Cell culture 1

Cell culture

Cell culture in a Petri dish

Epithelial cells in culture, stained for keratin (red)and DNA (green)

Cell culture is the complex process by which cells are grown undercontrolled conditions, generally outside of their natural environment.In practice, the term "cell culture" now refers to the culturing of cellsderived from multi-cellular eukaryotes, especially animal cells.However, there are also cultures of plants, fungi and microbes,including viruses, bacteria and protists. The historical development andmethods of cell culture are closely interrelated to those of tissue cultureand organ culture.

Animal cell culture became a common laboratory technique in themid-1900s,[1] but the concept of maintaining live cell lines separatedfrom their original tissue source was discovered in the 19th century.[]

History

The 19th-century English physiologist Sydney Ringer developed saltsolutions containing the chlorides of sodium, potassium, calcium andmagnesium suitable for maintaining the beating of an isolated animalheart outside of the body.[2] In 1885, Wilhelm Roux removed aportion of the medullary plate of an embryonic chicken and maintainedit in a warm saline solution for several days, establishing the principleof tissue culture.[] Ross Granville Harrison, working at Johns HopkinsMedical School and then at Yale University, published results of hisexperiments from 1907 to 1910, establishing the methodology of tissueculture.[3]

Cell culture techniques were advanced significantly in the 1940s and 1950s to support research in virology. Growingviruses in cell cultures allowed preparation of purified viruses for the manufacture of vaccines. The injectable poliovaccine developed by Jonas Salk was one of the first products mass-produced using cell culture techniques. Thisvaccine was made possible by the cell culture research of John Franklin Enders, Thomas Huckle Weller, andFrederick Chapman Robbins, who were awarded a Nobel Prize for their discovery of a method of growing the virusin monkey kidney cell cultures.

Concepts in mammalian cell culture

Isolation of cellsCells can be isolated from tissues for ex vivo culture in several ways. Cells can be easily purified from blood;however, only the white cells are capable of growth in culture. Mononuclear cells can be released from soft tissuesby enzymatic digestion with enzymes such as collagenase, trypsin, or pronase, which break down the extracellularmatrix. Alternatively, pieces of tissue can be placed in growth media, and the cells that grow out are available forculture. This method is known as explant culture.Cells that are cultured directly from a subject are known as primary cells. With the exception of some derived fromtumors, most primary cell cultures have limited lifespan. After a certain number of population doublings (called theHayflick limit), cells undergo the process of senescence and stop dividing, while generally retaining viability.

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An established or immortalized cell line has acquired the ability to proliferate indefinitely either through randommutation or deliberate modification, such as artificial expression of the telomerase gene. Numerous cell lines arewell established as representative of particular cell types.

Maintaining cells in cultureCells are grown and maintained at an appropriate temperature and gas mixture (typically, 37 °C, 5% CO2 formammalian cells) in a cell incubator. Culture conditions vary widely for each cell type, and variation of conditionsfor a particular cell type can result in different phenotypes.Aside from temperature and gas mixture, the most commonly varied factor in culture systems is the cell growthmedium. Recipes for growth media can vary in pH, glucose concentration, growth factors, and the presence of othernutrients. The growth factors used to supplement media are often derived from animal blood, such as calf serum.One complication of these blood-derived ingredients is the potential for contamination of the culture with viruses orprions, particularly in medical biotechnology applications. Current practice is to minimize or eliminate the use ofthese ingredients wherever possible and use chemically defined media, but this cannot always be accomplished.Alternative strategies involve sourcing the animal blood from countries with minimum BSE/TSE risk, such asAustralia and New Zealand, and using purified nutrient concentrates derived from serum in place of whole animalserum for cell culture.[4] Also the use of recently developed universal, fully defined and animal free alternatives likeSerum-Free avoids these complications.Plating density (number of cells per volume of culture medium) plays a critical role for some cell types. Forexample, a lower plating density makes granulosa cells exhibit estrogen production, while a higher plating densitymakes them appear as progesterone-producing theca lutein cells.[5]

Cells can be grown either in suspension or adherent cultures. Some cells naturally live in suspension, without beingattached to a surface, such as cells that exist in the bloodstream. There are also cell lines that have been modified tobe able to survive in suspension cultures so they can be grown to a higher density than adherent conditions wouldallow. Adherent cells require a surface, such as tissue culture plastic or microcarrier, which may be coated withextracellular matrix components to increase adhesion properties and provide other signals needed for growth anddifferentiation. Most cells derived from solid tissues are adherent. Another type of adherent culture is organotypicculture, which involves growing cells in a three-dimensional (3-D) environment as opposed to two-dimensionalculture dishes. This 3D culture system is biochemically and physiologically more similar to in vivo tissue, but istechnically challenging to maintain because of many factors (e.g. diffusion).

