eultre sealed a cell strain, 2. Secondary cultures recine| each chia tected by sector or doing, Normal cet lines: They were spontaneous oe immortalized (eg.Cardio-myocytes from rl) © Finae Ife span dh vivo, - " Immortalized: Transfected with some sort of “ies ecange ‘cucogene; SV40 (Simian virus), Hybridomas, ‘+ Exhibit contact inhibition. > Contact inhibRion &s 2 growth mechanism, In most eS anon bo cals ie ey aa ert decent nod Rare Scanned with CamScanner316/202 Tine is @ OF cell that ean ge Stet ples oa defined population tte mean Beiod of time, rete ited in ealtroforan extended || iD functions Carieeatin Abily of con pene see wnetions, Cel ‘sually clonal, meaning that the entice Prt ns pets Adherent cells Surface to grow, ‘8 cells which must be attached to a Cell cultures can be grown as: {+ Monolayers or as, 2, Suspension Cultures knee to RE TIES se tiea patacrvousnes a tabcal iow? the culture eos pers a i Nee ely cle est ie), which has chz shut im 2 monclaye al Mssrsscincaliae Oluteh A ‘fil suspension or suspension cul is pe of cal ale in which Sel ll or sll ages of cells are allowed to fantion and imaiply in an agitated growth medium, thus forming a suspensioa, F cece ic Ee indaiot vee Sispenin clue is on of he raced sn ee Ecermee inion cell culture, the other being adherent culture. SPM AeE RBI UF gil volo: Cell Sub-Culture Remove,spént- media froin the cultire vessel. presvarnied dissociation reagent such a trypiim, Geatly rock the Anite contiiiner to get complete coverage of the cell layer. Tnubat the cure wessel room temperatre for approximately 3 minutes: re swarined Eom 7 iste gi ing Over the: aay ell layer: surface Scanned with CamScannerMedia and supplements used in fish cell culture ‘The nutrient media used for culture of snimal cells and tissues must be able to support their survival as well as growth, oe sa nutritional, hormonal and stromal factors. The various types of media used for tissue culture may be groped into two broad cttezorie; 1, Natural Media and 2. antifcial Media, ‘These media consist solely of naturally o€curting biological fluids and are of the following three types: (U cagula of cots, 2) biological Muids and @) tissue extracts, The various artifical media developed for cell cultures may be grouped at: serum containing media (i) serum-fiee media Different artificial media have been devised to serve one of Basic Constituents of media the following purposes: : (1) immediate survival (a balanced salt solution, with + Inorganic salts specified pH and osmotic pressure is adequate), + Carbohydrates @) prolonged survival (a balanced salt solution + Amino Acids ‘supplemented with serum, or with suitable formulation of + Vitamins 7 organic compounds), + Fatty acids and lipids G) indefinite growth, and + Proteins and peptides (A) specialized functions. Buffering Systems Most cells require pH conditions in the range 7.2 - 7.4 and close control of pH is essential for optimum culture conditions. There are major variations to this optimum, Fibroblasts prefer a higher pH (7.4 - 7.7) whereas, continuous transformed cell lines Tequire more acid conditions pH (7.0 - 7.4). Regulation of pH is particularly important immediately following cell seeding when a new culture is establishing and is usually achieved by one of two buffering systems; 1, a “natural” buffering system where gaseous CO2 balances with the CO3 / HCO3 content of the culture medium and 2. chemical buffering using a zwitterion called HEPES Basic aseptic conditions to be maintained in the cell culture labs + Ifworking on the bench use a Bunsen flame to heat the air surrounding the Bunsen, + Swab all bottle tops é& necks with 70% ethanol, + Flame all bottle necks & pipette by passing very quickly through the hottest part of the flame, + Avoiding placing caps & pipettes down on the bench; practice holding bottle tops with the little finger. ; + Work either let to right or vice vérsa, so that all material goes to one side, once finished, Clean Scanned with CamScanneree i Working with cryopreserved ia siowously uot medium i quid nitrogen is placed into 37°C water bath, agitate via! © supernatant, Resuspends th labeled culture pine fer to pellet in 1 mt of complete medium with 20% FBS and transfer to Properiy Culture plate containing the appropriate amount of mediu™ Check the cultures after 24 