Mammalian cell lines used in antibody engineering and expression system


1. Mammalian cell lines

1.1. Mouse myeloma and hybridoma cell lines derived from MOPC21 and used as fusion partners

Among the cell lines derived from MOPC21, the SP2/0 (hybridoma), P3-X63-Ag8.653 (myeloma) and NS0 (myeloma) cell lines are frequently used as fusion partners in the generation of hybridomas. The table below shows their clone origin and their characteristics.

Cell line name Other cell line names Species and strain Cell type and cell lineage Clone origin IG production Cell line characteristics ECACC ATCC IMGT references
MOPC21 MOPC 21, MOPC21 (mineral oil induced plasmacytoma number 21) Mus musculus BALB/c (inbred female mouse) myeloma, B cell secreting IgG1-kappa (H+L+) Horibata and Harris 1970 [1]
P3K Mus musculus BALB/c myeloma, B cell derived from MOPC21 secreting IgG1-kappa (H+L+) suspension, heterogeneous population Horibata and Harris 1970 [1]
P3-X27 Mus musculus BALB/c myeloma, B cell cloned from P3K secreting IgG1-kappa (H+L+) Ramasamy et al. 1974 [2]
NSI/1 289-16 Mus musculus BALB/c myeloma, B cell cloned from P3-X27 non secreting, synthesing kappa (H-L+ intracellular) suspension Ramasamy et al. 1974 [2], Cowan et al. 1974 [7]
NS-1 P3/NSI/1-Ag4-1, NS-1-Ag4-1, P3-NS-1/1-Ag4-1 Mus musculus BALB/c myeloma, B cell cloned from NSI/1 non secreting, synthesing kappa (H-L+ intracellular) suspension, resistant to 8-azaguanine 85011427 TIB-18™ Köhler et al. 1976 [8]
NS0 NS/0, NS0/1 Mus musculus BALB/c myeloma, B cell cloned from NS-1 non-IG secreting, non-IG synthetising (H-L-) suspension, resistant to 8-azaguanine 85110503 Galfrè and Milstein 1981 [9]
P3-X63 Mus musculus BALB/c myeloma, B cell cloned from P3X27 secreting IgG1-kappa (H+L+) Ramasamy et al. 1974 [2]
P3-X63-Ag8 P3X63Ag8, P3/X63Ag8, X63 Mus musculus BALB/c myeloma, B cell cloned from P3X27 secreting IgG1-kappa (H+L+) suspension, resistant to 8-azaguanine 85011401 TIB-9™ Köhler et Milstein 1975 [3]
P3-X63-Ag8-U1 P3X63Ag8U.1, P3X63Ag8U1, P3U1 Mus musculus BALB/c myeloma, B cell variant of P3-X63-Ag8.653 non-IG secreting, synthesing kappa (H-L+) suspension 94120802 CRL-1597™ Yelton D.E. et al. 1979 [5]
P3-X63-Ag8.653 P3X63Ag8.653, P3/X63Ag8.653, X63-Ag8.653, X63Ag8.653, X63.653, Ag8 Mus musculus BALB/c myeloma, B cell cloned from P3-X63-Ag8 non-IG secreting, non-IG synthetising (H-L-) suspension 85011420 CRL-1580™ Kearney J.F. et al. 1979 [6]
Sp2/0 Sp2/0-Ag-14, Sp2/0-Ag14, SP-2 Mus musculus BALB/c hybridoma, B cell obtained by fusion with P3X63Ag8 non-IG secreting, non-IG synthetising (H-L-) suspension, resistant to 8-azaguanine 85072401 CRL-1581™ CRL-8287™ Shulman M. et al. 1978 [4]
YB2/0 YB2/3HL.P2.G11.16Ag.20 Rattus norvegicus hybridoma, B lymphoblast obtained by fusion with Y3/Ag1.2.3 non-IG secreting suspension, resistant to 8-azaguanine CRL-1662™ Kilmartin JV, et al. 1982 [10]

YB2/0 was obtained by fusing Y3/Ag1.2.3 (ATCC CRL-1631) cells with spleen cells from an AO strain rat. YB2/0 can be used as a fusion partner for rat B cells to make rat - rat hybridomas.

1.2. Rat hybridoma cell line used as fusion partner

Cell line Other cell line names Species and strain Cell type and cell lineage Clone origin IG production Cell line characteristics ECACC ATCC IMGT references
YB2/0 YB2/3HL.P2.G11.16Ag.20 Rattus norvegicus hybridoma, B lymphoblast obtained by fusion with Y3/Ag1.2.3 non-IG secreting suspension, resistant to 8-azaguanine CRL-1662™ Kilmartin JV, et al. 1982 [10]

The cell lines used as fusion partners do not grow in HAT medium (they die in the presence of HAT, they are HAT or HAz-sensitive). They lack a functional HGPRT, i.e., they are resistant to 8-azaguanine (the Ag in the cell names stands azaguanine).


