Susceptibility of Common Fibroblast Cell Lines to Transmissible Spongiform Encephalopathy Agents
- 1Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana;
- 2Department of Laboratory Medicine, University of Washington, Seattle, Washington
- Reprints or correspondence: Dr. Ina Vorberg, Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 903 S. 4th St., Hamilton, MT 59840 (vorberg{at}lrz.tum.de)
Abstract
The risk of contamination of tissue culture cells with transmissible spongiform encephalopathy (TSE) agents as a result of the use of animal products as medium components has been considered to be low, in part, because only a few brain-derived cell lines have been reported to be susceptible to TSE infection. In the present study, we demonstrate that the common laboratory fibroblast cell lines NIH/3T3 and L929, which express low levels of cellular mouse prion protein, are susceptible to infection with mouse-adapted scrapie. Our results show that the susceptibility of a cell line to TSE infection cannot be predicted on the basis of its tissue origin or its level of expression of the cellular prion protein, and they suggest that any cell line expressing normal host prion protein could have the potential to support propagation of TSE agents. Thus, testing of cells for TSE susceptibility might be necessary for all cell lines that are routinely used in vaccine production and in other medical applications
Transmissible spongiform encephalopathies (TSEs) are fatal degenerative disorders of the central nervous system (CNS), and they include Creutzfeldt-Jakob disease in humans, scrapie in sheep and goats, and bovine spongiform encephalopathy (BSE). TSEs are characterized by the accumulation of an abnormal form (PrP-res) of the normal host prion protein (PrP-sen). PrP-res is posttranslationally generated from PrP-sen. The function of the normal prion protein is unknown. No major chemical differences have been identified in comparisons of PrP-sen and PrP-res, which indicates that both forms differ mainly in their polypeptide fold. Whereas PrP-sen is rich in α-helical structure and is protease sensitive, PrP-res has a high β-sheet content and is partially resistant to proteolysis [1, 2]. PrP-res is the only identified constituent of the infectious particle of the TSE agent, and it has been proposed to act as a protein-only agent [3]. Although the exact nature of the TSE agents is still a subject of debate [4, 5], PrP-res has been used extensively as a surrogate marker for TSE infection
Although PrP-sen is most prominent in the brain, it is also expressed in other tissues, such as the heart, lungs, pancreas, spleen, kidneys, and skin [6–8]. Despite the widespread distribution of PrP-sen, PrP-res accumulation and, thus, infection appear to be primarily restricted to the nervous system and, in some species, the lymphoreticular system of the TSE-infected host [9, 10]. Restricted susceptibility to TSE agents has also been observed in cell-infection experiments performed in vitro. Even though almost all previously tested cell lines express PrP-sen, only a few appear to be susceptible to TSE agents [11–18]
Although infection of cell cultures with TSE agents appears to be difficult [14, 19], the wide use of bovine-derived products as tissue culture supplements has raised concerns that the contamination of these products with TSE agents could lead to persistent infection of tissue culture cells. Indeed, demonstration that TSE infectivity can be transmitted through blood transfusion [20, 21] raises the possibility that low levels of TSE agents may be present in tissue culture media supplements that contain blood components. This is of particular concern because bovine materials are used in the manufacture of most medical products produced by tissue culture cells, including vaccine-producing cell lines