Cell line cross-contaminationCell line cross-contamination can be a problem for scientists working with cultured cells. Studies suggest anywherefrom 15–20% of the time, cells used in experiments have been misidentified or contaminated with another cellline.[6][7][8] Problems with cell line cross-contamination have even been detected in lines from the NCI-60 panel,which are used routinely for drug-screening studies.[][9] Major cell line repositories, including the American TypeCulture Collection (ATCC), the European Collection of Cell Cultures (ECACC) and the German Collection ofMicroorganisms and Cell Cultures (DSMZ), have received cell line submissions from researchers that weremisidentified by them.[][] Such contamination poses a problem for the quality of research produced using cell culturelines, and the major repositories are now authenticating all cell line submissions.[10] ATCC uses short tandem repeat(STR) DNA fingerprinting to authenticate its cell lines.[]

To address this problem of cell line cross-contamination, researchers are encouraged to authenticate their cell lines atan early passage to establish the identity of the cell line. Authentication should be repeated before freezing cell linestocks, every two months during active culturing and before any publication of research data generated using the celllines. Many methods are used to identify cell lines, including isoenzyme analysis, human lymphocyte antigen (HLA)typing, chromosomal analysis, karyotyping, morphology and STR analysis.[]

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One significant cell-line cross contaminant is the immortal HeLa cell line.

Other technical issuesAs cells generally continue to divide in culture, they generally grow to fill the available area or volume. This cangenerate several issues:•• Nutrient depletion in the growth media• Accumulation of apoptotic/necrotic (dead) cells• Cell-to-cell contact can stimulate cell cycle arrest, causing cells to stop dividing, known as contact inhibition.• Cell-to-cell contact can stimulate cellular differentiation.• Genetic and epigenetic alterations, with a natural selection of the altered cells potentially leading to overgrowth of

abnormal, culture-adapted cells with decreased differentiation and increased proliferative capacity.[11]

Manipulation of cultured cellsAmong the common manipulations carried out on culture cells are media changes, passaging cells, and transfectingcells. These are generally performed using tissue culture methods that rely on sterile technique. Sterile techniqueaims to avoid contamination with bacteria, yeast, or other cell lines. Manipulations are typically carried out in abiosafety hood or laminar flow cabinet to exclude contaminating micro-organisms. Antibiotics (e.g. penicillin andstreptomycin) and antifungals (e.g.amphotericin B) can also be added to the growth media.As cells undergo metabolic processes, acid is produced and the pH decreases. Often, a pH indicator is added to themedium to measure nutrient depletion.

Media changes

In the case of adherent cultures, the media can be removed directly by aspiration, and then is replaced. Mediachanges in non-adherent cultures involve centrifuging the culture and resuspending the cells in fresh media.

Passaging cells

Passaging (also known as subculture or splitting cells) involves transferring a small number of cells into a newvessel. Cells can be cultured for a longer time if they are split regularly, as it avoids the senescence associated withprolonged high cell density. Suspension cultures are easily passaged with a small amount of culture containing a fewcells diluted in a larger volume of fresh media. For adherent cultures, cells first need to be detached; this iscommonly done with a mixture of trypsin-EDTA; however, other enzyme mixes are now available for this purpose.A small number of detached cells can then be used to seed a new culture.

Transfection and transduction

Another common method for manipulating cells involves the introduction of foreign DNA by transfection. This isoften performed to cause cells to express a protein of interest. More recently, the transfection of RNAi constructshave been realized as a convenient mechanism for suppressing the expression of a particular gene/protein. DNA canalso be inserted into cells using viruses, in methods referred to as transduction, infection or transformation. Viruses,as parasitic agents, are well suited to introducing DNA into cells, as this is a part of their normal course ofreproduction.

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Established human cell linesCell lines that originate with humans have been somewhat controversial in bioethics, as they may outlive their parentorganism and later be used in the discovery of lucrative medical treatments. In the pioneering decision in this area,the Supreme Court of California held in Moore v. Regents of the University of California that human patients haveno property rights in cell lines derived from organs removed with their consent.[12]

Generation of hybridomasIt is possible to fuse normal cells with an immortalised cell line. This method is used to produce monoclonalantibodies. In brief, lymphocytes isolated from the spleen (or possibly blood) of an immunised animal are combinedwith an immortal myeloma cell line (B cell lineage) to produce a hybridoma which has the antibody specificity of theprimary lymphoctye and the immortality of the myeloma. Selective growth medium (HA or HAT) is used to selectagainst unfused myeloma cells; primary lymphoctyes die quickly in culture and only the fused cells survive. Theseare screened for production of the required antibody, generally in pools to start with and then after single cloning.

