Home Health The Impact of Food Bioactives on Health
Stability, Consistency and Reproducibility of the System
THP-1 is an immortalized cell line that can be cultured in vitro up to passage 25 (approx. 3 months) without changes of cell sensitivity and activity. As far as our information reaches, U937 is used also at higher passage numbers (see e.g. Strefford et al. 2001). THP-1 as well as U937 cells can be stored for a number of years and, provided an appropriate protocol is followed, the cell lines can be recovered without any obvious effects on monocyte-macrophage features and cell viability (Chanput et al. 2014 and references therein).
Relevance to Human In Vivo Situation
Cell lines always have a malignant background, which presents a significant risk of experimental bias. The cultivation of cells under controlled conditions and outside their natural environment possibly results in different sensitivity and responses compared to normal somatic cells in their natural environment (Schildberger et al. 2013).
Also, possibly relevant interactions between the target cells and surrounding cells, as in natural tissues, cannot be easily mimicked. In vitro co-cultivation of THP-1 or U937 cells with neighbouring cells might be an option to make this drawback less pronounced (Chanput et al. 2014).
Other Models with the Same Applicability
Next to THP-1 and U937 cells, ML-2, HL-60 and Mono Mac 6 cells are used in biomedical research. U937 cells are the most frequently used. The basic difference between U937 and THP-1 cells is the origin and maturation stage. U937 cells are of tissue origin, thus at more mature stage, whereas THP-1 cells originate from a blood leukaemia origin at less mature stage (Chanput et al. 2014). Because Mono Mac 6 is able to phagocytose antibody-coated erythrocytes (Ziegler-Heitbrock et al. 1988) and mycobacteria (Friedland et al. 1993; Shattock et al. 1994) it is thought more suitable for the study of phenotypic and functional features of in vivo mature monocytes. Also, it expresses mature monocyte markers that cannot be found on the THP-1 and U937 cell lines, such as M42, LeuM3, 63D3, Mo2 and UCHMI. As THP-1 and U937 are very frequently used, we focus here on these two cell lines.
General Protocol of Culturing THP-1 Cells
Roswell Park Memorial Institute (RPMI) 1640 medium is a commonly used medium for THP-1 as well as for U937 cells. Alternatively, DMEM (Dulbecco's Modified Eagle's Medium; Morton 1970), also supplemented with 10 % FBS, is used to grow U937 cells. RPMI 1640 medium was originally developed by Moore et al. (1967), at Roswell Park Memorial Institute to culture human leukemic cells in suspension and as a monolayer. The formulation is based on RPMI-1630 medium and uses a bicarbonate buffering system and has alterations in the amounts of amino acids and vitamins. When RPMI is properly supplemented, it has a wide applicability for supporting growth of many types of cell cultures including HeLa, Jurkat, MCF-7, PC12, PBMC, astrocytes and carcinomas.
In most of the studies, the RPMI medium is supplemented with a combination of foetal bovine serum (FBS) and antibiotics. FBS is also known as foetal calf serum (FCS) and is obtained from whole blood by removing blood cells, platelets and fibrinogen. Serum includes all proteins not involved in blood clotting and all electrolytes, antibodies, antigens, hormones and exogenous substances. Foetal bovine serum is obtained via collection at a slaughterhouse. In some studies, foetal bovine serum is heat-inactivated in order to destroy heat-labile complement proteins (Biowest).
In many studies, the antibiotics penicillin and streptomycin are used to supplement the medium in order to prevent bacterial contamination of cell cultures due to their effective combined action against gram-positive and gram-negative bacteria. Penicillin, originally purified from the fungus Penicillium, acts directly and indirectly by interfering with the turnover of the bacterial cell wall and by triggering the release of enzymes that further alter the cell wall respectively. Streptomycin was originally purified from Streptomyces griseus and acts by binding to the 30S subunit of the bacterial ribosome, which leads to inhibition of protein synthesis and death in susceptible bacteria (Waksman 1953).
In some studies, cells were also supplemented with HEPES (4-(2-hydroxyethyl)1-piperazineethanesulfonic acid). HEPES is a zwitterionic organic chemical buffer and is widely used in many biochemical reactions and as a buffering agent in some cell culture media. These buffers have pKa values between 6.0 and 8.0, high solubility, limited effect on biochemical reactions, membrane permeability, are chemically and enzymatically stable and easy to prepare.
|< Prev||CONTENTS||Next >|