Volume 9 Supplement 9

24th European Society for Animal Cell Technology (ESACT) Meeting: C2P2: Cells, Culture, Patients, Products

Open Access

Establishment and characterization of new human cell lines for recombinant therapeutic protein production

  • Yuichi Inoue1,
  • Akira Iwamoto2Email author,
  • Aiko Inoue3 and
  • Hiroharu Kawahara1
BMC Proceedings20159(Suppl 9):P3

https://doi.org/10.1186/1753-6561-9-S9-P3

Published: 14 December 2015

Background

Recombinant therapeutic proteins are increasingly requested with advances in tissue engineering using stem cells. Human cell line is an attractive host for the production of such glycoprotein, but there are few reports on human cells for a commercial production [1]. In this study, we established new human lymphoid cell lines from peripheral blood mononuclear cells (PBMCs) by treatment with phorbol 12-myristate 13-acetate (PMA) under a non-GMP condition, and characterized them by gene and protein expression analyses.

Experimental Approach

The human PBMCs (2 × 106 cells/ml) were cultured in 24 well plates in 12.5%FBS-ERDF medium supplemented with 10 ng/ml of IL-4 and/or IL-6 and 1 µg/ml of PMA for three months. The medium was changed every two or three days.

The gene expression of telomerase reverse transcriptase (TERT), a marker of immortalization, was examined by RT-PCR. The immunoglobulin (Ig) isotype was confirmed by ELISA. The CD markers on cell surface were detected by flow cytometry.

Results and Discussion

Although normal human cells are difficult to be transformed by chemical reagents such as PMA, we succeeded in obtaining three human cell clones under above condition (Table 1). The key point in our transformation may be continuous stationary culture without passage. All obtained clones were able to be subcultured over one year and were found to express TERT. In addition, they produced any isotype of immunoglobulin in the medium, indicating B lymphocytes. The results of flow cytometry also supported it. Therefore, these clones may be suitable for the production of secreted human glycoprotein because mature B lymphocytes have the extensive endoplasmic reticulum. In the next step, we will establish GMP compliant new human lymphoid cell lines.
Table 1

Summary of cell establishment and characterization.

 

Clone1

Clone2

Clone3

Culture medium for transformation

12.5%FBS-ERDF +IL-4+IL-6+PMA

12.5%FBS-ERDF +IL-6+PMA

12.5%FBS-ERDF +IL-4+IL-6+PMA

Culture medium for maintenance

12.5%FBS-ERDF

(+PMA)

12.5%FBS-ERDF

(+PMA)

12.5%FBS-ERDF

(+PMA)

Possible period of subculture

Over 1 year

Over 1 year

Over 2 years

Mean doubling time

30 hours

30 hours

30 hours

TERT expression

Positive

Positive

Positive

Produced Immunoglobulin

IgG

IgM

IgG

B cell marker (CD19)

Positive

Positive

Positive

Acknowledgements

This work was supported by JSPS KAKENHI Grant Number 26460167.

Authors’ Affiliations

(1)
National Institute of Technology, Kitakyushu College
(2)
Department of Bioscience and Biotechnology, Kyushu University
(3)
Department of Clinical Laboratory, Kyurin Corporation

References

  1. Swiech K, Picanco-Castro V, Covas DT: Human cells: New platform for recombinant therapeutic protein production. Protein Expr Purifi. 2012, 84: 147-153.View ArticleGoogle Scholar

Copyright

© Inoue et al. 2015

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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