Development of a Serial Dilution Technique for Obtaining Monoclonal Cell Populations


 Single cell-based techniques have drawn the attention of researchers, because they provide invaluable information of various domains ranging from genomics to epigenetics, transcriptomics, and proteomics. Single cell-derived clones provide a reliable and sustainable source of genetic information due to the homogeneity of the cell population. Aiming to obtain single-cell clones, several approaches were engineered, among which, the Limiting dilution approach stands out as a cost-effective and unsophisticated, and easy-to-apply method. Here, we demonstrate how to acquire single cell-derived clones through a simple 1:10 diluting from genetically modified heterogeneous cell populations.


Introduction
Thus far, researchers have strived to envision innovating ways of sustainable systems of genetically modifying technologies, arming us to ght diseases, modulate cells, or rectifying mistakes in the genetic material of the cell (1). To this date, the various systems have broadened our understanding of the cell; these approaches include Zinc Finger Nucleases (ZFN), Transcription Activator-Like Effector Nucleases (TALEN), and the paradigm-shifting Clustered Regularly Interspaced Palindromic Repeats/ CRISPRassociated protein 9 (CRISPR/Cas9). CRISPR/Cas9 is genuinely one of the greatest recent developments in molecular biology and genetics which holds great promises in advancing our understating of the biology and developing new therapeutic options in clinical medicine. Those discoveries have collectively empowered researchers to manipulate genetic material to correct inherited genetic errors, create genetically modi ed animal models, and study cellular pathways (2)(3)(4).
Researchers have successfully diversi ed the mechanism of actions and applications of the aforementioned-engineered nucleases. However, they usually function via on of the two highly mechanisms of DNA repair; the Non-Homologous End Joining (NHEJ) and Homology Directed Repair (HDR) (5). Introduction of Double Strand Break (DSB) mediated by the programmed-nucleases triggers the activation of either NHEJ or HDR, recruits the components of the repair system to the targeted site, and restores the wild-type genotype. These strategies generate heterogeneous populations of cells.
Impreciseness and off-target effect of the systems on top of generating heterogeneous cells hampers the e cacy of these systems and delays their application. Therefore, a procedure that would allow us to separate analyze single cells, scrutinizing for the desired genotype, would come to prove highly useful (6).
Numerous strategies including Fluorescent-Activated Cell Sorting (FACS), limiting dilutions, and cloning cylinders, have been developed to solve the issues caused by the generation of heterogeneous cell population. These techniques are successful to tackle the task of nding the cells carrying the desired genetic modi cations to some extent. However, an ideal approach should be straightforward, e cient, high throughput, and compatible with the nature of the cellular manipulations. Cost-effectiveness is another limiting factor that is of utmost importance (7). Approaches such as FACS are expensive and require high-tech facilities, which is highly unfavorable. Moreover, the excessive stress induced on cells would result in a decreased growth rate and threaten their viability. The Cloning cylinders method is restricted to adherent cells, is incompatible with suspensions cells cultures and demands further exhaustive aseptic techniques (8).
Other methods including Magnetic-Activated Cell Sorting (MACS) and Laser-Capture Microdissection (LCM), operating based on cell characterization, have been devised to compensate for the shortcomings of the previous systems. However, these systems are also costly; MACS require labeling antibodies, and LCM demands trained operators. Aiming to resolve the highly important element of cost-effectiveness, the limiting Dilutions system was introduced which is further branched into approaches including low-density seeding, array dilution, and serial dilution. The simplicity is the main idea behind these methods, making them highly desirable (6).
In this study, we employed the Limiting dilutions system to isolate single cells from a heterogeneous HEK293 cell population, subjected to genetic modi cation via the CRISPR/Cas9 technique.