Cholesterol requirements
NS-1, P3-X63-Ag8, P3-X63-Ag.653 are cholesterol auxotrophs (requires cholesterol). In contrast, hybridoma Sp2/0 do not require neither LDL nor cholesterol (the fusion partner bringing the necessary genes) [11].



mouse cells

Figure 1. Myeloma cell lines derived from MOPC21 showing SP2/0 (hybridoma), P3-X63-Ag8.653 (myeloma) and NS0 (myeloma) used as fusion partners in the generation of hybridomas.

The original hybridoma Sp2/3-3 was obtained by fusion of BALB/c spleen cells producing an anti-SRBC mAb Sp2 (from mouse immunized with sheep RBC) with the P3-X63-Ag8 (X63) myeloma [3].

1.3. Hamster cell lines used as protein expression system

Cell line Other cell line names Species and strain Cell type and cell lineage Clone origin IG production Cell line characteristics ECACC ATCC Companies IMGT references
CHO CHO-ori, Chinese Hamster Ovary Cricetulus griseus epithelial adherent 85050302 Puck TT et al. 1958 [12]
FreeStyle™ CHO-S Cells CHO-derived suspension culture Cricetulus griseus derived from CHO-S suspension Life Technologies:R800-07
CHO-K1 CHOK1, CHO K1, CHO cell clone K1 Cricetulus griseus epithelial derived from CHO adherent 85051005 CCL-61™ Kao FT and Puck TT 1968 [13]
POTELLIGENT® CHOK1SV CHO-K1SV, suspension variant of CHO-K1 Cricetulus griseus derived from CHO-K1 suspension Lonza
CHO/dhFr- Cricetulus griseus epithelial adherent, dhFr- 94060607
CHO-DUK-B11 CHO duk-, DXB11, CHO-DUKX Cricetulus griseus epithelial derived from CHO-K1 adherent, dhFr- CRL-9096™ Urlaub G et al. 1980 [14]
CHO-DG44 Cells (cGMP banked) CHO DG44 Cricetulus griseus derived from CHO-K1 suspension, dhFr- Life Technologies:A11000-01 Urlaub G et al. 1983 [15]


1.4. Human cell lines

HEK293 cell line derived from human embryonic kidney cells transformed with sheared fragments of adenovirus type 5 DNA has been widely used in the production of monoclonal antibody. Some derivatives were further transformed either with the simian virus 40 (SV40) large T antigen, termed HEK293T, or with the Epstein Barr virus (EBV) nuclear antigen 1 (EBNA1), termed HEK293E, using an origin of replication (ori) of SV40 or EBV, respectively.

PER.C6® cell lines are derived from human embryonic retina cells that have been immortalized by transfecting the E1 genes from adenovirus 5 DNA.19. PER.C6® cells can proliferate indefinitely in suspension under serum-free conditions.


2. Selection

2.1. HAT

Hybridoma cells are selected using HAT (Hypoxanthine Aminopterin Thymidine) medium.

HAT selection depends on the fact that mammalian cells can synthesize nucleotides by two different pathways: the de novo and the salvage pathways. Aminopterin (a folic acid analog) blocks the de novo pathway, while hypoxanthine and thymidine allow growth via the salvage pathway.

Indeed, the DNA de novo synthesis (in which a methyl or formyl group is transferred from an activated from of tetrahydrofolate) is blocked by Aminopterin. When the de novo pathway is blocked, cells utilize the salvage pathway, which bypasses the aminopterin block by converting purines and pyrimidines directly into DNA. The enzymes catalyzing the salvage pathway include hypoxanthine-guanine phosphorribosyl transferase (HGPRT) and thymidine kinase (TK). This pathway can convert hypoxanthine in IMP, a reaction catalysed by HGPRT. It can also convert thymidine in dTMP, a reaction catalysed by TK.

On this medium, only cells which have functionnal HGPRT and TK will grow via the salvage pathway.