hrs to ensure that they are attached to the plate ra Change medium as the color changes, use 20% FBS until the cells a7° establi Freezing cells for storage Remove the growth medium, wash the cells by PBS and remove the PBS b: Dislodge the cells by trypsin-versene Dilute the cells with growth medium ‘Transfer the cell suspension to a 15 ml conical tube, centri remove the growth medium by aspiration Resuspend the cells in 1-2ml of freezing medium ‘Transfer the cells to cryovials, incubate the cryovials at -80 C overnight ‘Next day transfer the cryovials to Liguid nitrogen yy aspiration sige at 200g for $ mts at RT and Isolation of cell lines for in vitro culture: Tissue of interest 4 Cell or tissué culturein vitro Primary culture 4 Svb-cuttire ‘Secondary culture Ysup-cutture Cell Line ‘Single cell isolation a 4 “ immortalization J Lote orcontet ‘of cell growth - Senescence ‘Transformed cell line Immortalised cellline (caricerous cells) ‘Successive ‘sub-culture Clonal cell line Scanned with CamScanneree Cell Culture: Flow Chart 1291 Filter tw] ~qhesue explants are excised using sharp scalpel. } [GR are counted on a Haemiogytometer, 122° 1045 cells mis seeded in tothe] nei ss ‘Sm of cells is suspended into 25cm*2 flask. ‘The Masks are incubated in a CO2 incubator. Se i ‘The flasks are observed daily for th Cell viability + Cell viability is determined by staining the cells with trypan blue + As trypan blue dye is permeable to non-viable cells or death cells whereas it is impermeable to viable cells. + The cells are stained with trypan dye and loaded to haemocytometer and calculation of % of viable cells is done. - % of viable cells= Number of unstained cells x 100 total number of cells Contamination ‘A cell culture contaminant can be defined as some element in the culture system that is undesirable because of its possible adverse effects on either the system or its use. ‘Jwo types of contaminants 1. Chemicals - difficult to detect caused by impurities in media, sera, water, endotoxins, plasticizers, metal ions and traces of detergent that are invisible, 2. Biologleal ~ causes visible effects on the culture they are different microorganisms or also from cross-contamination of cells from other cell lines, Cell Toxicity + Cytotoxicity causes inhibition of cell growth. + Observed effect on the morphological alteration in the cell layer or cell shape, + Characteristics of abnormal morphology is the giant cells, multinucleated cells, a granular bumpy appearance, vacuoles in the cytoplasm or nucleus + Cytotoxicity is determined by substituting materials such as medium, serum, supplements flasks etc. at Scanned with CamScannerPop TT VI. Examples in Fish Cell Technology Pete be =a Isolate cells and Place in culture Call profiferation dish ‘Acell line is bom je use of fish cell lines in geno-ecotoxicology assessment crucial awarenes: . “Man-made contamination of aquatic environment raises the necessity {0 assess hazards and risks for aquatic organisms with fish -Many levels should be covered: Basic (eco)toxicological research Environmental surveys & monitoring Regulatory toxicity tests Thus, ethical, technical, scientific and economic reasons support the development of in vitro methods for ecotoxicology studies Fish cell lines : ALTERNATIVE SYSTEMS IN ECOTOXICOLOGY ‘eeslogy Fish cell ting Development Four pillars of fish cell line ws in environmental toxiotogy ‘Bela o a 2008) Use of teh cols inthe toxicology and ecatoxeology ofS Piscine cet tng 4 Blochemety and Molecular Biology of Fishes, Vol PB A483, ‘environmental toxicolony Scanned with CamScannerity testing warrant inclusion assessment processes ? Why does genotoxic hazard and risk saants in the aquatic environment are PNA Potential target for xenobiotics About a thd of contaminate oy genotoxic rectly OF : eae ee eel? material at non-lethal and non- aoe i trations : rotate concent cetaed eects (Month, yea...) which are crucially important at population and community levels GENOTOXINS have high ecotoxicological relevance in situation of chronic exposure to low doses and to multiple = contaminants ae one {oydaton, abo searangene) decane ‘oman {dou bri) desi ‘Quantification ofthe level of DNA breaks by Image analysis Genotoxicity Biomarkers ‘DNA repair activities Primary DNA damages = ~ Genotoxicity zap | Pvigenetic damages