Safety Considerations for ECM-based Biomaterials

LDEV – A Threat to Biomedical Research Results
Lactate dehydrogenase elevating virus (LDEV) is a single-strand RNA virus that infects mice and mouse cell cultures. These are the only hosts; rats are not susceptible to LDEV [1]. As its name implies, the diagnostic hallmark of LDEV infection is elevation of lactate dehydrogenase levels in serum.

Although natural infection in mice is generally subclinical, the major importance of LDEV is as a contaminant of biological material used in biomedical research, such as transplantable tumors, cell lines, antibodies, serum or viral stock, where it may seriously compromise research results. Indeed, LDEV alters several bodily and cellular functions in mice, notably those of macrophages, where the virus replicates [2–4]. Detrimental effects include prolonged survival of allografts, increased or suppressed growth of both spontaneous and transplanted tumors [1], the development of antinuclear antibodies [6], decreased neutrophil migration [7], and, more generally, the modification of cytokine profiles and alteration of cellular and humoral immune responses [3].

Therefore, it is critical that LDEV contamination of cell cultures or laboratory mice be avoided, notably in studies of immune function or oncogenesis, of which it may alter the outcome. Using only biological material that is guaranteed LDEV-free for mouse cell culture or administration to mice is thus of paramount importance.

Matrigel Basement Membrane Matrix
In vivo, so-called basement membranes are indispensable to cellular growth and to the organization of tissues and organs. Basement membranes are highly specialized sheets of extracellular matrices (ECM) that provide a mechanical support for cell layers and play a key role in diverse biological processes.
Today, ECM-rich basement membrane mimics are widely used by cell biologists as substrates for cell culture to recreate in vitro the complex extracellular environment found in tissues in vivo.

Matrigel Basement Membrane Matrix is a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma, a tumor rich in ECM proteins. At room temperature, the matrix polymerizes to produce biologically active matrix material that resembles the mammalian cellular basement membrane and promotes the attachment and differentiation of normal and transformed anchorage-dependent epithelioid and other cell types. Cells cultured on the matrix demonstrate complex cellular behavior that is very difficult or even impossible to observe under “classical” culture conditions.

For example, the matrix will influence gene expression in adult rat hepatocytes [8] as well as three dimensional culture in mouse [9–12] and human [13, 14] mammary epithelial cells. It will support in vivo peripheral nerve regeneration [15, 16], can be used for metabolism and toxicology studies [17], and is the basis for several types of tumor cell invasion assays [18–19]. The matrix provides the substrate necessary for the study of angiogenesis both in vitro [20–21] and in vivo [22–24], and supports in vivo propagation of human tumors in immunosupressed mice [25–27].
Matrigel is the most extensively referenced basement membrane matrix, with over 13,000 citations in various in vitro and in vivo applications, including the in vivo Plug Assay [28–30].

Certified LDEV-free Products
In 2007, BD Biosciences acknowledged the presence of LDEV in several Matrigel products. After conducting a thorough investigation, we confirmed the presence of LDEV in the murine EHS tumor from which the products are derived.

Quality Control procedures were improved immediately to make sure that all products released for sales were tested negatively for LDEV. Our QC testing has been expanded to include testing for over 30 (both bacterial and viral) pathogens. Since mid-2008, the product is now derived from an EHS tumor source that has been tested and found negative for LDEV and for a number of other pathogens. Extensive functional validation demonstrated that the improved product shows equivalent performance as our formerly offered product. Guaranteed LDEV-free, Matrigel is the matrix of choice for all in vivo assays in mice.

As science progresses and technological knowledge grows, QC and QA procedures will further be adapted to the latest developments and new tests will be introduced to ensure pathogen-free products and to continue to meet the high quality expected. It is important to note that LDEV cannot be practically cultured in vitro and is not known to affect other species than mouse.

Testing for LDEV
The MAP Test
LDEV causes persistent viremia in mice, which induces antiviral antibodies. The Mouse Antibody Production (MAP) test was developed in the sixties to screen biological material for these antiviral antibodies [31–32]. The test is an indirect assay that relies on the immune response of the mouse as an indicator of virus contamination. It involves inoculating the material into naive mice and serologically testing the inoculated animals 30 days later for antibodies specific to the viral pathogen.