HAT

Figure: Principle of HAT selection (origin: http://nfs.unipv.it/nfs/minf/dispense/immunology/lectures/files/monoclonal_antibodies.html)





More information: Monoclonal antibodies are commonly assumed to be monospecific, but anecdotal studies have reported genetic diversity in antibody heavy chain and light chain genes found within individual hybridomas. In order to evaluate the prevalence of such diversity, 185 random hybridomas were analysed in a large multicenter dataset [16]. Bradbury ARM, Trinklein ND, Thie H, Wilkinson IC, Tandon AK, Anderson S, Bladen CL, Jones B, Aldred SF, Bestagno M, Burrone O, Maynard J, Ferrara F, Trimmer JS, Görnemann J, Glanville J, Wolf P, Frenzel A, Wong J, Koh XY, Eng HY, Lane D, Lefranc M-P, Clark M, Dübel S. When monoclonal antibodies are not monospecific: hybridomas frequently express additional functional variable regions MAbs. 2018 May/Jun;10(4):539-546.doi: 10.1080/19420862.2018.1445456. Epub 2018 Mar 29.PMID: 29485921 Free PMC Article.


References:
[1] Horibata K. and Harris A.W. Exp Cell Res. 60(1):61-77 (1970) PMID: 5439579
[2] Ramasamy R. et al. Nature. 249(457):573-4 (1974) PMID: 4545851
[3] Köhler G. and Milstein C. Nature. 256(5517):495-7 (1975) PMID: 1172191
[4] Shulman M. et al. Nature. 276(5685):269-70 (1978) PMID: 714156
[5] Yelton D.E. et al. Curr Top Microbiol Immunol. 81:1-7 (1978) PMID: 567551
[6] Kearney J.F. et al. J Immunol. 123(4):1548-50 (1979) PMID: 113458
[7] Cowan N.J.et al. J Mol Biol. 90(4):691-701 (1974) PMID: 4449137
[8] Köhler G. et al. Eur J Immunol. 6(4):292-5 (1976) PMID: 825374
[9] Galfrè G. and Milstein C. Methods Enzymol. 73(Pt B):3-46 (1981) PMID: 730068
[10] Kilmartin J.V. et al. J Cell Biol. 93(3):576-82 (1982) PMID: 6811596
[11] Sato J.D. et al. J Exp Med. 165(6):1761-6 (1987) PMID: 3585251
[12] Puck T.T. et al. J Exp Med. 108(6):945-56 (1958) PMID: 13598821
[13] Kao FT and Puck TT. Proc Natl Acad Sci U S A. 60(4):1275-81 (1968) PMID: 5244736
[14] Urlaub G. et al. Proc Natl Acad Sci U S A. 77(7):4216-20 (1980) PMID: 6933469
[15] Urlaub G. et al. Cell. 33(2):405-12 (1983) PMID: 6305508
[16] Bradbury A.R.M. et al. MAbs. 10(4):539-546 (2018) PMID: 29485921

Software material and data coming from IMGT server may be used for academic research only, provided that it is referred to IMGT®, and cited as "IMGT®, the international ImMunoGeneTics information system® http://www.imgt.org (founder and director: Marie-Paule Lefranc, Montpellier, France)." References to cite: Lefranc, M.-P. et al., Nucleic Acids Res., 27:209-212 (1999); doi: 10.1093/nar/27.1.209 Full text Cover; Ruiz, M. et al., Nucleic Acids Res., 28:219-221 (2000); doi: 10.1093/nar/28.1.219 Full text; Lefranc, M.-P., Nucleic Acids Res., 29:207-209 (2001); doi: 10.1093/nar/29.1.207 Full text; Lefranc, M.-P., Nucleic Acids Res., 31:307-310 (2003); doi: 10.1093/nar/gkg085 Full text; Lefranc, M.-P. et al., In Silico Biol., 5, 0006 (2004) [Epub], 5:45-60 (2005); Lefranc, M.-P. et al., Nucleic Acids Res., 33:D593-597 (2005); doi: 10.1093/nar/gki065 Full text; Lefranc, M.-P. et al., Nucleic Acids Res., 37:D1006-1012 (2009); doi: 10.1093/nar/gkn838 Full text; Lefranc, M.-P. et al., Nucleic Acids Res., 43:D413-422 (2015); doi: 10.1093/nar/gku1056 Full text.
For any other use please contact Marie-Paule Lefranc Marie-Paule.Lefranc@igh.cnrs.fr.

CNRS Université de Montpellier European Commission
IMGT® Founder and Executive Director Emeritus:
Marie-Paule Lefranc Marie-Paule.Lefranc@igh.cnrs.fr
IMGT® Director:
Sofia Kossida Sofia.Kossida@igh.cnrs.fr
Bioinformatics manager:
Véronique Giudicelli Veronique.Giudicelli@igh.cnrs.fr
Computer manager:
Patrice Duroux Patrice.Duroux@igh.cnrs.fr
Webmaster:
Amélie Houles

Citing IMGT | Warranty disclaimer and copyright notice | Privacy policy and advertisement policy

© Copyright 1995-2019 IMGT®, the international ImMunoGeneTics information system®