The risk of replication of TSE agents in tissue culture cells cultivated in the presence of bovine serum potentially contaminated with TSE agents is considered to be low, in part, because propagation of TSE agents appears to be restricted to neurons or brain-derived cell cultures [12, 13, 15, 16, 18, 22, 23]. In 2 cases, however, TSE infectivity has been reported to have been transmitted to cells that were not derived from the CNS. Studies performed in the late 1970s and early 1980s suggested that scrapie infectivity replicated in L fibroblast cell cultures exposed to scrapie agents [11, 24], but such experiments have not been repeated. Most recently, formation of PrP-res and scrapie infectivity were detected in rabbit epithelial cells exposed to sheep scrapie agent, which demonstrates that nonneuronal cells can support TSE infection [17]. However, these cells had been genetically engineered to express significant levels of sheep prion protein. Because it has been reported that over-expression of cellular prion protein in cell culture increases the susceptibility of cells to TSE infection [25], it is unclear whether epithelial cells expressing endogenous, wild-type levels of PrP are naturally susceptible to TSE infection
To evaluate the susceptibility of nonneuronal laboratory cell lines to TSE agents, we tested whether the mouse fibroblast cell lines NIH/3T3 and L929 could be infected with mouse-derived scrapie. In the present study, we demonstrate that unmodified, wild-type NIH/3T3 and L929 cells can be persistently infected with mouse scrapie. Furthermore, a significant percentage of cells in both fibroblast cell lines continuously produced high levels of PrP-res after infection. Thus, commonly used nonneuronal tissue culture cell lines can be highly susceptible to TSE infection. Our data demonstrate that neither cell type nor levels of expression of PrP-sen can be used as an indicator of whether a particular cell line will be susceptible to TSE infection
Materials and Methods
AntibodiesThe mouse monoclonal antibody (MAb) 3F4 and the rabbit polyclonal antiserum R30 used in the present study have been described elsewhere [26, 27]
Cell cultureMedia and fetal bovine serum (FBS) were purchased from Gibco Invitrogen. Mouse fibroblast cells ψ2, PA317, NIH/3T3, and L929 (ATCC CCL-1) were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS. The ψ2 and PA317 cells are retroviral packaging cell lines that have been generated to produce helper-free defective retrovirus [28, 29]. A mixed culture of ψ2 and PA317 cells, as well as L929 cells that stably express 3F4 epitope–tagged mouse PrP-sen, was generated as described elsewhere [30]
Scrapie strains and preparation of inoculaScrapie strains 22L, ME7, and 87V [31] were a gift of Dr. James Hope (Lasswade Veterinary Laboratory, Penicuik, Midlothian, UK). Scrapie strain RML was from the Rocky Mountain Laboratories (National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT). Strains were passaged as described elsewhere [32]. In C57BL/10 mice, the titer of the 22L scrapie agent, as determined by end-point titration, was 2×108.5 ID50/g of infected brain. Ten-percent brain homogenates were prepared in Dulbecco’s PBS by use of a Dounce homogenizer, and cell debris were removed by low-speed centrifugation (for 10 min at 4°C and at 2000 g). Cleared homogenate was sonicated for 2 min before a final dilution in Opti-MEM
Cell infectionsInoculation of cells with scrapie agents was done as described elsewhere [32]. In a 24-well plate, 5×104 cells were plated in DMEM supplemented with 10% FBS. One day later, cell medium was removed and replaced by 200 μL of a 1% brain homogenate in Opti-MEM. After 4 h at 37°C, 400 μL of DMEM with 10% FBS was added, and the cells were incubated for an additional 92 h. Cells were then continuously passaged in DMEM with 10% FBS
Metabolic labeling and immunoprecipitationMetabolic labeling and immunoprecipitation of PrP-sen were performed as described elsewhere [33]. PrP-sen was immunoprecipitated using the mouse MAb 3F4 or the polyclonal antiserum R30 and protein A sepharose beads. Samples were subjected to gel electrophoresis, and radiolabeled proteins were visualized by autoradiography
Detection of PrP-resConfluent cell monolayers were lysed in lysis buffer, and cell debris were removed by centrifugation (at room temperature for 5 min at 16,000 g). For detection of PrP-sen, 1/50 of the lysate was mixed with 2× sample buffer (5% sodium dodecyl sulfate, 6 mmol/L EDTA, 4% β-mercaptoethanol, 10% glycerol, 0.04% bromphenol blue, and 126 mmol/L Tris-HCl [pH 6.8]) and was assayed by Western blot. The remaining lysate was incubated with 20 μg/mL proteinase K for 40 min at 37°C. Proteolysis was stopped by the addition of 2 mmol/L phenylmethylsulfonyl fluoride. Samples were subjected to centrifugation (at 4°C for 1 h at 200,000 g), and pellets were sonicated into 1× sample buffer. After electrophoresis, proteins were transferred to polyvinylidine fluoride membrane, and PrP-res was detected using either the anti-PrP mouse MAb 3F4 or the anti-PrP polyclonal antiserum R30, by use of enhanced chemiluminescence, according to the manufacturer’s instructions (Amersham Pharmacia Biotech AB)
