Generation of Epstein-Barr Virus Immortalized-B Lymphocytes Cell Line of Orang Asli

Obtaining sustainable source of cells or DNA from a single individual has always been a constraint in biomedical research. Generation of lymphoblastoid cell lines (LCLs) as a surrogate for replacement of isolated peripheral blood mononuclear cells (PBMCs) has substantially accelerated the process of biological investigations. LCLs can be established by transfection of B lymphocytes from peripheral blood with Epstein Barr Virus (EBV), bearing minor genetic and phenotypic alterations. Here we report the generation of LCLs for the indigenous population (Orang Asli) from Peninsular Malaysia. The establishment of cell line is crucial for such isolated populations since the population size is small and sample collections are often challenging. We applied an alternative approach in isolation of PBMCs before proceeding to EBV transfection of Orang Asli samples, of which the BD Vacutainer CPT tubes were used to replace the conventional protocol such as Ficoll-paque or histopaque. We called this approach the “modified method of CPT” (MMC). A total of 59 out of the 64 samples (92%) of Orang Asli were successfully transformed into LCLs by transfecting with EBV supernatant in a single attempt. To verify the success of LCLs transformation and confirm the presence of the human DNA, FCGR3B gene copy number variations of the DNA extracted from the LCLs were typed using Paralogue ratio test-restriction enzyme digest variant ratio (PRT-REDVR) analysis. The genotype results were compared between the DNA obtained from the LCLs and the original PBMCs from the identical individuals, and results revealed 100% concordance rate. The success of the transformation for LCL for the indigenous populations will eventually allow a more comprehensive and advanced biomedical research to be carried out in the next future without the exhaustion of the biomaterials.

Many of these Orang Asli tribes are undergoing drastic transformation of their lifestyle due to urbanization.On a separate note, some tribes for examples the Negritos, have been experiencing isolation and inbreeding resulted a smaller gene pool and population size [2,4].Therefore they are thought to be an ideal model for the population genetic studies and mapping for complex diseases due to the fact that they are less admixed, less exposed to massive environmental changes and long period of isolation [5,6].However, accessing the Negrito populations and obtaining sufficient biomaterials for any biomedical related research has been a major hindrance.Thus establishing the immortalized lymphoblastoid cell lines (LCLs) is of crucial to provide infinite resources of Orang Asli population such as Negritos for future sustainable genomic studies.
LCL is not only capable to extend the proliferation to avoid the exhaustion of storage of precious DNA samples thus minimizes the need of resampling, but also it possesses identical genotypes and expression to their parental cells.Therefore it has been widely used as the resources in many biomedical studies, ranging from gene expression and functionality studies, proteomics and metabolomics, mutational analysis, to the population based genomic studies including Human Genome Diversity Project, HapMap and 1000 Genome Project [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21].
We report in this paper the generation of the immortalized LCL for the Orang Asli population from Peninsular Malaysia using the modified method called MMC and subsequently verified the integrity of genomic DNA extracted from the immortalized LCL samples by typing the FCGR3B copy number variation.

Introduction
Orang Asli is a generic Malaysian term used officially to name the indigenous people of Peninsular Malaysia.They are believed to be the The cells were transferred into a 25 cm 2 tissue culture flask to induce virus production.B95-8 cell line at 1 × 10 6 cells/ml was stimulated with 40 ng/ml tetradecanoylphorbol acetate (TPA) (Sigma) for 1 hour in a CO 2 incubator to produce EBV supernatant.After 1 hour, the cells in the flask were collected by using 10 ml of sterile serological pipettes and transferred into 15 ml Falcon tubes.Cells were washed three times with PBS to remove TPA.Cells were re-suspended in 5 ml of CM1 and placed the flask in a CO 2 incubator for 96 hours.After that, the cell was centrifuged at 300 × g for 10 min at 4 °C to separate EBV particle from B95-8 cells.The supernatant was filtered through a 0.20-micron filter (Sigma) to unsure no remaining of B95-8 marmoset cells was mixed with EBV supernatant.EBV supernatant was aliquot in 1 ml cryopreservation vials and labeled appropriately.The cryopreserved vial can be stored at -80 °C for maximum of one year.