Applications of cell cultureMass culture of animal cell lines is fundamental to the manufacture of viral vaccines and other products ofbiotechnologyBiological products produced by recombinant DNA (rDNA) technology in animal cell cultures include enzymes,synthetic hormones, immunobiologicals (monoclonal antibodies, interleukins, lymphokines), and anticancer agents.Although many simpler proteins can be produced using rDNA in bacterial cultures, more complex proteins that areglycosylated (carbohydrate-modified) currently must be made in animal cells. An important example of such acomplex protein is the hormone erythropoietin. The cost of growing mammalian cell cultures is high, so research isunderway to produce such complex proteins in insect cells or in higher plants, use of single embryonic cell andsomatic embryos as a source for direct gene transfer via particle bombardment, transit gene expression and confocalmicroscopy observation is one of its applications. It also offers to confirm single cell origin of somatic embryos andthe asymmetry of the first cell division, which starts the process.

Cell culture in two dimensionsResearch in tissue engineering, stem cells and molecular biology primarily involves cultures of cells on flat plasticdishes. This technique is known as two-dimensional (2D) cell culture, and was first developed by Wilhelm Rouxwho, in 1885, removed a portion of the medullary plate of an embryonic chicken and maintained it in warm salinefor several days on a flat glass plate. From the advance of polymer technology arose today's standard plastic dish for2D cell culture, commonly known as the Petri dish. Julius Richard Petri, a German bacteriologist, is generallycredited with this invention while working as an assistant to Robert Koch. Various researchers today also utilizeculturing laboratory flasks, conicals, and even disposable bags like those used in single-use bioreactors.Aside from Petri dishes, scientists have long been growing cells within biologically-derived matrices such ascollagen or fibrin, and more recently, on synthetic hydrogels such as polyacrylamide or PEG. They do this in orderto elicit phenotypes that are not expressed on conventionally rigid substrates. There is growing interest in controllingmatrix stiffness,[13] a concept that has led to discoveries in fields such as:• Stem cell self-renewal[14][15]

• Lineage specification[16]

• Cancer cell phenotype[17][18][19]

• Fibrosis[20][21]

• Hepatocyte function[22][23][24]

• Mechanosensing[25][26][27]

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Cell culture in three dimensionsCell culture in three dimensions has been touted as "Biology's New Dimension".[28] Nevertheless, the practice of cellculture remains overwhelmingly based on rigid, 2D substrates. That being said, there is an increase in use of 3D cellcultures in research areas including drug discovery, cancer biology, regenerative medicine and basic life scienceresearch. There are a variety of platforms used to facilitate the growth of 3 dimensional cellular structures such asnanoparticle facilitated magnetic levitation,[29] gel matrices scaffolds, and hanging drop plates.[30]

Tissue culture and engineeringCell culture is a fundamental component of tissue culture and tissue engineering, as it establishes the basics ofgrowing and maintaining cells in vitro. The major application of human cell culture is in stem cell industry, wheremesenchymal stem cells can be cultured and cryopreserved for future use.

VaccinesVaccines for polio, measles, mumps, rubella, and chickenpox are currently made in cell cultures. Due to the H5N1pandemic threat, research into using cell culture for influenza vaccines is being funded by the United Statesgovernment. Novel ideas in the field include recombinant DNA-based vaccines, such as one made using humanadenovirus (a common cold virus) as a vector,[][31] and novel adjuvants.[32]

Culture of non-mammalian cells

Plant cell culture methodsPlant cell cultures are typically grown as cell suspension cultures in a liquid medium or as callus cultures on a solidmedium. The culturing of undifferentiated plant cells and calli requires the proper balance of the plant growthhormones auxin and cytokinin.

Insect cell cultureCells derived from Drosophila melanogaster (most prominently, Schneider 2 cells) can be used for experimentswhich may be hard to do on live flies or larvae, such as biochemical studies or studies using siRNA. Cell linesderived from the army worm Spodoptera frugiperda, including Sf9 and Sf21, and from the cabbage looperTrichoplusia ni, High Five cells, are commonly used for expression of recombinant proteins using baculovirus.