BioassaysCells exposed to brain homogenates were passaged 13 times (for Mo3F4-Ψ2/PA317 cells) or 27 times (for NIH/3T3 cells) before being harvested for inoculation. Cells were resuspended in DMEM supplemented with 10% FBS. After 5 consecutive cycles of freeze-thawing, lysates were sonicated, and mice were inoculated intracranially with ∼5×105 cells (Mo3F4-Ψ2/PA317 cells) or ∼2×105 cells (NIH/3T3 cells) per C57BL/10 weanling mouse. Fifty microliters of a 1% brain homogenate of a 22L-infected mouse was used as a control. At least twice a week, mice were observed for the onset of neurological disease, and mice showing signs of scrapie disease were killed. All animal experiments were approved by the Rocky Mountain Laboratory Animal Care and Use Committee. The Rocky Mountain Laboratories are fully accredited by the American Association for Laboratory Animal Care
TitrationThe titer of the 22L scrapie brain homogenate and the titer of NIH/3T3 cells infected with 22L scrapie were determined for C57BL/10 mice. Groups of 4–8 mice were inoculated intracranially with serial dilutions of brain homogenates or cell lysates, and animals showing signs of scrapie disease were killed. Titer calculations were performed according to the method of Spearman and Kaerber
Determination of the number of infected cells per culture NIH/3T3 cells or L929 cells that had been exposed to 22L scrapie agent were passaged 12–14 times, and single cell clones were established from these cultures by limiting dilution cloning. Single cell clones were expanded into 24-well plates, and lysates of these cells were tested for their PrP-res content
Results
Persistent formation of PrP-res in mouse ψ2/PA317 fibroblast cells expressing epitope-tagged mouse PrP-senTo study whether fibroblast cells can be infected with scrapie agent and can accumulate PrP-res, we initially used a mixed culture of ψ2 and PA317 mouse fibroblast cells that were originally derived from NIH/3T3 cells [28, 29]. Mouse PrP-sen that was tagged with the epitope for the MAb 3F4 (Mo3F4) was over-expressed in these cells to increase the sensitivity of PrP-sen detection and to monitor the production of new PrP-res over the background of PrP-res originally present in the scrapie brain homogenate. A previous unpublished study demonstrated that the introduction of the epitope for the MAb 3F4 does not inhibit continuous PrP-res formation in cell culture (S. Priola, unpublished data) [34]
For 4 days, cells were exposed to a 1% brain homogenate from either an uninfected mouse or a mouse infected with scrapie strain 22L, and they were then subsequently passaged. Newly formed PrP-res could be detected in cells exposed to scrapie-infected brain homogenate, whereas no PrP-res was observed in cells incubated with normal brain homogenate (figure 1). Furthermore, PrP-res was present even after 15 passages in cells exposed to 22L scrapie, indicating that the formation of PrP-res in these cells was not transient (data not shown). Thus, Mo3F4-ψ2/PA317 mouse fibroblast cells were capable of persistently producing PrP-res
Figure 1
Accumulation of 3F4-positive PrP-res (i.e., an abnormal form of the 3F4 epitope–tagged normal prion protein PrP-sen) in a mixed culture of ψ2/PA317 fibroblast cells expressing Mo3F4 PrP-sen (Mo3F4-ψ2/PA317 cells.) A Western blot of the first 2 passages after infection is shown. The blots were developed with monoclonal antibody 3F4–positive protease-resistant prion protein (PK+) was detectable only in cell lysates that had been exposed to scrapie brain homogenate. The band at 34 kDa, which is also present in mock-infected cells, is not prion protein specific. Molecular mass markers (in kilodaltons) are shown. Unglycosylated (*), partially glycosylated (•), and fully glycosylated (▴) PrP-res is shown. Mock, cells incubated with brain homogenate from an uninfected mouse
