BIOTARGET™-1 Xeno-Free Medium for Immunology
Transcript of BIOTARGET™-1 Xeno-Free Medium for Immunology
BIOTARGET™-1 Xeno-Free Medium for Immunology
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BIOTARGETTM is optimized for cell growth, viability and functionality
BIOTARGETTM delivers consistent results under serum-free conditions
BIOTARGETTM is xeno-free, reducing the risk of immunogenicity
BIOTARGETTM eases the transition to clinical research
BIOTARGETTM is the cost-effective alternative for growing human mononuclear cells.
BIOTARGETTM medium is available in many customized packaging configurations,
including Sartorius-Stedim Cultibags
BIOTARGET™-1: Advancing the field of Immunotherapy
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Standardizing immunology research and elucidating the complex cellular interactions
involved in immune response, requires effective and consistent tools.
BIOTARGET™-1 medium is one such tool, developed and optimized specifically for
culturing peripheral blood mononuclear cells (lymphocytes and monocytes).
Currently, mononuclear cells are grown in standard media and supplemented with
either human serum (A, AB) or foetal bovine serum. To eliminate the drawbacks
involved in serum supplementation, BI has developed BIOTARGET™-1, a serum-free
alternative.
The advantages of animal component-free media in culturing mononuclear cells are:
Elimination of non-specific growth factors and/or inhibitors, which interfere with ּ
cell activation
Elimination of potential introduction of pathogens
Allowing a reliable evaluation of the antigenic reaction (e.g. quantity of the
lymphokines)
Lot-to-lot consistency
During development, we screened hundreds of possible formulations variations,
and compared them (for viability, induction by mitogens, and other key parameters)
to serum supplemented media, as well as to a leading competition’s serum-free
medium.
Introduction
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• Xeno-free
• Chemically defined
• No added cytokines
• Comprehensive validation studies
• CE Mark
• Manufactured in cGMP compliant, ISO 13485:2003 and ISO 9001:2008 certified
facility
1. Mitogen mediated activation of mononuclear cells (PHA, CON.A, OKT-3)
2. Lymphoid cell mediated activation of mononuclear cells (RAJI, PEER, BA,
MOLT-4, JURKAT)
3. IL-2 and IL-3 production from mononuclear cells
4. Long-term post- activation culture of mononuclear cells
5. LAK / TIL cell generation by mononuclear cell interleukin-2 mediated activation
6. Natural killer cell (NK) generation by mononuclear cell activation
7. Cytotoxic T cell generation by mononuclear cell activation
8. Activation of macrophages
9. Investigating various cytokines’ influence on the production of mononuclear cell
sub-populations
10. HIV proliferation
11. Retrovirus proliferation in T cells for vaccine development
12. Cytokine production in human mixed leukocyte reaction (MLR), BIOTARGET™-1
was found as effective as serum containing medium in supporting leukocyte
proliferation and IL-2 secretion7
BIOTARGET™-1 advantages:
Applications
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Multiple evaluation protocols were used in the development of BIOTARGET™-1.
For evaluation, mononuclear cells were prepared by pooling peripheral blood
received from Israel’s Central Blood Bank. After stringent testing for Hepatitis B and
HIV viruses, cells were centrifuged in Ficoll-paque containing tubes. Mononuclear
cells were thus concentrated in a layer between the Ficoll and the plasma. Cells
were then washed 3x in PBS and divided into medium containing microwells at a
concentration of 2x105 cells per well.
