Cell culture


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 under controlled conditions. In practice, the term "cell culture" has come to refer to the culturing of cells derived from singlecellular eukaryotes, especially animal cells. However, there are also cultures of plants, fungi and microbes, including viruses, bacteria and protists. The historical development and methods of cell culture are closely interrelated to those of tissue culture and organ culture.

Animal cell culture began when scientists removed a small amount of monkey kidney from a host organism and maintained it in a warm saline solution for several days, establishing the principle of tissue culture.[1]

Contents

Concepts in mammalian cell culture

Isolation of cells

Cells 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 tissues by enzymatic digestion with enzymes such as collagenase, trypsin, or pronase, which break down the extracellular matrix. Alternatively, pieces of tissue can be placed in growth media, and the cells that grow out are available for culture. 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 from tumors, most primary cell cultures have limited lifespan. After a certain number of population doublings (called the Hayflick limit), cells undergo the process of senescence and stop dividing, while generally retaining viability.

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

Maintaining cells in culture

Cells are grown and maintained at an appropriate temperature and gas mixture (typically, 37°C, 5% CO2 for mammalian cells) in a cell incubator. Culture conditions vary widely for each cell type, and variation of conditions for a particular cell type can result in different phenotypes being expressed.

Aside from temperature and gas mixture, the most commonly varied factor in culture systems is the growth medium. Recipes for growth media can vary in pH, glucose concentration, growth factors, and the presence of other nutrients. 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 or prions, particularly in medical biotechnology applications. Current practice is to minimize or eliminate the use of these 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 as Australia and New Zealand, and using purified nutrient concentrates derived from serum in place of whole animal serum for cell culture.[2]

Plating density (number of cells per volume of culture medium) plays a critical role for some cell types. For example, a lower plating density makes granulosa cells exhibit estrogen production, while a higher plating density makes them appear as progesterone-producing theca lutein cells.[3]

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

Cell line cross-contamination

Cell line cross-contamination can be a problem for scientists working with cultured cells. Studies suggest anywhere from 15–20% of the time, cells used in experiments have been misidentified or contaminated with another cell line.[4][5][6] 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.[7][8] Major cell line repositories, including the American Type Culture Collection (ATCC) and the German Collection of Microorganisms and Cell Cultures (DSMZ), have received cell line submissions from researchers that were misidentified by them.[7][9] Such contamination poses a problem for the quality of research produced using cell culture lines, 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.[11]

To address this problem of cell line cross-contamination, researchers are encouraged to authenticate their cell lines at an early passage to establish the identity of the cell line. Authentication should be repeated before freezing cell line stocks, every two months during active culturing and before any publication of research data generated using the cell lines. Many methods are used to identify cell lines, including isoenzyme analysis, human lymphocyte antigen (HLA) typing, chromosomal analysis, karyotyping, morphology and STR analysis.[11]

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

Manipulation of cultured cells

As cells generally continue to divide in culture, they generally grow to fill the available area or volume. This can generate several issues:

Among the common manipulations carried out on culture cells are media changes, passaging cells, and transfecting cells. These are generally performed using tissue culture methods that rely on sterile technique. Sterile technique aims to avoid contamination with bacteria, yeast, or other cell lines. Manipulations are typically carried out in a biosafety hood or laminar flow cabinet to exclude contaminating micro-organisms. Antibiotics (e.g. penicillin and streptomycin) 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 the medium to measure nutrient depletion.

Media changes

In the case of adherent cultures, the media can be removed directly by aspiration, and then is replaced.

Passaging cells

Passaging (also known as subculture or splitting cells) involves transferring a small number of cells into a new vessel. Cells can be cultured for a longer time if they are split regularly, as it avoids the senescence associated with prolonged high cell density. Suspension cultures are easily passaged with a small amount of culture containing a few cells diluted in a larger volume of fresh media. For adherent cultures, cells first need to be detached; this is commonly 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 is often performed to cause cells to express a protein of interest. More recently, the transfection of RNAi constructs have been realized as a convenient mechanism for suppressing the expression of a particular gene/protein. DNA can also 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 of reproduction.

