An efficient method for immortalization of amniocytes with chromosome abnormalities for renewable application of rare disease sources

Objective: We describe a new method of immortalizing amniocytes with chromosome abnormalities to generate renewable resources for studying rare diseases. Methods: Three methods were investigated by randomly dividing 180 cases of adherent amniocytes into groups A, B, and C. Group A cells were digested with 0.25% trypsin for 10, 20, 30, 60, 90, and 120 mins. Group B and C cells were digested with 0.25% trypsin for 3–5 minutes. Group A and B cells were then transfected with PT67 cell-produced SV40LT. Group C digested cells underwent liposome-mediated SV40LT-transfection. Results: The percentage of clones immortalized by SV40LT transfection in groups A, B, and C were 18.3, 5.0, and 16.7%, respectively, after G418 screening. Group A produced a higher percentage of immortalized clones than did groups B and C, but only significantly differed from group B. The karyotypes and chromosome 13, 18, 21, X, and Y fluorescence signals in transfected cells of all groups were identical to those of the primary cells after passaging for 10–15 generations. Conclusions: The trypsin-mediated SV40LT transfection used in group A can be applied for routine establishment of amniocyte lines to obtain a renewable resource for the study of rare diseases and as quality control materials for prenatal diagnosis.


Introduction
Ideal cell transfection methods should combine both high efficiency and low cytotoxicity. Liposome-mediated simian virus 40 large T antigen (SV40LT) transfection is superior to calcium phosphate coprecipitation and other cell 3 transfection methods [1,2] and is widely used because of its ease and high transfection efficiency. However, liposome-mediated SV40LT transfection results in deregulated gene expression [3], which causes cytotoxicity and impedes its application in the establishment of cell lines. To overcome this, researchers have started to explore new non-liposomal transfection agents [4], but have not yet found more efficient alternatives to liposome-mediated transfection. Transfection of cells with SV40LT alone can lead to cell immortalization and this method has already been widely used to establish tissue cell lines [5]. However, immortalization efficiency using this approach is low [6] and the method has not been used to establish amniocyte lines with chromosome abnormalities. To address this, we conducted in-depth studies to identify an efficient method for the establishment of amniocyte lines with chromosome abnormalities to generate a renewable resource for the study of rare diseases.

Fluorescence in situ hybridization (FISH) detection reagents
The FISH kit was purchased from Beijing JinPuJia Medical Technology. The 13/21 dual-color probe (GLP) provided with this kit includes GLP 13 and GLP 21. GLP 13 hybridizes to 13q14 and produces a green signal. GLP 21 hybridizes to 21q22 and produces a red signal. The 18/X/Y tri-color probe (CSP) hybridizes to the centromeric regions of chromosomes 18, X, and Y and produces sky-blue, green, and red signals, respectively. The fixing solution used consists of 70% methanol and 30% acetic acid.

Recombinant vector SV40LT/pLXSN and expression of SV40LT
SV40 DNA (strain 776) was used as a template to amplify the SV40LT gene by highfidelity PCR [7]. The amplified sequence was digested with EcoRI and BamHI restriction endonucleases and ligated into the pLXSN vector. The constructed recombinant retrovirus vector SV40LT/pLXSN was confirmed by two-way sequencing (forward sequencing primer pLXSN(nt1398-1420) 5′-CCCTTGAACCTCCTCGTTCGACC-3′ and reverse sequencing primer pLXSN(nt1537-1515) 5′-GAGCCTGGGGACTTTCCACACCC-3′). The recombinant SV40LT/pLXSN plasmid was transformed into logarithmically growing PT67 cells (a retroviral packaging cell line (Clontech)) using the calcium phosphate coprecipitation method [8]. After culturing for 72 h in the presence of 500 mg/L G418 (Sigma) for selection, a single clone was picked and amplified to obtain the PT67 packaging cell line containing SV40LT.
Finally, the PT67 culture medium was harvested and NIH3T3 fetal fibroblast cell lines (preserved by our laboratory) were used to evaluate the SV40LT viral titer.