Propagation of TSE infectivity in Mo3F4-ψ2/PA317 fibroblast cellsThe presence of scrapie infectivity in passage 13 of Mo3F4-ψ2/PA317 fibroblasts was determined by intracranial inoculation of lysates of ∼5×105 cells/C57BL/10 mouse. Passage 13 was chosen to eliminate the possibility that detectable scrapie infectivity was due to residual inoculum that was still present in the cell culture from the original scrapie brain homogenate. After injection of scrapie-exposed cells, all mice died of disease (table 1). By contrast, control animals inoculated with cells that were exposed to normal uninfected brain homogenate showed no signs of neurological disease for >600 days. All mice that were clinically positive for scrapie accumulated substantial amounts of PrP-res in their brains, a finding that indicates that the neurological signs resulted from transmission of 22L scrapie infectivity (data not shown). Thus, exposure to 22L scrapie agent led to the persistent infection of Mo3F4-ψ2/PA317 cells, thereby demonstrating that murine fibroblast cells are susceptible to TSE agent
Efficient PrP-res formation in cell clones expressing high or low levels of Mo3F4 PrP-senIt has been reported that the formation of PrP-res is facilitated by the expression of high levels of PrP-sen [25]. To determine whether the amount of PrP-res that was formed was dependent on the level of expression of Mo3F4 PrP-sen, ψ2 cell clones expressing different levels of Mo3F4 PrP-sen (figure 2A) were exposed to 22L brain homogenate and were tested for the accumulation of 3F4-positive PrP-res. Of interest, cell lines expressing high or low amounts of Mo3F4 PrP-sen accumulated comparable levels of PrP-res (figure 2B). These results demonstrate that there is not necessarily a correlation between the level of expression of PrP-sen and the amount of PrP-res that accumulates. Furthermore, these data suggest that the level of expression of PrP-sen cannot be used as an indicator to predict whether a cell can produce PrP-res and, thus, would be susceptible to TSE infection
Figure 2
Formation of abnormal 3F4 epitope–tagged prion protein (PrP-res) in ψ2 clones expressing low (ψ2C2) or high (ψ2C4) levels of 3F4 epitope–tagged normal prion protein (PrP-sen). Western blots of PrP-sen (A) and PrP-res (B) of the first 2 passages are shown. All blots were developed with monoclonal antibody 3F4. Clones exposed to 22L scrapie–infected brain homogenate accumulated comparable levels of 3F4-positive PrP-res, independent of the level of expression of Mo3F4 PrP-sen. Molecular mass markers (in kilodaltons) are shown. Unglycosylated (*), partially glycosylated (•), and fully glycosylated (▴) PrP-res is shown. Mock, cells incubated with brain homogenate from an uninfected mouse; PK+, protease-resistant prion protein; PK−, protease-sensitive prion protein
Persistent infection of NIH/3T3 fibroblasts with mouse scrapieThe aforementioned experiments demonstrated that Mo3F4-ψ2/PA317 cells could be infected with scrapie and could produce high levels of PrP-res. Because both ψ2 and PA317 cells have been modified to express retroviral genes that could confound the results, we tested whether the progenitor wild-type cell line NIH/3T3 was susceptible to scrapie infection. These cells express levels of endogenous mouse PrP-sen that are similar to those expressed by mouse neuroblastoma cells, a cell line susceptible to scrapie agents (figure 3A) [13, 25]. NIH/3T3 fibroblast cells were exposed to a 22L scrapie brain homogenate or an uninfected brain homogenate. PrP-res formation was detected in all passages of NIH/3T3 cells exposed to scrapie brain homogenate (figure 3B), whereas no PrP-res was present in cell lysates exposed to uninfected brain homogenate (i.e., mock-infected cells). After intracranial inoculation of mice with cell lysates from NIH/3T3 cells that had been exposed to 22L scrapie brain homogenate, all animals died of disease (table 1). The amount of scrapie infectivity present in the cells was determined by titration of lysates of 22L-infected NIH/3T3 cells in C57BL/10 mice and revealed a titer of ∼2.5×105 ID50/mL of cell lysate. Mice that were inoculated with mock-infected NIH/3T3 cells showed no signs of scrapie infection. Although PrP-res was readily detectable in mice inoculated with 22L-infected cells, no PrP-res was present in the brains of mice inoculated with mock-infected cells (data not shown). Thus, normal NIH/3T3 cells expressing wild-type levels of mouse PrP-sen are susceptible to scrapie
Figure 3