Established human cell lines

Cultured HeLa cells have been stained with Hoechst turning their nuclei blue, and are one of the earliest human cell lines descended from Henrietta Lacks, who died of cervical cancer from which these cells originated.

Cell lines that originate with humans have been somewhat controversial in bioethics, as they may outlive their parent organism 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 have no property rights in cell lines derived from organs removed with their consent.[12]

Generation of hybridomas

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

Applications of cell culture

Mass culture of animal cell lines is fundamental to the manufacture of viral vaccines and other products of biotechnology

Biological 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 are glycosylated (carbohydrate-modified) currently must be made in animal cells. An important example of such a complex protein is the hormone erythropoietin. The cost of growing mammalian cell cultures is high, so research is underway to produce such complex proteins in insect cells or in higher plants, use of single embryonic cell and somatic embryos as a source for direct gene transfer via particle bombardment, transit gene expression and confocal microscopy observation is one of its applications. It also offers to confirm single cell origin of somatic embryos and the asymmetry of the first cell division, which starts the process.


Cell culture in two dimensions

Research in tissue engineering, stem cells and molecular biology primarily involves cultures of cells on flat plastic dishes. This technique is known as two-dimensional (2D) cell culture, and was first developed by Wilhelm Roux who, in 1885, removed a portion of the medullary plate of an embryonic chicken and maintained it in warm saline for several days on a flat glass plate. With the advance of polymer technology, today's standard plastic dish for 2D cell culture, commonly known as the Petri dish, was invented by Julius Richard Petri, a German bacteriologist, who is generally credited with inventing the Petri dish while working as assistant to Robert Koch.

Tissue culture and engineering

Cell culture is a fundamental component of tissue culture and tissue engineering, as it establishes the basics of growing and maintaining cells ex vivo. The major application of human cell culture is in stem cell industry, where mesenchymal stem cells can be cultured and cryopreserved for future use.

Vaccines

Vaccines for polio, measles, mumps, rubella, and chickenpox are currently made in cell cultures. Due to the H5N1 pandemic threat, research into using cell culture for influenza vaccines is being funded by the United States government. Novel ideas in the field include recombinant DNA-based vaccines, such as one made using human adenovirus (a common cold virus) as a vector,[13][14] and novel adjuvants.[15]

Culture of non-mammalian cells

Plant cell culture methods

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

Bacterial and yeast culture methods

For bacteria and yeasts, small quantities of cells are usually grown on a solid support that contains nutrients embedded in it, usually a gel such as agar, while large-scale cultures are grown with the cells suspended in a nutrient broth.

Viral culture methods

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

Common cell lines

Human cell lines
Primate cell lines
  • Vero (African green monkey Chlorocebus kidney epithelial cell line initiated in 1962)
Rat tumor cell lines
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