Bacterial and yeast culture methodsFor bacteria and yeasts, small quantities of cells are usually grown on a solid support that contains nutrientsembedded in it, usually a gel such as agar, while large-scale cultures are grown with the cells suspended in a nutrientbroth.

Viral culture methodsThe culture of viruses requires the culture of cells of mammalian, plant, fungal or bacterial origin as hosts for thegrowth and replication of the virus. Whole wild type viruses, recombinant viruses or viral products may be generatedin cell types other than their natural hosts under the right conditions. Depending on the species of the virus, infectionand viral replication may result in host cell lysis and formation of a viral plaque.

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Common cell linesHuman cell lines•• HeLa• National Cancer Institute's 60 cancer cell lines• ESTDAB database [33]

• DU145 (prostate cancer)• Lncap (prostate cancer)• MCF-7 (breast cancer)• MDA-MB-438 (breast cancer)• PC3 (prostate cancer)• T47D (breast cancer)• THP-1 (acute myeloid leukemia)• U87 (glioblastoma)• SHSY5Y Human neuroblastoma cells, cloned from a myeloma• Saos-2 cells (bone cancer)Primate cell lines• Vero (African green monkey Chlorocebus kidney epithelial cell line initiated in 1962)Rat tumor cell lines• GH3 (pituitary tumor)• PC12 (pheochromocytoma)Mouse cell lines• MC3T3 (embryonic calvarium)Plant cell lines• Tobacco BY-2 cells (kept as cell suspension culture, they are model system of plant cell)Other species cell lines• Zebrafish ZF4 and AB9 cells• Madin-Darby canine kidney (MDCK) epithelial cell line• Xenopus A6 kidney epithelial cells

List of cell linesThis list is incomplete; you can help by expanding it [34].

Cell line Meaning Organism Origin tissue Morphology Link

293-T Human Kidney (embryonic) Derivative of HEK 293ECACC [35] Cell LinesData Base (CLDB) [36]

3T3 cells "3-day transfer,inoculum 3 x 10^5cells"

Mouse Embryonic fibroblast Also known as NIH3T3 ECACC [37].Search for the many3T3 cells [38] in theCLDB.

721 Human Melanoma

9L Rat Glioblastoma

Cell culture 7

A2780 Human Ovary Ovarian cancer ECACC [39], CLDB[40]

A2780ADR Human Ovary Adriamycin-resistantderivative

ECACC [41]

A2780cis Human Ovary Cisplatin-resistantderivative

ECACC [42]

A172 Human Glioblastoma Malignant glioma ECACC [43]

A20 Murine B lymphoma B lymphocyte

A253 Human Head and neck carcinoma Submandibular duct

A431 Human Skin epithelium Squamous cell carcinoma ECACC [44]CLDB[45]

A-549 Human Lungcarcinoma Epithelium DSMZ [46]ECACC[47]

ALC Murine Bone marrow Stroma PubMed [48]

B16 Murine Melanoma ECCAC [49]

B35 Rat Neuroblastoma ATCC [50]

BCP-1 cells Human PBMC HIV+ lymphoma ATCC [51]

BEAS-2B Bronchial epithelium +Adenovirus 12-SV40virus hybrid(Ad12SV40)

Human Lung Epithelial ATCC [52]

bEnd.3 Brain endothelial Mouse Brain/cerebral cortex Endothelium ATCC [53]

BHK-21 Baby hamster kidneyfibroblast cells

Hamster Kidney Fibroblast ECACC [54]Olympus[55]

BR 293 Human Breast Breast cancer

BxPC3 Biopsy xenograph ofpancreatic carcinomaline 3

Human Pancreatic adenocarcinoma Epithelial ATCC [56]

C2C12 Mouse Myoblast cell line ECACC [57]

C3H-10T1/2 Mouse Embryonic mesenchymalcell line

ECACC [58]

C6/36 Asian tigermosquito

Larval tissue ECACC [59]

Cal-27 Human Tongue Squamous cell carcinoma

CHO Chinese hamster ovary Hamster Ovary Epithelium ECACC [60]ICLC [61]

COR-L23 Human Lung ECACC [62]

COR-L23/CPR Human Lung ECACC [63]

COR-L23/5010 Human Lung ECACC [64]

COR-L23/R23 Human Lung Epithelial ECACC [65]

Cell culture 8

COS-7 Cercopithecusaethiops,origin-defective SV-40

Ape -Cercopithecusaethiops(Chlorocebus)

Kidney Fibroblast ECACC [66]ATCC[67]