Persistent formation of an abnormal form of the host prion protein (PrP-res) in NIH/3T3 cells expressing wild-type levels of normal mouse prion protein (PrP-sen). A Levels of expression of endogenous mouse PrP-sen in NIH/3T3 cells and mouse neuroblastoma cells (MNB) are comparable. Cells were metabolically labeled, and endogenous PrP-sen was immunoprecipitated from the cell lysate by use of the polyclonal antiserum R30. B Accumulation of protease-resistant prion protein (PK+) in NIH/3T3 cells after exposure to brain homogenate of a mouse infected with the scrapie strain 22L. A Western blot of multiple cell passages tested for PrP-res accumulation is shown. The blots were developed with the polyclonal antiserum R30. Molecular mass markers (in kilodaltons) are indicated. Unglycosylated (*), partially glycosylated (•), and fully glycosylated (▴) PrP-res is shown. Mock, cells incubated with brain homogenate from an uninfected mouse; PK+, protease-resistant prion protein; PK−, protease-sensitve prion protein
Persistent formation of PrP-res is not restricted to fibroblasts of the NIH/3T3 lineageThat Mo3F4-ψ2 /PA317 cells and their progenitor cell line NIH/3T3 could be infected with 22L scrapie raised the possibility that other fibroblast cells would also be susceptible to TSE agents. Previous studies reported that mouse L fibroblast cells accumulated titers of scrapie agent on exposure to the mouse scrapie strains ME7 and Chandler [11, 24], suggesting that fibroblast cells in general may be susceptible to TSE infection. L cells, however, have not been tested for PrP-res production. To investigate the ability of fibroblast cells of the L lineage to persistently produce PrP-res, we exposed wild-type L929 cells expressing endogenous mouse PrP-sen (figure 4A, left lane) to 22L scrapie brain homogenate or uninfected brain homogenate (i.e., “mock”). Cells were checked for protease-resistant prion protein after 2, 3, and 9 passages. PrP-res was readily detectable in all passages tested, whereas cells inoculated with uninfected brain homogenate did not exhibit any detectable levels of PrP-res (figure 4B). Thus, wild-type L929 cells are capable of sustaining PrP-res formation. In addition, exposure of L929 cells stably over-expressing Mo3F4 PrP-sen to 22L scrapie (figure 4A) also led to the production of 3F4-tagged PrP-res (figure 4C). In conjunction with the previously reported propagation of scrapie infectivity in L cells, the results of our experiments suggest that infection of fibroblast cells with scrapie agent and production of PrP-res are not restricted to a specific fibroblast cell line
Figure 4
Accumulation of abnormal prion protein (PrP-res) in L929 cells expressing wild-type (WT) normal host prion protein (PrP-sen) or Mo3F4 PrP-sen. A Level of endogenous expression of mouse PrP-sen in WT L929 cells (left lane) or total level of expression of PrP-sen in L929 cells that express Mo3F4 PrP-sen (right lane). Cells were metabolically labeled, and total PrP-sen was immunoprecipitated using the polyclonal rabbit antiserum R30. B Persistent production of PrP-res in WT L929 cells after exposure to 22L scrapie–infected brain homogenate. C L929 cells expressing high levels of Mo3F4 PrP-sen continuously produce PrP-res on exposure to scrapie agent. The Western blot in panel B was developed with the polyclonal antiserum R30, and the blot in panel C was developed with the monoclonal antibody 3F4. Molecular mass markers (in kilodaltons) are indicated. Unglycosylated (*), partially glycosylated (•), and fully glycosylated (▴) PrP-res is shown. Mock, cells incubated with brain homogenate from an uninfected mouse; PK+, protease-resistant prion protein; PK−, protease-sensitive prion protein
The high rate of infection of mouse fibroblast cell cultures Previous studies of scrapie infection demonstrated that at least some cell lines did not produce detectable levels of PrP-res, even though they replicated TSE infectivity [13]. Cloning of these cells revealed that <1% of the cells accumulated PrP-res. The finding that PrP-res formation was easily detectable in uncloned populations of NIH/3T3 and L929 cells suggested that a higher percentage of these cells might be infected than previously has been described for scrapie-infected mouse neuroblastoma cells [25]. Cloning of both NIH/3T3 cells and L929 cells after 14 and 12 passages, following exposure to scrapie, revealed that ∼12% of NIH/3T3 cells (7 of 60 clones tested) and 47% of