List of cell lines

Cell line Meaning Organism Origin tissue Morphology Link
293-T Human Kidney (embryonic) Derivative of HEK 293 ECACC Cell Lines Data Base (CLDB)
3T3 cells "3-day transfer, inoculum 3 x 10^5 cells" Mouse Embryonic fibroblast Also known as NIH 3T3 ECACC. Search for the many 3T3 cells in the CLDB.
721 Human Melanoma
9L Rat Glioblastoma
A2780 Human Ovary Ovarian cancer ECACC, CLDB
A2780ADR Human Ovary Adriamycin-resistant derivative ECACC
A2780cis Human Ovary Cisplatin-resistant derivative ECACC
A172 Human Glioblastoma Malignant glioma ECACC
A20 Murine B lymphoma B lymphocyte
A253 Human Head and neck carcinoma Submandibular duct
A431 Human Skin epithelium Squamous cell carcinoma ECACCCLDB
A-549 Human Lungcarcinoma Epithelium DSMZECACC
ALC Murine Bone marrow Stroma PubMed
B16 Murine Melanoma ECCAC
B35 Rat Neuroblastoma ATCC
BCP-1 cells Human PBMC HIV+ lymphoma ATCC
BEAS-2B Bronchial epithelium + Adenovirus 12-SV40 virus hybrid (Ad12SV40) Human Lung Epithelial ATCC
bEnd.3 Brain endothelial Mouse Brain/cerebral cortex Endothelium ATCC
BHK-21 Baby hamster kidney fibroblast cells Hamster Kidney Fibroblast ECACCOlympus
BR 293 Human Breast Breast cancer
BxPC3 Biopsy xenograph of pancreatic carcinoma line 3 Human Pancreatic adenocarcinoma Epithelial ATCC
C2C12 Mouse Myoblast cell line ECACC
C3H-10T1/2 Mouse Embryonic mesenchymal cell line ECACC
C6/36 Asian tiger mosquito Larval tissue ECACC
Cal-27 Human Tongue Squamous cell carcinoma
CHO Chinese hamster ovary Hamster Ovary Epithelium ECACCICLC
COR-L23 Human Lung ECACC
COR-L23/CPR Human Lung ECACC
COR-L23/5010 Human Lung ECACC
COR-L23/R23 Human Lung Epithelial ECACC
COS-7 Cercopithecus aethiops, origin-defective SV-40 Ape - Cercopithecus aethiops (Chlorocebus) Kidney Fibroblast ECACCATCC
COV-434 Human Ovary Metastatic granulosa cell carcinoma [1]ECACC
CML T1 Chronic myeloid leukaemia T lymphocyte 1 Human CML acute phase T cell leukaemia Blood
CMT Canine mammary tumor Dog Mammary gland Epithelium
CT26 Murine Colorectal carcinoma Colon
D17 Canine Osteosarcoma ECACC
DH82 Canine Histiocytosis Monocyte/macrophage ECACC

J Vir Meth

DU145 Human Androgen insensitive carcinoma Prostate
DuCaP Dura mater cancer of the prostate Human Metastatic prostate cancer Epithelial PubMed

{Ehrlich ascites carcinoma} mice

EL4 Mouse T cell leukaemia ECACC
EM2 Human CML blast crisis Ph+ CML line CLDB
EM3 Human CML blast crisis Ph+ CML line CLDB
EMT6/AR1 Mouse Breast Epithelial-like ECACC
EMT6/AR10.0 Mouse Breast Epithelial-like ECACC
FM3 Human Metastatic lymph node Melanoma
H1299 Human Lung Lung cancer
H69 Human Lung ECACC
HB54 Hybridoma Hybridoma Secretes L243 mAb (against HLA-DR) Human Immunology
HB55 Hybridoma Hybridoma secretes MA2.1 mAb (against HLA-A2 and HLA-B17) Journal of Immunology
HCA2 Human Fibroblast Journal of General Virology
HEK-293 Human embryonic kidney Human Kidney (embryonic) Epithelium ATCC
HeLa "Henrietta Lacks" Human Cervical cancer Epithelium DSMZECACC
Hepa1c1c7 Clone 7 of clone 1 hepatoma line 1 Mouse Hepatoma Epithelial ECACC

ATCC

HL-60 Human leukemia Human Myeloblast Blood cells ECACCDSMZ
HMEC Human mammary epithelial cell Human Epithelium ECACC
HT-29 Human Colon epithelium Adenocarcinoma ECACC

CLDB

Jurkat Human T cell leukemia white blood cells ECACC

DSMZ

JY cells Human Lymphoblastoid EBV immortalised B cell
K562 cells Human Lymphoblastoid CML blast crisis ECACC
Ku812 Human Lymphoblastoid Erythroleukemia ECACC