COV-434 Human Ovary Metastatic granulosa cellcarcinoma

[68]ECACC [69]

CML T1 Chronic myeloidleukaemia Tlymphocyte 1

Human CML acute phase T cell leukaemia Blood [70]

CMT Canine mammarytumor

Dog Mammary gland Epithelium

CT26 Murine Colorectal carcinoma Colon

D17 Canine Osteosarcoma ECACC [71]

DH82 Canine Histiocytosis Monocyte/macrophage ECACC [72] J VirMeth [73]

DU145 Human Androgen insensitivecarcinoma

Prostate

DuCaP Dura mater cancer ofthe prostate

Human Metastatic prostate cancer Epithelial PubMed [74] {Ehrlichascites carcinoma} mice

EL4 Mouse T cell leukaemia ECACC [75]

EM2 Human CML blast crisis Ph+ CML line CLDB [76]

EM3 Human CML blast crisis Ph+ CML line CLDB [77]

EMT6/AR1 Mouse Breast Epithelial-like ECACC [35]

EMT6/AR10.0 Mouse Breast Epithelial-like ECACC [78]

FM3 Human Metastatic lymph node Melanoma

H1299 Human Lung Lung cancer

H69 Human Lung ECACC [79]

HB54 Hybridoma Hybridoma Secretes L243 mAb(against HLA-DR)

Human Immunology[80]

HB55 Hybridoma Hybridoma secretes MA2.1 mAb(against HLA-A2 andHLA-B17)

Journal of Immunology[81]

HCA2 Human Fibroblast Journal of GeneralVirology [82]

HEK-293 Human embryonickidney

Human Kidney (embryonic) Epithelium ATCC [83]

HeLa "Henrietta Lacks" Human Cervical cancer Epithelium DSMZ [84]ECACC[85]

Hepa1c1c7 Clone 7 of clone 1hepatoma line 1

Mouse Hepatoma Epithelial ECACC [86] ATCC[87]

High Five cells Insect (moth) -Trichoplusia ni

Ovary

Cell culture 9

HL-60 Human leukemia Human Myeloblast Blood cells ECACC [88]DSMZ[89]

HMEC Human mammaryepithelial cell

Human Epithelium ECACC [90]

HT-29 Human Colon epithelium Adenocarcinoma ECACC [91] CLDB[92]

HUVEC Human umbilical veinendothelial cell

Human Umbilical vein endothelium Epithelial ECACC [93] CLDB[94]

Jurkat Human T cell leukemia white blood cells ECACC [95] DSMZ[96]

J558L cells Mouse Myeloma B lymphocyte cell ECACC [97]

JY cells Human Lymphoblastoid EBV immortalised B cell ECACC [97]

K562 cells Human Lymphoblastoid CML blast crisis ECACC [97]

Ku812 Human Lymphoblastoid Erythroleukemia ECACC [98]

LGCstandards [99]

KCL22 Human Lymphoblastoid CML

KG1 Human Lymphoblastoid AML

KYO1 Kyoto 1 Human Lymphoblastoid CML DSMZ [100]

LNCap Lymph node cancer ofthe prostate

Human Prostatic adenocarcinoma Epithelial ECACC [101]ATCC[102]

Ma-Mel 1, 2,3....48

Human A range of melanoma celllines

MC-38 Mouse Adenocarcinoma

MCF-7 Michigan CancerFoundation-7

Human Mammary gland Invasive breast ductalcarcinoma

ER+, PR+

MCF-10A Michigan CancerFoundation

Human Mammary gland Epithelium ATCC [103]

MDA-MB-231 M.D. Anderson -metastatic breast

Human Breast Cancer ECACC [104]

MDA-MB-468 M.D. Anderson -metastatic breast

Human Breast Cancer ECACC [104]

MDA-MB-435 M.D. Anderson -Metastatic Breast

Human Breast Melanoma or carcinoma(disputed)

Cambridge Pathology[105] ECACC [104]

MDCK II Madin Darby caninekidney

Dog Kidney Epithelium ECACC [106] ATCC[107]

MDCK II Madin Darby caninekidney

Dog Kidney Epithelium [106] ATCC [107]

MG63 Human Bone Osteosarcoma

MOR/0.2R Human Lung ECACC [108]

MONO-MAC 6 Human WBC Myeloid metaplasic AML CLDB [109]

MRC5 Human (foetal) Lung Fibroblast]

Cell culture 10

MTD-1A Mouse Epithelium

MyEnd Myocardial endothelial Mouse Endothelium

NCI-H69/CPR Human Lung ECACC [110]

NCI-H69/LX10 Human Lung ECACC [111]

NCI-H69/LX20 Human Lung ECACC [112]

NCI-H69/LX4 Human Lung ECACC [113]

NIH-3T3 NIH, 3-day transfer,inoculum 3 x 105 cells

Mouse Embryo Fibroblast ECACC [114]ATCC[115]

NALM-1 Peripheral blood Blast-crisis CML Cancer Genetics andCytogenetics [116]

NW-145 Melanoma ESTDAB [117]

OPCN / OPCTcell lines

Onyvax [118] prostatecancer....