L929 cells (9 of 19 clones tested) accumulated detectable levels of PrP-res. Thus, for NIH/3T3 cells (of which 12% of the cells were infected), there were ∼2 infected cells/1 ID50. These experiments demonstrate that, although there appears to be a difference in the susceptibility of NIH/3T3 and L929 cells to 22L scrapie, a higher percentage of cells is susceptible to TSE infection in both cell lines, when compared with the percentage of scrapie-positive cells in other cell lines usually used for scrapie infections [13, 25]. Thus, our data suggest that NIH/3T3 and L929 fibroblast cells are particularly susceptible to scrapie, even though they are of nonneuronal origin
L929 cells generate PrP-res on exposure to different scrapie strainsEarlier studies indicated that L fibroblasts were capable of propagating scrapie infectivity on inoculation with the scrapie strains ME7 and Chandler [11, 24]. To test whether L929 cells were also susceptible to different scrapie strains, Mo3F4-expressing L929 cells (Mo3F4-L929) were exposed to brain homogenates from uninfected or scrapie-infected mice and were continuously passaged. Newly formed PrP-res was present in cells exposed to the mouse-adapted scrapie strains ME7, 22L, and RML (figure 5), whereas no newly formed PrP-res could be detected in cells exposed to uninfected or 87V-infected brain homogenate. PrP-res production was detected even after 5 passages and was independent of the expression of 3F4 epitope–tagged PrP-sen (data not shown). Thus, L929 cells are capable of persistently accumulating PrP-res on exposure to ⩾3 different mouse-adapted scrapie strains, which suggests that these cells are particularly susceptible to TSE infection
Figure 5
Generation of abnormal prion protein (PrP-res) in L929 cells expressing Mo3F4 prion protein. Cells were incubated with brain homogenate from either uninfected mice or mice infected with scrapie strains 87V, ME7, 22L, and RML. Cell lysates were tested for their PrP-res content by use of the monoclonal antibody 3F4. Molecular weight markers (in kilodaltons) are indicated. Unglycosylated (*), partially glycosylated (•), and fully glycosylated (▴), PrP-res is shown. Mock, cells incubated with brain homogenate from an uninfected mouse; PK+, protease-resistant prion protein
Table 1
Propagation of transmissible spongiform encephalopathy infectivity in Mo3F4-ψ2/PA317 cells and NIH/3T3 cells
Discussion
We have demonstrated that the commonly used mouse fibroblast cell lines NIH/3T3 and L929 can support PrP-res formation and can be efficiently infected with scrapie agent. Little information is available with regard to the susceptibility of fibroblast cells to TSE infection in vivo. In vivo, replication of TSE infectivity and accumulation of PrP-res appear to be restricted to cells of the nervous system, such as neurons and astrocytes, and, in some cases, to cells of the lymphoreticular system. An infectivity study that used tissues from cattle inoculated with BSE revealed no infectivity in the skin, a tissue rich in fibroblast cells [35]. Furthermore, human skin fibroblast cells have been reported to express PrP-sen, although no PrP-res was detected in such cells in patients with Creutzfeldt-Jakob disease [36]. Thus, that replication of TSE infectivity has not been reported in fibroblasts in vivo argues that the susceptibility of tissue culture cells to TSE agent cannot be predicted on the basis of their tissue of origin
Previous experiments with mouse neuroblastoma cells have demonstrated that the rate of infection of these cells was <1%, unless they were cloned, which suggests that only a small percentage of the cells were capable of replicating scrapie infectivity [13, 14, 37, 38]. Infection rates of 13% could be achieved by over-expression of mouse PrP-sen in mouse neuroblastoma cells; this finding suggests that high levels of expression of PrP-sen increased the susceptibility of tissue culture cells to scrapie agent [25]. In the present study, we have shown that ∼47% of wild-type L929 cells and 12% of wild-type NIH/3T3 cells, both of which express low levels of PrP-sen, efficiently accumulated PrP-res on exposure to 22L scrapie. Furthermore, titration of 22L scrapie–infected NIH/3T3 cells revealed a titer of 0.06 ID50/inoculated cell. Thus, high levels of PrP-sen expression are not a prerequisite for efficient PrP-res formation and agent replication in all cell lines. Furthermore, the expression of elevated levels of epitope-tagged mouse PrP-sen did not lead to elevated levels of PrP-res formation in fibroblast cell clones, demonstrating that increased expression of PrP-sen does not necessarily correlate with increased PrP-res formation. Overall, our data strongly suggest that the assumptions of a cell line’s susceptibility to TSE infection that are based on the level of expression of PrP-sen are invalid