LGCstandards

KCL22 Human Lymphoblastoid CML
KG1 Human Lymphoblastoid AML
KYO1 Kyoto 1 Human Lymphoblastoid CML DSMZ
LNCap Lymph node cancer of the prostate Human Prostatic adenocarcinoma Epithelial ECACCATCC
Ma-Mel 1, 2, 3....48 Human A range of melanoma cell lines
MC-38 Mouse Adenocarcinoma
MCF-7 Michigan Cancer Foundation-7 Human Mammary gland Invasive breast ductal carcinoma ER+, PR+
MCF-10A Michigan Cancer Foundation Human Mammary gland Epithelium ATCC
MDA-MB-231 M.D. Anderson - metastatic breast Human Breast Cancer ECACC
MDA-MB-468 M.D. Anderson - metastatic breast Human Breast Cancer ECACC
MDA-MB-435 M.D. Anderson - Metastatic Breast Human Breast Melanoma or carcinoma (disputed) Cambridge Pathology ECACC
MDCK II Madin Darby canine kidney Dog Kidney Epithelium ECACC ATCC
MDCK II Madin Darby canine kidney Dog Kidney Epithelium [2] ATCC
MG63 Human Bone Osteosarcoma
MOR/0.2R Human Lung ECACC
MONO-MAC 6 Human WBC Myeloid metaplasic AML CLDB
MRC5 Human (foetal) Lung Fibroblast]
MTD-1A Mouse Epithelium
MyEnd Myocardial endothelial Mouse Endothelium
NCI-H69/CPR Human Lung ECACC
NCI-H69/LX10 Human Lung ECACC
NCI-H69/LX20 Human Lung ECACC
NCI-H69/LX4 Human Lung ECACC
NIH-3T3 NIH, 3-day transfer, inoculum 3 x 105 cells Mouse Embryo Fibroblast ECACCATCC
NALM-1 Peripheral blood Blast-crisis CML Cancer Genetics and Cytogenetics
NW-145 Melanoma ESTDAB
OPCN / OPCT cell lines Onyvax [3] prostate cancer.... Range of prostate tumour lines Asterand
Peer Human T cell leukemia DSMZ
PNT-1A / PNT 2 Prostate tumour lines ECACC
RenCa Renal carcinoma Mouse Renal carcinoma
RIN-5F Mouse Pancreas
RMA/RMAS Mouse T cell tumour
Saos-2 cells Human Osteosarcoma ECACC
Sf-9 Spodoptera frugiperda Insect - Spodoptera frugiperda (moth) Ovary DSMZECACC
SiHa Human Cervical cancer Epithelium ECACC
SkBr3 Human Breast carcinoma
T2 Human T cell leukemia/B cell line hybridoma DSMZ
T-47D Human Mammary gland Ductal carcinoma
T84 Human Colorectal carcinoma / Lung metastasis Epithelium ECACCATCC
THP1 cell line Human Monocyte AML ECACC
U373 Human Glioblastoma-astrocytoma Epithelium
U87 Human Glioblastoma-astrocytoma Epithelial-like Abcam
U937 Human Leukaemic monocytic lymphoma ECACC
VCaP Vertebra prostate cancer Human Metastatic prostate cancer Epithelial ECACC ATCC
Vero cells Vero (truth) African green monkey Kidney epithelium ECACC
WM39 Human Skin Primary melanoma
WT-49 Human Lymphoblastoid
X63 Mouse Melanoma
YAC-1 Mouse Lymphoma CLDB ECACC
YAR Human B cell EBV transofrmed [4] Human Immunology