The PCR Test
Polymerase Chain Reaction (PCR) technology can detect the presence of viruses and other microbial contaminants directly in biological samples by amplifying their DNA or RNA. PCR-based testing is rapid, specific, and highly sensitive. Furthermore, this method eliminates the need for using animals to screen for murine viruses in biological material. It is also flexible in that it allows testing for pathogens from multiple species simultaneously (e.g., pathogen screening) [33].

A Wide Range of Applications
In vivo Angiogenesis Studies and Augmentation of Tumors in Immunosuppressed Mice
The high Concentration of the matrix is suited for in vivo applications where a high protein concentration augments growth of tumors. It can be used to assess in vivo angiogenic activity of different compounds via the so-called Matrigel Plug Assay.

Cell growth and differentiation – the differentiation of many cells types is promoted by the matrix, including hepatocytes, endothelial cells, smooth muscle cells, and neurons. It is especially suited for the culture of polarized cells, such as epithelial cells.

Metabolism/toxicology studies – the matrix has been used to successfully construct in vitro models of liver cells for drug toxicity studies.

Invasion assays – the matrix provides a biologically active basement membrane model for in vitro invasion assays.

In vitro angiogenesis assays – the matrix serves as a substrate for in vitro endothelial cell invasion and tube formation assays.

Human embryonic stem cell culture – hESC-Qualified matrix offers an optimal surface for long-term propagation of human Embryonic Stem (hES) cells. Combined with mTeSR1 high quality medium (StemCell Technologies) hESC-Qualified matrix supports feeder-free expansion of hES cells.

References
[1] Baker D.G.: Clin Microbiol Rev 11, 231-266 (1998)
[2] Baker D.G.: Clin Microbiol Rev 11, 231-266 (1998)
[3] National Research Council: (1991)
[4] Zitterkopf N.L. et al.: Virus Res 106, 35-42 (2004)
[5] Isakov N. et al.: Cancer Res 41, 667-672 (1981)
[6] Musaji A. et al.: Autoimmun Rev 4, 247-252 (2005)
[7] Hayashi T. et al.: J Comp Pathol 104, 161-170 (1991)
[8] Page J.L. et al.: Toxicol Sci 97, 384-397 (2007)
[9] Barcellos-Hoff M.H. et al.: Development 105, 223-235 (1989)
[10] Li M.L. et al.: PNAS 84, 136-140 (1987)
[11] Roskelley C.D. et al.: PNAS 91, 12378-12382 (1994)
[12] Xu R. et al.: J Biol Chem 282, 14992-14999 (2007)
[13] Debnath J. et al.: Methods 30, 256-268 (2003)
[14] Muthuswamy S.K. et al.: Nat Cell Biol 3, 785-792 (2001)
[15] Fouad K. et al.: J Neurosci 25, 1169-1178 (2005)
[16] Madison R. et al.: Exp Neurol 88, 767-772 (1985)
[17] Bi Y.a. et al.: Drug Metab Dispos 34, 1658-1665 (2006)
[18] Albini A. et al.: Cancer Res 47, 3239-3245 (1987)
[19] Terranova V.P. et al.: PNAS 83, 465-469 (1986)
[20] Kubota Y. et al.: J Cell Biol 107, 1589-1598 (1988)
[21] Maeshima Y. et al.: J Biol Chem 276, 15240-15248 (2001)
[22] Isaji M. et al.: Br J Pharmacol 122, 1061-1066 (1997)
[23] Kisucka J. et al.: PNAS 103, 855-860 (2006)
[24] Passaniti A. et al.: Lab Invest 67, 519-528 (1992)
[25] Albini A. et al.: Int J Cancer 52, 234-240 (1992)
[26] Angelucci A. et al.: Endocr Relat Cancer 13, 197-210 (2006)
[27] Yue W. et al.: J Steroid Biochem Mol Biol 44, 671-673 (1993)
[28] Albini A. et al.: Int J Cancer 52, 234-240 (1992)
[29] Angelucci A. et al.: Endocr Relat Cancer 13, 197-210 (2006)
[30] Yue W. et al.: J Steroid Biochem Mol Biol 44, 671-673 (1993)
[31] Parker J.C. et al.: Am J Epidemiol 88, 112-125 (1968)
[32] Rowe W.P. et al.: J Exp Med 109, 379-391 (1959)
[33] Bootz F. et al.: Lab Anim 37, 341-351 (2003)