1. Mitogenic Activation of Mononuclear Cells (Figures 1-9)
Activation was evaluated using different mitogens such as PHA, CON.A and
OKT-3. Proliferation determined through radioactive thymidine uptake. In order
to provide comprehensive measurements, several mitogen concentrations were
tested and thymidine uptake was determined over several days
Figures 1-2: Mitogenic activation of mononuclear cells by varying concentrations of
CON.A
Evaluation Protocol
Figure 2: Day 7Figure 1: Day 5
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Evaluation Protocol
Figures 3-4: Effects of CON.A concentration on mononuclear cell activation
Figure 9: Effects of OKT-3 concentration on mononuclear cell activation
Figure 4: Day 7
Figure 6: Day 5
Figure 8: Day 8
Figure 3: Day 5
Figure 5: Day 3
Figure 7: Day 7
Figures 5-8: Effects of PHA concentration on mononuclear cell activation
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Evaluation Protocol
2. Lymphoid Cell activation of Mononuclear Cells (Figures 10-11)
Mononuclear cell were activated using various lymphoid cells, such as:
JURKAT, RAJI, MOLT-4, and BA. A number of tumor:mononuclear
cell ratios were examined, and proliferation was measured through
radioactive thymidine uptake.
Figures 10 and 11 Activation of mononuclear cells with lymphoid cells
Figure 12: CON. A
Figure 10: (RAJI) Figure 11: (BA)
Figure 13: PHA
3. Production of Lymphokines by Activated Mononuclear Cells (Figures 12-15)
The levels of lymphokines IL-2 and IL-3 were measured in mononuclear cell
cultures after activation with various mitogens. Relative IL-2 concentrations
were estimated using CTLL-2 cells, mouse cytotoxic T-cells that grow only in the
presence of IL-2.
Figures 12 and 13: Production of IL-2 by CON.A / PHA activated mononuclear cells (Supernatant on CTLL cells)
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Evaluation Protocol
Figures 14 and 15: Production of IL-3 by CON.A / PHA activated mononuclear cells
(supernatant on 32 D-CL-23 cells)
Figure 15: PHAFigure 14: CON. A
Tables 1 & 2. RAJI cell mediated activation of mononuclear cells (5 days)
Table 1. Proliferation of the mononuclear cells.
Relative proliferation denoted in counts per minute of radioactive Thymidine.
Mononuclear cells only Mononuclear cellsRatio RAJI cells
1/5 1/10FBS 3193 1154 4190
COMPETITOR A 361 314 2519BIOTARGET™-1 2939 771 5680
Table 2. Measurement of Cytotoxicity using RAJI cells as target cells.
Results expressed as percentage of specific release of radioactive
Chromium (Total release minus spontaneous release).
Mononuclear cells Ratio RAJI cells
1/5 1/10FBS 3.0 21
COMPETITOR A 2.5 6.0BIOTARGET™-1 12 11
4. Cytotoxicity (see Tables 1 and 2) Mononuclear cells were seeded
at a concentration of 106 cells per well together with RAJI cells treated with mitomycin C. Varying ratios of the two cell types were examined. Following activation (5-7 days), lymphocytes were collected, centrifuged, suspended in medium and seeded in microwells in order to measure proliferation and cytotoxicity. RAJI cells were labeled with radioactive chromium (100 ci in a volume of 0.2 ml), washed three times, suspended at a concentration of 105 cells per ml, and divided into microwells containing the activated lymphocytes. Cytolytic activity was evaluated after 18 hours of incubation by measuring the radioactive chromium released from the target (RAJI) cells.
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Custom Manufacturing and Formulation Services
Biological Industries (BI) is positioned to become your product formulation
and manufacturing partner.
Our extensive experience in liquid manufacturing and packaging for the
biological and biopharmaceutical markets makes partnering with BI an
obvious choice as your products progress from the laboratory and pilot
scales to full scale production.