The lack of a tissue culture system to titer the infectivity from multiple TSE agents means that analysis of agent replication must be done using time-consuming in vivo bioassays. The availability of a cell culture line that is highly susceptible to TSE infectivity would be valuable in the development of an in vitro assay for replication of the TSE agent. In the present study, a high percentage of L929 cells were infected with mouse scrapie strain 22L, which suggests that these fibroblast cells are particularly susceptible to TSE agents. This hypothesis is further supported by our data demonstrating that incubation of L929 cells with ME7 and RML scrapie can lead to persistent formation of PrP-res. Furthermore, L fibroblasts have been reported to be capable of replicating ME7 and Chandler scrapie agents [11, 24]. Thus, because of their apparent susceptibility to different TSE agents, L929 cells may also be a valuable indicator cell culture system for TSE infectivity
Susceptibility of commonly used tissue culture lines, such as fibroblasts, to TSE infection is of particular importance, given the use of tissue culture cells for the production of vaccines and for other medical applications. For example, NIH/3T3 cells are used as feeder cell layers for keratinocyte cultures in cutaneous gene-therapy applications [39, 40]. Thus, TSE contamination of tissue cultures used in medical production—for example, through potentially contaminated bovine serum—could pose a threat to humans. The risk of TSE contamination in bovine media supplements has been considered to be theoretical and remote [41]. Nevertheless, the United States Food and Drug Administration (FDA; Rockville, MD) requests that bovine-derived materials from cattle that have resided in or originated from countries where BSE has been diagnosed not be used in the manufacture of FDA-regulated products, including those produced by tissue cultures [41]. A similar precautionary measure was taken by the Department of Health of the United Kingdom, where a polio vaccine was withdrawn because the manufacturer had not followed the European recommendations on the use of bovine products of British origin during manufacturing [42]. Our findings demonstrating that cell lines of nonneuronal origin can be highly susceptible to infection with TSE agents support these precautionary measures and raise the possibility that other commonly used cell lines may also be susceptible to TSE agents, such as scrapie or BSE
It should be noted, however, that, although BSE has been transmitted to multiple animal species, little information is available regarding the susceptibility of mammalian tissue culture cell lines to the BSE agent. Transmission of TSE infectivity from one species to another is usually inefficient and is accompanied by a prolonged incubation time, a phenomenon referred to as the “species barrier” [43]. Because most cell lines used in research and drug development originate from mammals other than cattle, it is questionable whether these cells can become infected with BSE agent that is potentially present in serum samples or other media nutrients derived from bovine sources. However, infection of rat PC12 cells with mouse-adapted scrapie demonstrates that cross-species transmission of TSE agents in cell culture is theoretically possible [12]. Thus, further experiments are needed to evaluate the potential susceptibility of human or other mammalian laboratory cell lines to TSE agents, such as BSE, and may be advisable for cell lines that are routinely used in the manufacture of medical products
Acknowledgments
We thank Lisa Kercher, Rachel LaCasse, and Rick Race, for critical reading of the manuscript, and Anita Mora, for assistance with the graphics
- Received April 29, 2003.
- Accepted August 14, 2003.
- © 2004 by the Infectious Diseases Society of America
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