Range of prostate tumourlines

Asterand [119]

Peer Human T cell leukemia DSMZ [120]

PNT-1A / PNT2

Prostate tumour lines ECACC [35]

Raji human B lymphoma lymphoblast-like

RBL cells Rat BasophilicLeukaemia

Rat Leukaemia Basophil cell ECACC [97]

RenCa Renal carcinoma Mouse Renal carcinoma

RIN-5F Mouse Pancreas

RMA/RMAS Mouse T cell tumour

Saos-2 cells Human Osteosarcoma ECACC [121]

Sf21 Spodoptera frugiperda Insect (moth) -Spodopterafrugiperda

Ovary DSMZ [122]ECACC[123]

Sf9 Spodoptera frugiperda Insect (moth) -Spodopterafrugiperda

Ovary DSMZ [124]ECACC[125]

SiHa Human Cervical cancer Epithelium ECACC [126]

SKBR3 Sloan-Kettering HER23+ Breast Cancer

Human Breast carcinoma [127]

SKOV-3 Sloan-Kettering HER23+ Ovarian Cancer

Human ovary adenocarcinoma [128]

T2 Human T cell leukemia/B cell linehybridoma

DSMZ [129]

T-47D Human Mammary gland Ductal carcinoma

T84 Human Colorectal carcinoma /Lung metastasis

Epithelium ECACC [130]ATCC[131]

THP1 cell line Human Monocyte AML ECACC [132]

U373 Human Glioblastoma-astrocytoma Epithelium

Cell culture 11

U87 Human Glioblastoma-astrocytoma Epithelial-like Abcam [133]

U937 Human Leukaemic monocyticlymphoma

ECACC [35]

VCaP Vertebra prostatecancer

Human Metastatic prostate cancer Epithelial ECACC [134] ATCC[135]

Vero cells Vero (truth) African greenmonkey

Kidney epithelium ECACC [136]

WM39 Human Skin Primary melanoma

WT-49 Human Lymphoblastoid

X63 Mouse Melanoma

YAC-1 Mouse Lymphoma CLDB [137] ECACC[138]

YAR Human B cell EBV transofrmed [139] HumanImmunology [140]

References and notes[2] http:/ / www. whonamedit. com/ synd. cfm/ 2119. html[3] Schiff, JudithAnn. Yale Alumni Magazine, February 2002.[13] http:/ / www. sciencemag. org/ content/ 310/ 5751/ 1139. abstract[14] Gilbert, P.M. et al. Substrate elasticity regulates skeletal muscle stem cell self-renewal in culture. Science 329, 1078–1081 (2010).http:/ /

www. sciencemag. org/ content/ 329/ 5995/ 1078. short[15] Chowdhury, F. et al. Soft substrates promote homogeneous self-renewal of embryonic stem cells via downregulating cell-matrix tractions.

PLoS ONE 5, e15655 (2010).http:/ / www. plosone. org/ article/ info%3Adoi%2F10. 1371%2Fjournal. pone. 0015655[16] Engler, A.J., Sen, S., Sweeney, H.L. & Discher, D.E. Matrix elasticity directs stem cell lineage specification. Cell 126, 677-689 (2006).http:/

/ www. cell. com/ retrieve/ pii/ S0092867406009615[17] Paszek, M.J. et al. Tensional homeostasis and the malignant phenotype. Cancer Cell 8, 241-254 (2005).http:/ / www. cell. com/ cancer-cell/

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jcb. rupress. org/ content/ 190/ 4/ 693. short[21] Wipff, P.-J., Rifkin, D.B., Meister, J.-J. & Hinz, B. Myofibroblast contraction activates latent TGF-beta1 from the extracellular matrix. J.

Cell Biol 179, 1311–1323 (2007).http:/ / jcb. rupress. org/ content/ 179/ 6/ 1311. short[22] Georges, P.C. et al. Increased stiffness of the rat liver precedes matrix deposition: implications for fibrosis. Am. J. Physiol. Gastrointest.

