Introducció

The Cell Immortalization Unit of the Josep Carreras Leukaemia Research Institute offers Infection of B-cells with Epstein–Barr virus (EBV) leads to more and subsequent immortalization. This is considered as the method of choice for generating lymphoblastoid cell lines (LCLs). Cell culture is an essential tool to study the fundamentals of genetic background variables. With the development of personalized medicine, this applies increasingly to the development and safety testing of drugs. 

BACKGROUND

Infection of B-cells with Epstein–Barr virus (EBV) leads to more and subsequent immortalization. This is considered as the method of choice for generating lymphoblastoid cell lines (LCLs). Producing LCLs, although very useful but is very time consuming and troublesome, drives the requirement for quicker and more reliable methods for EBV-driven B-cell transformation.

The immortalized cells are derived from primary cells, thereby establishing an immortalized cell line to achieve the cells cultured in vitro. The purpose of unlimited proliferation and no difference between cells.

CELL IMMORTALIZATION

The primary cells only undergo a predetermined and limited number of cell division in the process of culture, however, after a limited population multiplication (the number varies according to species, cell types and culture conditions), the primary cells enter a state called replication aging, in which they no longer divide. So, scientists often need to recreate new cultures – a complex and lengthy process. Compared with primary cells, immortalized cells can solve this problem to a certain extent.

The exogenous immortalized genes were introduced into the target cells by gene transfection technology to establish immortalized cell lines, so as to make the cells cultured in vitro have the ability of proliferation and there is no difference between cells. Immortalized cells can be passed on continuously, enabling scientists to use the same consistent cells throughout the research project.

Summary protocol of Cell Immortalization Technology:

Peripheral Blood B Lymphocyte Immortalization with Epstein Barr Virus (EBV) Protocol

After successfully production of LCLs, different parameters including temperature, serum concentration, type of culture medium, and CO₂ concentration have to be evaluated on EBV-transformed B-cells. Our unit are able to produce LCLs and optimize condition.

APPLICATIONS

- This immortalization technology enables rapid, efficient, and reliable production of unlimited numbers of personalized cells.

- To produce control material for rare genetic disorders.

- Lymphocyte Immortalization technique let to preserve of DNA, RNA and proteins samples, that appears to be a valid strategy for further studies.

- To determine optimized condition for reliable and reproducible LCLs from different sources.

- Testing drugs analysis.

- Allows us to have enough biological sample without having to access the patient again. 

EBV immortalized B lymphocyte cell lines have been extensively used as a source of biological material for functional and molecular studies and represent a potentially limitless source of genomic DNA, expression RNA, and protein and system biology analysis.

OUR AVANTAGES

The Cell Immortalization Unit works closely with you in an integrated way beginning with the discussion of the project (including project outline and experimental design). we are continuously working to optimize the standard operation procedures and implementing the latest methodologies. 

With our custom service, you can get our high-quality customer service experience with high success rates, frequent communication, and fast timelines.

CELL IMMORTALIZATION UNIT TEAM

The Cell Immortalization Unit has a broad scientific and technological experienced to setup successfully immortalize Lymphocyte cells with the function you need. Our custom immortalization service can significantly extend replicative capacity of your target cells, which saves your time and money over trying by yourself. 

Carolina De La Torre
Manager of the Cell Immortalization Unit 

Fernando Setien
Specialist in Cell Immortalization technology  

We look forward to collaborating with you!

PUBLICATIONS USING THE CELL IMMORTALIZATION TECHNOLOGY

Minority monogenic diseases:

The Cell Immortalization Unit exhibits a great experience in the establishment of EBV-transformed B-cells from a wide spectrum of rare disorders, such as Rett syndrome, Sotos syndrome, Rubinstein-Taybi syndrome, Ohdo syndrome, MECP2 duplication syndrome, Werner syndrome, Dyschromatosis Symmetrica, Paroxysmal Nocturnal Hemoglobinuria, Non Ketotic Hyperglycinemia and Zellweger syndrome, among others.

Selected articles:

- The impact of MECP2 mutations in the expression patterns of Rett syndrome patients. Ballestar E, Ropero S, Alaminos M, Armstrong J, Setien F, Agrelo R, Fraga MF, Herranz M, Avila S, Pineda M, Monros E, Esteller M. Hum Genet. 2005 Jan;116(1-2):91-104. doi: 10.1007/s00439-004-1200-0. Epub 2004 Nov 11.