See also

References and notes

  1. ^ {{cite web|url=http://caat.jhsph.edu/pubsthe virus in monkey kidney cell cultures.
  2. ^ "LipiMAX purified lipoprotein solution from bovine serum". Selborne Biological Services. 2006. http://www.selbornebiological.com/products/lipimax.htm. Retrieved 2010-02-02. 
  3. ^ Portela VM, Zamberlam G, Price CA (April 2010). "Cell plating density alters the ratio of estrogenic to progestagenic enzyme gene expression in cultured granulosa cells". Fertil. Steril. 93 (6): 2050–5. doi:10.1016/j.fertnstert.2009.01.151. PMID 19324349. 
  4. ^ Drexler, HG; Dirks, WG; Macleod, RA (Oct 1999). "False human hematopoietic cell lines: cross-contaminations and misinterpretations". Leukemia 13 (10): 1601–7. doi:10.1038/sj/leu/2401510. ISSN 0887-6924. PMID 10516762. 
  5. ^ Drexler, HG; Macleod, RA; Dirks, WG (Dec 2001). "Cross-contamination: HS-Sultan is not a myeloma but a Burkitt lymphoma cell line" (Free full text). Blood 98 (12): 3495–6. doi:10.1182/blood.V98.12.3495. ISSN 0006-4971. PMID 11732505. http://www.bloodjournal.org/cgi/pmidlookup?view=long&pmid=11732505. 
  6. ^ Cabrera, CM; Cobo, F; Nieto, A; Cortés, JL; Montes, RM; Catalina, P; Concha, A (Jun 2006). "Identity tests: determination of cell line cross-contamination". Cytotechnology 51 (2): 45–50. doi:10.1007/s10616-006-9013-8. ISSN 0920-9069. PMID 19002894. 
  7. ^ a b Chatterjee, R (Feb 2007). "Cell biology. Cases of mistaken identity.". Science 315 (5814): 928–31. doi:10.1126/science.315.5814.928. ISSN 0036-8075. PMID 17303729. 
  8. ^ Liscovitch, M; Ravid, D (Jan 2007). "A case study in misidentification of cancer cell lines: MCF-7/AdrR cells (re-designated NCI/ADR-RES) are derived from OVCAR-8 human ovarian carcinoma cells.". Cancer letters 245 (1-2): 350–2. doi:10.1016/j.canlet.2006.01.013. ISSN 0304-3835. PMID 16504380. 
  9. ^ Macleod, RA; Dirks, WG; Matsuo, Y; Kaufmann, M; Milch, H; Drexler, HG (Nov 1999). "Widespread intraspecies cross-contamination of human tumor cell lines arising at source.". International journal of cancer. Journal international du cancer 83 (4): 555–63. doi:10.1002/(SICI)1097-0215(19991112)83:4<555::AID-IJC19>3.0.CO;2-2. ISSN 0020-7136. PMID 10508494. 
  10. ^ Masters, JR (Apr 2002). "HeLa cells 50 years on: the good, the bad and the ugly.". Nature reviews. Cancer 2 (4): 315–9. doi:10.1038/nrc775. ISSN 1474-175X. PMID 12001993. 
  11. ^ a b Dunham, J.H.; Guthmiller, P. (2008). "Doing good science: Authenticating cell line identity" (PDF). Cell Notes 22: 15–17. http://www.promega.com/cnotes/cn022/cn022_15.pdf. 
  12. ^ Ceb.com
  13. ^ Reuters (2006-01-26). Wired.com "Quickie Bird Flu Vaccine Created". Wired. http://wired.com/news/wireservice/0,70102-0.html?tw=wn_index_7 Wired.com. Retrieved 2010-01-31. 
  14. ^ Gao W, Soloff AC, Lu X, Montecalvo A, Nguyen DC, Matsuoka Y, Robbins PD, Swayne DE, Donis RO, Katz JM, Barratt-Boyes SM, Gambotto A. (February 2006). "Protection of mice and poultry from lethal H5N1 avian influenza virus through adenovirus-based immunization". Journal of Virology (United States: American Society for Microbiology) 80 (4): 1959–1964. doi:10.1128/JVI.80.4.1959-1964.2006. ISSN 0022-538X. PMC 1367171. PMID 16439551. http://jvi.asm.org/cgi/content/abstract/80/4/1959. Retrieved 2010-01-31. 
  15. ^ "NIAID Taps Chiron to Develop Vaccine Against H9N2 Avian Influenza". National Institute of Allergy and Infectious Diseases (NIAID). 2004-08-17. http://www3.niaid.nih.gov/news/newsreleases/2004/h9n2.htm. Retrieved 2010-01-31. [dead link]

External links


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