Liver Physiol 293, G1147-1154 (2007).http:/ / ajpgi. physiology. org/ content/ 293/ 6/ G1147. short[23] Li, L. et al. Functional modulation of ES-derived hepatocyte lineage cells via substrate compliance alteration. Ann Biomed Eng 36, 865-876

(2008).http:/ / www. springerlink. com/ content/ n101pmj76k162433/[24] Semler, E.J., Lancin, P.A., Dasgupta, A. & Moghe, P.V. Engineering hepatocellular morphogenesis and function via ligand-presenting

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[25] Friedland, J.C., Lee, M.H. & Boettiger, D. Mechanically Activated Integrin Switch Controls α5β1 Function. Science 323, 642 -644(2009).http:/ / www. sciencemag. org/ content/ 323/ 5914/ 642. short

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[28] http:/ / www. nature. com/ drugdisc/ news/ articles/ 424870a. html[29] Three-dimensional tissue culture based on magnetic cell levitation Glauco R. Souza1,9, Jennifer R. Molina2, Robert M. Raphael3, Michael

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abs/ nnano. 2010. 23. html[30] Amy Y. Hsaio, Yi-Chung Tung, Xianggui Qu, Lalit R. Patel, Kenneth J. Pienta, Shuichi Takayma. 384 Hanging Drop Arrays Give Excellent

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Cell culture 13

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Cell culture 14

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• Hpacultures.org.uk (http:/ / www. hpacultures. org. uk/ ), Health Protection Agency Culture Collections(ECACC)

• MacLeod, R. A. F.; Dirks, Wilhelm G.; Matsuo, Yoshinobu; Kaufmann, Maren; Milch, Herbert; Drexler, Hans G.(1999). "Widespread intraspecies cross-contamination of human tumour cell lines". International Journal ofCancer 83 (4): 555–563. doi: 10.1002/(SICI)1097-0215(19991112)83:4<555::AID-IJC19>3.0.CO;2-2 (http:/ / dx.doi. org/ 10. 1002/ (SICI)1097-0215(19991112)83:4<555::AID-IJC19>3. 0. CO;2-2). PMID  10508494 (http:/ /www. ncbi. nlm. nih. gov/ pubmed/ 10508494).

• Masters, John R. (2002). "HeLa cells 50 years on: the good, the bad and the ugly". Nature Reviews Cancer 2 (4):315–319. doi: 10.1038/nrc775 (http:/ / dx. doi. org/ 10. 1038/ nrc775). PMID  12001993 (http:/ / www. ncbi. nlm.nih. gov/ pubmed/ 12001993).

• Recently invented was the 3D Petri dish, the first 3D cell culture (http:/ / www. microtissues. com) offering.

External links• Table of common cell lines from Alberts 4th ed. (http:/ / www. ncbi. nlm. nih. gov/ books/ bv. fcgi?rid=mboc4.

table. 1515)• Cancer Cells in Culture (http:/ / users. rcn. com/ jkimball. ma. ultranet/ BiologyPages/ C/ CancerCellsInCulture.

html)• Hypertext version of the Cell Line Data Base (http:/ / bioinformatics. istge. it/ hypercldb/ )• Cell Culture Basics (http:/ / www. invitrogen. com/ site/ us/ en/ home/ References/ gibco-cell-culture-basics.

html) - Introduction to cell culture, covering topics such as laboratory set-up, safety and aseptic techniqueincluding basic cell culture protocols and video training

• Database of Who's Who in Cell Culture and Related Research (http:/ / www. mavensemantic. com/ )• Witkowski JA. Experimental pathology and the origins of tissue culture: Leo Loeb's contribution. (http:/ / www.

pubmedcentral. gov/ articlerender. fcgi?artid=1139336) Med Hist. 1983 July; 27(3): 269–288.• Coriell Cell Repositories (http:/ / ccr. coriell. org/ )• The National Centre for Cell Science (http:/ / www. nccs. res. in/ ) (NCCS), Pune, India; national repository for

cell lines/hybridomas etc.• Neural Stem Cell Culture: Neurosphere generation, microscopical analysis and cryopreservation (a protocol)

(http:/ / www. natureprotocols. com/ 2006/ 08/ 25/ neural_stem_cell_culture_neuro. php)• Rat Chromaffin cells primary cultures: Standardization and quality assessment for single-cell assays (a protocol)

(http:/ / www. natureprotocols. com/ 2006/ 09/ 29/ rat_chromaffin_cells_primary_c. php)