Generation of lymphoblastoid cell line
This protocol was carried out according to Danjoh, et al. (2012) [23].The mixture of the PBMC in CM2 was prepared according to table 2 and cultured into 24-well plate after evenly mixed by eight figure swirling motion.The plate was placed in 5% CO 2 incubator (Galaxy R) at 37 °C for seven days without disturb or any manipulation.On day seven, 0.25 ml of complete media (CM2) was added and the culture was incubated for another seven days where the cell showed rosette morphology and cell clumping, indicating that the cells have reached the stage of exponential proliferation hence can be transferred into 25 cm 2 T-flask for further cell expansion.Then 1 ml of LCLs from 24 well plates was transferred into a 25 cm 2 T-flask.About 4 ml of the CM2 was added into the flask and kept upright in the incubator with 5% CO 2 at 37 °C.The feeding procedure for LCLs in the 25 cm 2 T-flask was continued every 5-7 days with the addition of 2 ml of CM2.This culture condition is biased towards B cells proliferation and limited the growth of other cells.After about 1 month, the LCLs are ready to be prepared for DNA extraction using Qiagen Mini Blood Extraction Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol or were frozen in cryopreserved for future usage.

Validation of the extracted genomic DNA with FCGR3B copy number variation genotyping
DNA was extracted on 14 identical samples of both peripheral blood (PB) and LCLs sample and the quality and purity of DNA extracted from both peripheral blood and LCL were measured by using nanospectrophotometer (Nanodrop, ND-1000).Then, FCGR3B was genotyped by using the paralogue ratio test-restriction enzyme digest variant ratio (PRT-REDVR) technique.The PRT-REDVR assays were described according to Hollox, et al. (2009) [24].The PRT analysis for copy number (CN) call was performed in combination with the REDVR analysis, using a maximum likelihood approach [25].

Sample recruitment and PBMC isolation
Peripheral blood samples were collected in ACD tubes and kept for 3 days at the sampling locations without proper storage equipment.The modified approaches were applied and demonstrated a better PBMCs separation, with minimal red blood cells (RBC) contamination.The cell count viability for Orang Asli PBMC's sample ranged from 30-91% cells/ml for all 64 samples, with the mean ± standard error of mean (SEM) of 7.8 ± 0.14 x 10 6 cells/ml of viable cell with more than 75% of the PBMCs samples show high cell viability ranging from 70-91%.
Department of Orang Asli Development (JAKOA) [Ref no: JHEOA.PP.30.052.Jld 5 (17)].The details of recruited samples are shown in table 1.The Orang Asli samples were collected from Gua Musang, Kelantan, located at the northern east of Peninsular Malaysia.Prior sample collection, the headmen of the tribes or the community members were first consulted in a customary courtesy visit and their permission were obtained.During sampling, all participants were interviewed and written informed consent was obtained.Interview was conducted in Malay language and witnessed by an officer from JAKOA.Only participants above 18 years old who gave consent were recruited in this study.The samples consisted of both males and females from Negrito sub-tribes.A total of 6 ml of peripheral blood (PB) was withdrawn from the 64 volunteers by using acid-citratedextrose (ACD) tube (BD vacutainer ® ) and the samples were kept in Styrofoam storage box and kept at room temperature for further action.

Peripheral blood mononuclear cell (PBMC) separation
We modified the sample isolation method by using the ACD tubes which prolonged the period of preservation especially for remote area sample collection, often without proper storage equipment for many days.PBMCs isolation was conducted using strict aseptic technique using CPT tube that reduced isolation steps greatly as compared to gradient density method using conventional protocol such as Ficollpaque or histopaque solution.We called this alternative approach the "modified method of CPT" (MMC).Firstly, the collected blood samples in ACD tube was transferred into CPT tubes (BD vacutainer ® ) as soon as we were back in the laboratory.The isolation was done by centrifuging (Multifuge 3 s-r, Heraeus) the CPT tubes at 1,750 × g for 30 min at room temperature.Then, the buffy coat (consisted mononuclear cell or PBMC) from CPT was transferred into a 15 ml falcon tube.The PBMCs were washed twice with the Phosphate Buffer Saline (PBS) (Sigma) and centrifuged for 300 × g for 10 min at room temperature.The supernatant was discarded and the pellet was stored in a cryopreserved vial (Greiner Bio-one) in FM media.The cryopreserved vial was stored in -152 °C freezer for longterm storage after performing differential tryphan blue for PBMCs recovery analysis.