- Epigenetic inactivation of the Sotos overgrowth syndrome gene histone methyltransferase NSD1 in human neuroblastoma and glioma. Berdasco M, Ropero S, Setien F, Fraga MF, Lapunzina P, Losson R, Alaminos M, Cheung NK, Rahman N, Esteller M. Proc Natl Acad Sci U S A. 2009 Dec 22;106(51):21830-5. doi: 10.1073/pnas.0906831106. Epub 2009 Dec 14.

- A novel Werner Syndrome mutation: pharmacological treatment by read-through of nonsense mutations and epigenetic therapies. Agrelo R, Sutz MA, Setien F, Aldunate F, Esteller M, Da Costa V, Achenbach R. Epigenetics. 2015;10(4):329-41. doi: 10.1080/15592294.2015.1027853.

Hereditary cancer: 

The Cell Immortalization Unit has a top-notch expertise in the development of immortalized B-cell lines from carriers of germline mutation in genetic predisposition syndromes, such as those ocurring in BRCA1 and BRCA1 families (breast and ovarian cancer) and MLH1 and MSH2 (colon, stomach and uterine cancer) families, plus other tumor-associated disorders.

Selected articles:

-  A mutation in the POT1 gene is responsible for cardiac angiosarcoma in TP53-negative Li-Fraumeni-like families. Calvete O, Martinez P, Garcia-Pavia P, Benitez-Buelga C, Paumard-Hernández B, Fernandez V, Dominguez F, Salas C, Romero-Laorden N, Garcia-Donas J, Carrillo J, Perona R, Triviño JC, Andrés R, Cano JM, Rivera B, Alonso-Pulpon L, Setien F, Esteller M, Rodriguez-Perales S, Bougeard G, Frebourg T, Urioste M, Blasco MA, Benítez J. Nat Commun. 2015 Sep 25; 6:8383. doi: 10.1038/ncomms9383.

- Whole-exome sequencing identifies MDH2 as a new familial paraganglioma gene. Cascón A, Comino-Méndez I, Currás-Freixes M, de Cubas AA, Contreras L, Richter S, Peitzsch M, Mancikova V, Inglada-Pérez L, Pérez-Barrios A, Calatayud M, Azriel S, Villar-Vicente R, Aller J, Setién F, Moran S, Garcia JF, Río-Machín A, Letón R, Gómez-Graña Á, Apellániz-Ruiz M, Roncador G, Esteller M, Rodríguez-Antona C, Satrústegui J, Eisenhofer G, Urioste M, Robledo M. J Natl Cancer Inst. 2015 Mar 11;107(5):djv053. doi: 10.1093/jnci/djv053. 

- Germline missense pathogenic variants in the BRCA1 BRCT domain, p. Gly1706Glu and p. Ala1708Glu, increase cellular sensitivity to PARP inhibitor olaparib by a dominant negative effect. Vaclová T, Woods NT, Megías D, Gomez-Lopez S, Setién F, García Bueno JM, Macías JA, Barroso A, Urioste M, Esteller M, Monteiro ANA, Benítez J, Osorio A.  Hum Mol Genet. 2016 Dec 15;25(24):5287-5299. doi: 10.1093/hmg/ddw343.

- Germline mutations in the spindle assembly checkpoint genes BUB1 and BUB3 are infrequent in familial colorectal cancer and polyposis. Mur P, De Voer RM, Olivera-Salguero R, Rodríguez-Perales S, Pons T, Setién F, Aiza G, Valdés-Mas R, Bertini A, Pineda M, Vreede L, Navarro M, Iglesias S, González S, Brunet J, Valencia A, Esteller M, Lázaro C, Kops GJPL, Urioste M, Puente XS, Capellá G, Valle L. Mol Cancer. 2018 Feb 15;17(1):23. doi: 10.1186/s12943-018-0762-8.

- Germline variation in O6-methylguanine-DNA methyltransferase (MGMT) as cause of hereditary colorectal cancer. Belhadj S, Moutinho C, Mur P, Setien F, Llinàs-Arias P, Pérez-Salvia M, Pons T, Pineda M, Brunet J, Navarro M, Capellá G, Esteller M, Valle L. Cancer Lett. 2019 Apr 10;447:86-92. doi: 10.1016/j.canlet.2019.01.019.