Article Sources and Contributors 15

Article Sources and ContributorsCell culture  Source: http://en.wikipedia.org/w/index.php?oldid=545257225  Contributors: 124Nick, 2001:700:303:1:3144:4029:7CA8:328D, 22Kartika, 3D Cell Culture, AManWithNoPlan,Aaron1214, Abstraktn, Al Lemos, AlanUS, Anat, Ariccapel, ArkadiRenko, Ashley Payne, Barbaraparodi, Bcary, Bdekker, Beliysh, Ben Ben, Benbest, Bioxpert, Bobo192, Boghog, Boothy443,Bowlhover, Bpavel, Catgut, Cclehnen, Ceyockey, Cfroberg, Charles Matthews, Chris Capoccia, ChrisGualtieri, Chrisjwmartin, Christian75, Ciar, Cinik, ClockworkSoul, Cmcnicoll,Colin.sanctuary, Colincbn, Coolcaesar, Cquan, Creidieki, Cybercobra, DO11.10, Daisy1620, Deli nk, Diederikklaassen, Dlindner, Dogaroon, DoktorDec, Dominiquewikki, Dr Aaron,Dysmorodrepanis, EJF, ElinneaG, Emote, Enozkan, Escape Orbit, Fences and windows, FiddleheadLP, Fvandrog, G716, Gabbe, Gaius Cornelius, GetAgrippa, Glane23, Graham87, Grumbler12,GrummelMC, Gökhan, Hakunamenta, HarlandQPitt, Heathhunnicutt, Hebrides, Hex, Hu12, Iamnotanorange, Int21h, Ixfd64, J04n, Jackhynes, Jacopo Werther, Jakob Suckale, JamesBWatson,Jauerback, Jesse V., Jim1138, John Vandenberg, Johni Bezuiden, JonHarder, KJS77, Ka Faraq Gatri, Kaarel, Kaisershatner, Kalaiarasy, Kaushal mehta, Kbir1, Kjkolb, Krickrack, Lcwilsie,Ligulem, LilHelpa, Ling.Nut, LittleWink, Lord Anubis, LostLucidity, Lotez, Lotje, Lscox, MTWEmperor, MacDaid, Mardueng, Marek69, Martious, MassimoScipio, Materialscientist, Matrigen,Matt.T, Maxxicum, Mcuddihy, Mhirschey, MichaK, Mikael Häggström, Mikeo, Mild Bill Hiccup, Moez, Moshe Constantine Hassan Al-Silverburg, Myseducatedgal, Nick Number,Occamsrazorwit, Oguk, Okruzhnoy, Onebravemonkey, Owenjm2, PDH, PaoloRomano, Pekaje, Pen1234567, Peter Znamenskiy, Ph.eyes, Philip Trueman, Pinethicket, Pmlineditor, Ppgardne,Prabhubct, Pwb, Ramubra, Reinoutr, Remuel, Rengachen27, Reo On, Rich Farmbrough, Richard Arthur Norton (1958- ), Rishi.bedi, Rjwilmsi, Ronhjones, RupertMillard, SchroCat, Seans PotatoBusiness, Selket, Shinryuu, Snowmanradio, Squidonius, Stephanelarre, Sumitdr, Tameeria, The Thing That Should Not Be, TheGrimReaper NS, Thingg, Tigerman20, TimVickers, Tmyara,Tpbradbury, Tree1983, Treyt021, Truthflux, UtherSRG, Vespristiano, Vhuang715, Victor D, Vogon77, Vsmith, Vssailu, WAS 4.250, Walkiped, Wasell, Why Not A Duck, Williamheyn,Wisdom89, Wobble, Woohookitty, WriterHound, Xtothel, Yvaud, ZePedroPONTO, Zeamays, Zephyris, 271 ,حامد صوفی anonymous edits

Image Sources, Licenses and ContributorsFile:Cell Culture in a tiny Petri dish.jpg  Source: http://en.wikipedia.org/w/index.php?title=File:Cell_Culture_in_a_tiny_Petri_dish.jpg  License: Creative Commons Attribution 2.0 Contributors: kaibara87File:Epithelial-cells.jpg  Source: http://en.wikipedia.org/w/index.php?title=File:Epithelial-cells.jpg  License: GNU Free Documentation License  Contributors: Dbc334, Dietzel65, Duesentrieb,GeorgHH, Helix84, JWSchmidt, Martin H., ViperSnake151, Was a bee, 2 anonymous edits

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