EBV stock preparation
The culturing method of B95-8 cell line was adapted from Hui-Yuen, et al. (2011) [22].B95-8 cells (ECACC Lot # 09H030) were cultured in 5 ml CM1 media in a T25 tissue culture flask at 5 × 10 5 cells/ml seeding density.The flask was incubated at 37 °C in the presence of 5% CO 2 .After 72 hours, all B95-8 cells in the flask were collected by using 10 ml of sterile serological pipettes (Greiner Bio-one) and transferred into 15 ml falcon tubes.The cell was then centrifuged at 300 × g for 10 min at room temperature and the pellet was resuspended in fresh 10 ml of CM1 at 1 × 10 6 cells/ml cell density.  of rosette shape (Figure 1).The cell elongation, cell trajectory, big clumping and rosette morphology of the cells observed indicated that the cell was successfully transformed.After 6 weeks, the bigger clumping of the cell can be seen by naked eyes and usually at this stage the LCLs were ready for DNA extraction or to be cryopreserved for future study.

Verification of the integrity of DNA extracted from LCL using PRT-REDVR
PRT-REDVR of FCGR3B CNV was performed on both DNA extracted from identical samples of LCL and peripheral blood samples.Comparison of the copy number calls between LCL and peripheral blood on 14 identical samples revealed 100% concordance of copy number calls, suggesting the success of DNA extraction from the LCL transformation with appropriate integrity (Table 3).

Isolation of PBMC by modified method of CPT (MMC)
The efficiency of establishing B-LCLs from freshly obtained blood samples are often great, however long storage duration in compromised with high temperature during sample collection in rural area is still the biggest hindrance for EBV transfection success story [23,26].The usage of acid-citrate-dextrose (ACD) tube consisting of trisodium citrate, citric acid and dextrose solution which serve as an anticoagulant, cell preservative and nutrient to the cell culture allows better survival of cell during storage by enhancing the vitality and recovery of the leukocytes for up to five days after blood collection [27,28].Thus, it is suitable for sample collection in rural area as demonstrated in this study where ACD tube has shown a better outcome for prolonged blood sample collection specifically in remote area compared to the EDTA and sodium heparin tubes [29].Nonetheless the vitality and recovery of the leukocytes will decrease drastically once all nutrients in the tube are consumed by cellular components.In this study we used the cell preparation tube (CPT) containing citrate anticoagulant with a gradient density Ficoll™ Hypaque™ fluid and polyester gel barrier which permits cell separation from whole blood being conducted in a single centrifugation step with minimal erythrocytes contamination during PBMCs fractionization step.Blood samples should be centrifuged or separated within 2 hours of blood drawing maintain great cell viability.Mononuclear cell recovery decreases with the increased of time delay before centrifugation, falling to 40-80% of PBMC recovery at 12 to 24 hours respectively.

Immortalization of PBMC of Orang Asli samples
Traditionally Ficoll-Paque is placed at the bottom of a conical tube, and blood is then slowly layered above Ficoll-Paque.After a minimum of 30 minutes uninterrupted centrifugation, the following layers will be visible in the conical tube, from top to bottom: (i) Plasma and other constituents, (ii) A layer of mononuclear cells called buffy coat (PBMC), (iii) Ficoll-Paque, and (iv) Erythrocytes and granulocytes which should be presented in pellet form.This separation allows efficient harvest of PBMC's.Point to note that, this technique requires high skilled operator especially when layering the blood above the Ficoll-Paque solution.Mistakes normally occur during this step which could cause damage to the blood sample.Furthermore, additional step where dilution of blood with phosphate buffer saline is needed to perform this technique thus making this technique lengthy and tedious.Our protocol offers more convenient and less exhausting steps by eliminating blood dilution and Ficoll-Paque layering steps during PBMC isolation.This is particularly convenient when involving large number of sampling in remote area with minimal storage equipment by effectively preserves high quality isolated cells even after a standard 48 hours cell isolation window period.