Interest ofa mult-biomarkers approach to optimizehe hazard and risk assessment in multi- contamination scenario ne Scanned with CamScannerEg. Fish Cell Culture row = Potential applications of germ cell isolation and culture ‘testes are removed from the fish body and dissociate, providing a cell suspension tht can be sorted using Teste a remtvated cll sorting of cetifugation in a deasity gradient; e.g in Percoll solution or by agnetic-acivated cell sorting. The purified germ cell suspension can be ‘characterized with respect to astucular charactcristics and morphology. Purified cells may be injected into a recipient to generate germ-line Thimera or amplified by in vitro culture. Cultured cells ean be employed in gene editing or ‘subjected to cryopreservation. Scanned with CamScannerPrimordial germ cells lo and te many genome editing t0 8° ection offspring to improve ine iii Kea So te intensity. coer B selon Phenotypes of ‘growing medaka stem fp cell lines in culture, All cells were grown on gelatin-coat cr layer cells. Similar phe are seen ‘ ted Th the absence of feeder leyer cells. Similar phenotypes fora he tes el ns. A) MESH Pehl ES ell ine fom friiation blastulae (B) SG3, 8 male = eat dul testi ‘gyno-genetic blastulae. Scale bars, (Om (top) and 10 Dm otion) (© HXI, haploid ES cell line from Scanned with CamScanner36/2 ish germ cell oo amin ston ot : transplantation i: = - ss Ret toll i eas ae Ee, Me Se ‘output. Red italics _— brackets: indicate the Anticipated time forthe step, \Waton species A Malureapcia A Meters epecien 8 ereprneration Applications for Fish cell cultures “To investigate the normal physiology or biochemistry of cells. For instance, studies of cell metabol ‘To test the effect of various chemical compounds or drugs on specific cell types. ‘To study the sequential or parallel combination of various cll types to generate artificial tissues. ‘Therapeutic proteins can be synthesized in large quantities by growing genetically engineered cells in large- seale cultures, Creation of viral vaccines from large scale cell cultures, Cytotoxicity and genotoxicity studies, Fish cell cultures prove a useful tol for the transfestion(gene delivery) studies, Scanned with CamScannerFish Coll Culture Advantages ‘The major advantage of using cell culture for any ofthe above tpplications isthe consistency and reproducibility of results that can be obtained from using a batch of clonal cells, the materials are cheap and easy to bt the experimental condition could be controlled accurately Fewer animals are harmed Can control all extemal factors Can easily test what the cells are doing Cells ae easy to manipulate and propagate All of the cells are the same hence results of experiments willbe consistent (Cheaper to maintain. Limitations of Animal/Fish Cell Culture + After a period of continuous growth, cell characteristics can change and may become quite different from those found in the starting population. Cells can also adapt to different culture environments (¢-8. different nutiens, temperatures, salt concentrations etc.) by varying the activities of their enzymes. «Tt necessitates expertise for handling and to check chemical contamination, microbial contamination and cross contamination + Require a control environment in the workplace; for incubation, pH control containment and disposal of biohazards + Quantity and cost involvement is more in capital equipment, consumables, medium, serum, plastics ‘which is ton times more costly than using animal itself. + Genetic instability like heterogeneity and variability may appear. It is a major problem with many ‘continuous cell lines resulting from their unstable aneuploid chromosomal constitution. Heterogeneity in growth rate and capacity to differentiate Within the population can produce variability from one passage to another, + Phenotypic instability: some jc characteristics of the tissue may get lost which is due to DEDIFFERENTIATION Great ene to be the reversal of differentiation) also due to overgrowth of undifferentiated cells, It also maybe due to adaptation. + Identification of the cell type: 1 the ifferentiated properties are los, i is difficult to relate the cultured I with the fumctional cell in tneng i aed nal cell in the tissue from where the tissue were derived. For this stable markers are Scanned with CamScanner