Sterile filtration and aseptic filling in a controlled environment
clean rooms (graded from ISO 8 up to ISO 5)
Flexible packaging in industrial single-use bags (0.5-2000 Liter)
- Consistent quality
- Rapid delivery
Upstream and downstream formulation (i.e. buffers)
Optimization of media and liquid formulations
Research and pilot scale freeze-drying
Extensive range of Quality Control services
Manufacturing under stringent quality system
- ISO 9001:2008 and ISO 13485:2003 certification
- cGMP compliant facility
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References
1. E. Elinav, N. Adam, T. Waks and Z. Eshhar. Amelioration of Colitis by genetically engineered murine regulatory T cells redirected by antigen-specific chimeric receptor. Gastroenterology, Vol. 136, Issue 5, pp. 1721-1731, 2009
2. Yang-Ming Tseng, Sheng-Yi Chen, Chien-Hung Chen, Yi-Ru Jin, Shih-Meng Tsai, Ing-Jun Chen, Jang-Hwa Lee, Chzng-Cheng Chiu and Li-Yu Tsai. Effects of Alcohol-Induced Human Peripheral Blood Mononuclear Cell (PBMC) Pretreated Whey Protein Concentrate (WPC) on Oxidative Damage. Journal of Agricultural and Food Chemistry, 56 (17), pp 8141–8147, 2008
3. Q. Leng, Z. Bentwich and G. Borkow. Increased TGF-ß, Cbl-b and CTLA-4 levels and immunosuppression in association with chronic immune activation. International Immunology 18(5):637-644, 2006
4. D. Melamed, O. Messika, L. Glass-Marmor and A. Miller. Modulation of matrix metalloproteinase-9 (MMP-9) secretion in B lymphopoiesis. International Immunology 18(9):1355-1362, 2006
5. C. Rabinowitz and B. Rinkevich. Epithelial cell cultures from Botryllus schlosseri palleal buds: accomplishments and challenges. Methods in Cell Science, Vol. 25, Numbers 3-4, 2004
6. F. Martí, E. Bertran, M. Llucià, E. Villén, M. Peiró, J. Garcia and F. Rueda. Platelet factor 4 induces human natural killer cells to synthesize and release interleukin-8. Journal of Leukocyte Biology 72:590-597, 2002
7. A. Bishara, R. Malka, C. Brautbar, V. Barak, I. Cohen and E. Kedar. Cytokine production in human mixed leukocyte reactions performed in serum-free media. Journal of Immunological Methods, Vol. 215, Issues 1-2, pp.187-190, 1998
8. G. Kampen , L. Poulsen, C. Reimert and P. Skov. A method for production and determination of histamine releasing activity from human
peripheral blood mononuclear cell. Journal of Immunological Methods, Vol. 210, Issue 2, pp. 185-193, 1997 9. S. Morecki, Y. Gelfand, S. Levi, A. Nagler, R. Condiotti, C. Nabet, A. Ackerstein, S. Slavin.
Activated long-term peripheral blood cultures as preparation for adoptive alloreactive cell therapy in cancer patients. Journal of Hematotherapy, 6 (2), pp. 115-124, 1997
10. Malka R.; Brautbar C.; Kedar E.; Cohen I.; Bishara A. Human mixed leukocyte reaction (MLR) performed in serum-free media and serum-containing medium. Human immunology, vol. 44, supp. 1, pp. 137, 1995
Product Name Catalogue
No.Unit Size
StorageTemp.
EZ Lympho-Sep™- Lymphocyte SeparationTubes
01-899-U 18 Or 30Tubes/Box
AMB
Human Serum Albumin (HSA Solution 10%),
05-720-1B 100ml -20ºC
05-720-1C 20ml -20ºC
05-720-1D 10ml -20ºC
Phytohemagglutinin-M (PHA-M), Lyophilized 12-006-1H 5ml 2-8ºC
Serum-Free Cell Freezing MediumProtein-free, Animal Component-Free
05-065-1A 500ml 2-8ºC
05-065-1C 20ml 2-8ºC
DCCM-1A high protein serum-free medium, designed for hybridoma cell growth and monoclonal antibody production.
05-010-1A 500ml 2-8ºC
05-010-1B 100ml 2-8ºC
DCCM-2A low protein serum-free medium, designed for hybridoma cell growth and monoclonal antibody production.
05-015-1A 500ml 2-8ºC
05-015-1B 100ml 2-8ºC
Low Protein Medium (LPM)Serum-free medium for the growth of a wide variety of hybridomas and other lymphocytes
05-040-1A 500ml 2-8ºC
05-040-1B 100ml 2-8ºC
BIOMPM-1, Multi-purpose SFMserum-free medium formulation for a wide variety of anchorage-dependent cells
05-060-1A 500ml 2-8ºC
05-060-1B 100ml 2-8ºC
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