Immortalization of amniocytes
Group A amniocyte samples with chromosomal abnormalities (n = 60) were grown to 5 approximately 80% confluence. These cells were then pre-transfected with culture medium from PT67 cells, containing SV40LT, and grown further for a further 24 h. Subsequently, the culture medium was removed and 3.0 mL 0.25% trypsin was added to each culture flask for digestion. Thereafter, 0.5 mL aliquots of digested cell suspensions were removed after 10, 20, 30, 60, 90, and 120 mins. After washing 2-3 times with phosphate buffered saline and centrifuging at 1000 rpm for 5 mins at room temperature(167.7×g), residual trypsin was removed, and the harvested cells were resuspended in 2 mL of PT67 cell culture medium containing SV40LT. The cells were grown in 6-well plates to approximately 60% confluence, before being screened with 50-400 mg/L G418 for 48 h. Once one or more positive clones were observed, it was assumed that cells were successfully transfected by SV40LT. In groups B and C, amniocyte samples with chromosomal abnormalities (n = 60, for each of B and C) were grown to approximately 80% confluence. Cells were then digested using 0.25% trypsin for 3-5 min and harvested to obtain cell suspensions. Group B cells were transfected with SV40LT from the PT67 cell culture medium [9] and group C cells underwent liposome-mediated SV40LT transfection [2,10]. Cells were inoculated into 6-well plates and positive clones were selected.
SV40LT transfection rates were calculated for groups A, B, and C.

Verification of aneuploidy of amniocyte lines
Amniocyte lines from the 10 th to 15 th generation of aneuploid primary cells (47, XY, +13 47, XXY) were harvested. The 13/21 dual-color and 18/X/Y tri-color probes were used to detect chromosomes 13, 18, 21, X, and Y in both the generated cell lines and primary cells [11,12]. The fluorescence signals in the generated cell lines and primary cells were compared.

Renewable application of amniocyte lines in FISH detection quality control
The concentration of cells in the 47, XY,+13, 47,XY,+18, 47,XY,+21, and 48,XXYY 10 th generation was calculated using cell counting plates, Four cell suspensions containing 2 × 10 5 cells/mL were prepared. To generate chimeric quality-control cell lines for FISH, equal volumes of the four cell suspensions were mixed. Theoretically, cells of each of the contributing karyotypes account for 25% of the total. Then, three blinded technicians were used for the FISH procedure [11,12]. Each of the technicians was unaware of the composition ratio of the chimeric cell lines used.
Each technician successively and confidentially counted 100 cells with obvious target chromosome fluorescence signals under a fluorescence microscope for five consecutive days. The results are presented as mean ± 1 SD. Inter-and intraobserver variability were analyzed using SPSS 17.0 statistical software.

Results
The effect of amniotic fluid cell morphology We found that spindle-shaped amniocytes (Fig 1 A-C) can be transfected by SV40LT, and that round-, elliptical-, and polygonal-shaped amniocytes (Fig 1 D-H) cannot be immortalized using SV40LT (Fig 1).