The generation of LCLs for Orang Asli negrito
Approaches for generation of EBV transformed B lymphocyte cell lines have been established since the last 30 years; whilst generation of the LCLs for numerous indigenous populations if the world has been previously reported.Danjoh, et al. (2011) [21] was one of the groups that established LCL on the indigenous ethnic population of South America and deposited in Japan (Sonoda-Tajima Cell Collection).In our study, LCL from 59 Orang Asli samples have been established, consisting of Mendriq (n = 23) and Bateq (n = 27) from Negrito tribe and some sample from Temiar (a Senoi sub-tribe) (N = 9).The success rate of 92.2 of LCL immortalization is comparable with other studies conducted in Japan and United State of America ranging from 90-94% [23,27].Several factors could be attributed to the failure of the transformation of LCL in this study: (i) Compromised sample collection tubes leading to contamination of PBMC; (ii) Low seeding density at initial culture stage; (iii) Long period of cryopreserved PBMCs before EBV transfection; (iv) Low number of PBMCs per sample collected; (v) High percentage of non-viable cells during proliferation stage which contribute to cytotoxic culture condition; and (vi) Suboptimal seeding density of PBMCs after EBV infection and EBV titre.
Our study has successfully demonstrated the transformation of EBV-B lymphocyte by discovering the formation of rosette shape morphology in culture plate as established by previous studies [7,30].The average time taken for the sign has been detected as early as 7-14 days, which in line with previous studies [27,28].Several approaches can be applied to increase the success rate of EBV-B lymphocytes such as increasing the number of PBMCs [31].EBV titer, viability of PBMCs, or the introductions of feeder cell, are among factors to increase the success of LCL generation.In this study we eliminated the cytotoxic effect of T cell by suppress the activity of T cell using cyclosporine A as suggested by previous study [21,23].FK 506 also known as Tacrolimus also can be used to eliminate the activity of T cell activation since this drug has similar function as cyclosporine A [22].

Consistency of using LCL for PRT-RDVR assay for FCGR3B CNV typing
Multiallelic the copy number variation of FCGR3B was significantly associated with a number of systemic autoimmune and infectious diseases [32][33][34][35][36]. Therefore on top of confirming the presence of the genomic DNA of the LCL generated, it is crucial to understand the nature of the gene copy number variation of this gene in immortalized LCL before a thorough investigation is carried out.PRT produces the sum of the copy number calls for FCGR3A and FCGR3B genes whilst the REDVR estimates the copy numbers of FCGR3A and based on the ratio determined.PRT produces the sum of the copy number calls for FCGR3A and FCGR3B genes whilst the REDVR estimates the copy numbers of FCGR3A and FCGR3B based on the ratio determined [32].LCLs are known to exist in two distinguishable forms namely, pre-and post-immortal.In the pre-immortal stage cells proliferate actively and maintain a diploid karyotype.These cells are non-tumorigenic and stable genetically and phenotypically [37,30].
In our study, the comparison of the copy number calls for FCGR3B between LCLs and peripheral blood revealed 100% concordance of copy number calls which prove the stability and integrity of genomic material isolated from the transformed LCLs and suitable to replace the limited peripheral blood.

Summary
We have successfully generated the lymphoblastoid cell lines of the Orang Asli samples using an alternative approach, and have further verified the integrity of the genomic DNA by characterizing the FCGR3B copy number distribution among Orang Asli Negrito using LCLs as starting biomaterial.LCL has been used for last few decades and their utility is gradually being recognized as an excellent surrogate for limited sources of isolated lymphocytes.However, one has to be aware of the few potential inherent limitations of LCLs use, which include potential chromosomal aberration in the transfected B-cells [38][39][40].The generation of LCL for indigenous population will allow more comprehensive advanced investigations to be carried out on these isolated populations in the next future without exhaustion of the biomaterials.

Table 1 :
Sample recruitment of Orang Asli in this study.Samples were collected from numerous locations at the district of Gua Musang, located at the north-east of Peninsular Malaysia.Bateq and Mendriq are the sub-tribe of Negritol; whilst the Temiar is a sub-tribe of Senoi.

Table 2 :
The formulation and counting of PBMC for EBV transfection per well.

Table 3 :
Comparison of copy number (CN) calls for FCGR3B on the genomic DNA extracted from lymphoblastoid cell line and peripheral blood using PRT-REDVR assay.CNV typing revealed 100% concordance rate.PB: peripheral blood; LCL: lymphoblastoid cell line.