Discussion
Recently, research focusing on the pathogenesis and prenatal diagnosis of birth defects is increasingly valued [14,15]. However, because birth defects are usually rare diseases, in-depth study is often hindered by insufficient samples. Therefore, there is an urgent need for a renewable source of samples with which to study birth defects [16].
The identification of SV40LT for cell line construction has been regarded a research highlight in the tissue engineering field since Gould first proposed the concept of "cell immortalization" [5,17,18]. SV40LT can immortalize most human cells [5], allowing them to retain the differentiated phenotype and biological characteristics of the primary cells [18,20] and to be passaged for more than 350 generations in vitro [ 19]. Transfection using SV40LT has been extensively applied to immortalize human gastric mucosal cells [21], nasopharyngeal cells [22], tracheal epithelial cells [21], and dermal and embryonic lung fibroblasts [23]. These cell lines are successfully constructed from surface cells of the respiratory and digestive tracts, which arise from the same source as do fetal amniocytes. Therefore, we speculated that SV40LT-transfection may be used to immortalize amniocytes with chromosomal abnormalities. To date, there are no reports detailing the establishment of SV40LTtransfected amniocyte lines with chromosomal abnormalities to generate a renewable resource of rare disease samples.
In this study, for the first time, we immortalized amniocytes from rare diseases using trypsin. At first, we used an SV40LT-transfected human hepatocyte cell line to establish aneuploid amniocyte lines [9,24]. As shown in Table 1, only three samples of amniotic fluid cells were transfected by SV40LT (in group B), and the observed transfection rate of 5.0% (3/60) is similar to that previously reported [6]. We speculate that the observed low transfection efficiency was related to difficulties in the passaging of primary amniocytes which consist of cells with different characteristics. Some of these component cells, susceptible to transfection by SV40LT, may have died before transfection. Moreover, the cell wall structure may also affect transfection efficiency. To solve these problems, and improve transfection efficiency, we introduced a pre-transfection step with SV40LT. We followed this with passage amplification. This benefits component cells by facilitating passage through SV40LT gene integration. We then introduced a new procedure, digesting pre-transfected amniotic fluid cells with 0.25% trypsin for 10, 20, 30, 60, 90, and 120 min following which cells were transfected with SV40LT.
Using this approach, we presume that cell wall proteins are trypsinized, making the cell wall more amenable to the transfection process. We found that establishment of cell lines from spindle-shaped primary amniotic fluid cells (Fig. 1A-B) was easier than the establishment of cell lines from cells of other shapes (Fig. 1D-H). SV40LT-pre-transformed adherent amniocytes transfected by SV40LT are sensitive to trypsin digestion and die easily. Therefore, trypsin digestion should be limited to 3-5 min in these cells. We also found that SV40LT transfection efficiency is affected by the concentration of G418 used for selection. For some SV40LT-transfected samples, selection using lower G418 concentrations (50 and 100 mg/L) resulted in positive colonies (Fig. 2B), while no positive colonies were obtained using higher G418 concentrations. These results indicate that when selecting for positive transfected cell clones, a G418 concentration gradient should be used. We performed SV40LT transfection in 60 samples of amniotic fluid cells with chromosomal abnormalities using each of the approaches detailed. The transfection rate (18.3%) obtained from trypsin-mediated SV40LT transfection, which does not cause cytotoxicity, in group A is consistent with that obtained using traditional liposome-mediated SV40LT transfection (16.7%) [1][2][3] in group C, and was superior to that of transfection mediated by PT67 cell culture medium in group B [9].
The immortal cell lines constructed in this study have stable karyotypes and can be used for quality-control in prenatal diagnosis. We randomly selected three samples of amniocyte lines after 10-15 generations for routine karyotype analysis. Although mutations occurred in chromosome 7, 18, 20, and 22 in 3% of amniocyte lines from one sample (Fig. 3D), the other karyotypes were consistent with those of their respective primary cells (Fig. 3). Consistent with previous reports [18,20], these results show that the karyotypes are stable, and can serve as quality-control material for karyotype analysis. The small level of chromosome variation observed could be explained by the mutation of some individual cells within the amniotic fluid. Therefore, it is important to evaluate the skill of the technicians in diagnosing abnormal karyotypes. Randomly selected amniocyte lines from generations 10-15, 13 and their respective primary cells, were tested using FISH and the 13/21 dual-color and 18/X/Y tri-color probes. The fluorescence signals of chromosomes 13, 18, 21, X, and Y were consistent in these cells. These results indicate that the fluorescence signal of the established cell lines was consistent with that of the corresponding primary cells, and can be used for quality assessment of FISH detection. Based on reports using 60% and 20% as judgment boundaries of aneuploidy and diploidy [25,26], four cell lines with abnormal chromosome numbers were mixed in equal proportions to generate quality-control cells for FISH. As shown in Table 2, The actual ratios of 47,XY,+18 and 25% 48,XXYY measured by technicians A and C significantly each differed from their theoretical ratios of 25% with P < 0.05 and P < 0.001, respectively. Quality evaluation of karyotype analysis showed that 47, XX, +mar was present 8% less frequently than predicted. Moreover, 47, XX, +mar was misdiagnosed as 47, XX, + 19 with a misdiagnosis rate of 5.0% (1/20) (Table 3).

Conclusion
Trypsin-mediated SV40LT transfection, as described here for the first time, is an ideal immortalization method for spindle-shaped amniocytes. This method can be applied for the routine establishment of amniocyte lines to obtain a renewable resource of rare disease samples and will prove useful as quality-control material for prenatal diagnosis.