Procedure
Ensuing transfection, Green Fluorescent Protein (GFP) serving as a screenable gene marker took roughly around 48 to 72 hours to reach a detectable threshold of a uorescent microscope. Several papers have reported that the transfection aid materials including the Lipofectamine 2000 and 3000 re ect a celldependent e cacy, which requires further optimization for each cell type (9). Studies demonstrate that the Cos-7 cell line holds the transfection e ciency of roughly 95%; other efforts cite that cells such as Caco-2 display a rather lower e ciency of 75%. Studies focusing on HEK293 cells have elucidated Lipofectamine 3000 results in higher cellular plasmid uptake when compared with other variants such as Lipofectamine 2000 (10). Furthermore, some cell types are intrinsically more resilient to obtaining foreign genetic materials from the environment, neuroblastoma cells and primary astrocyte cells were cited to be among such categories of cells with approximately 10-12% and 5-12% e ciency, respectively (11). Here, we observed that roughly 50 % of cells absorbed the plasmids containing the target and the reporter genes. To facilitate the transfection process we exploited the Lipofectamine 2000. Single cells were observed in 30 wells under a stereomicroscope, proliferated to constitute a clone of 40 cells after 10-12 days ( Figure 2). NOTE: Our results are in concordance with previous studies demonstrating that roughly, a third proportion of the entire wells contains a cell or scattered cells, ensuing the diluting process (7). NOTE: As cells' characteristics are distinctive to their type, they may differ in matters of cell-cell interactions; therefore, they may require more time to form. Our experiences unveiled that during the rst 5 to 6 days of incubation and frequent media change, the single cell-derived clone begins to form and is observable after 10 to 12 days. NOTE: To identify the desired genetic modi cations the automated Sanger sequencing method is recommended, for high-throughput sequencing analysis, derivative methods of Sanger sequencing including pyrosequencing and illumine-based sequencing have also proven to be highly effective approaches. TA cloning is the method of choice for separating the different alleles of genes to identify the heterogeneity.
NOTE: Sequence analysis without prior selection of single cells and the separation of alleles may lead to witnessing different peaks for the same spot. Interpreting such ndings would be an insurmountable task, therefore prior isolations are highly recommended. NOTE: Antibiotics similar to serum proteins may interfere with the transfection process. Additionally, antibiotics may lead to an increase in cell death (12).
2) Supplement 250μl Opti-MEM medium with 10μl Lipofectamine and 4μg of the desired vector in separate 1.5 ml microtubes and further incubated for 20-25 minutes at room temperature.
NOTE: For the majority of cases the appropriate DNA (μg) to Lipofectamine (μl) ratio is 1:2 to 1:3 (according to the manufacture's instruction). Nevertheless, due to the inherent toxicity of LFNs and distinct cellular characteristics, further optimization of the ratio for every cell line seems to be necessary (13).
3) Gently pipet and mix the vector and Lipofectamine, and further transfer the mixture into plates 4) Replace the Opti-MEM medium with complete growth medium (DMEM, 10% FBS, 1% penicillin/streptomycin) 4-6 hours following transfection.
NOTE: Cell viability would be compromised if the mixture remains in contact with cells for a longer period. Of note, incubations may slightly vary as a function of the district characteristics of each cell type (14).

Single-cell isolation:
Ensuing transfection, screen the cell populations based on the GFP protein expression and Puromycin serving as a selectable marker carried by the vector, identify and separate the cells for further incubation and expansion for 7 days.
1) Trypsinize the cells and transfer them to a sterile 15ml falcon.
2) Pellet the cells via centrifugation at 1500 rpm for 5 minutes.
3) Resuspend the cells in a 1 ml complete growth media.

4) Cellular viability analysis using the Trypan blue method 5) Counting viable cells via Neobar chamber
6) Isolate and resuspended 10 6 cells in 1 ml growth medium 7) In order to reach a single cell, a serial dilution should be carried out according to the following procedure: 100μl of the original cell suspension should be transferred to a new ask containing 900μl medium, this process dilutes the original sample containing 10 6 cells by 1:10 ratio, yielding a suspension of 10 5 populations. Next, 100μl of the ask encompassing 10 5 cells should be transferred to a subsequent ask and the process should continue accordingly until reaching a population of 10 3 cells. Next, 100μl of the previous culture should be conveyed to the next ask, however this time, instead of 900μl of media, cells should be supplied with 9 ml of medium, rendering a nal concentration of 10 cells per 1 ml medium. Every 100μl derived from the last ask would roughly contain one cell, which should be further transferred into 96-well plates. Figure 1 illustrates how the serial dilution process should be performed.
NOTE: Gentle pipetting throughout the entire process is essential to both preventing the formation of clumped cells and generation of foam. Furthermore, It ensures that cells are homogeneously dispersed in the solution.
NOTE: Proper mixing is a vital step to improve the reproducibility of the serial dilution procedure.
8) Transfer 100μl of the previous step's result to 96-well plates ( Figure 1B). 9) Incubate the cells at 37°C for 6-7 days without any disturbance (Figure 1.C (. NOTE: After 6-7 days, check the medium for any change in the color, which is a re ection of the pH. In case of acidi cation and shift in color, substitute half of the medium with fresh and pre-warmed medium to prevent cellular demise. Following reaching desired cellular con uency, cells should be passaged into 48-well plates and as this process further proceeds, the passaging should be carried out into a 24, 12, and 6 well plate in a step-wise manner (Figure 1.D).
Screening and analysis: 1) Extract DNA of each well, amplify DNA by PCR, and ligate PCR product into TOPO cloning vector to separate two alleles of every single cell.
2) Screen and analyze DNA fragments by sequencing. The advent of genome engineering tools such as CRISPR/Cas9, which marked a turning point in the eld of genetic manipulations of basically every organism presented a conundrum of trustworthiness (15) . As a way to address the issue, we envisioned that a uni ed protocol of isolating cells is required. One of the major issues associated with such techniques is the heterogeneity of the modi ed population of cells induced through indels and point mutations (16,17). Analysis of such a genetically diverse population of cells is an arduous task, and would generate and reliable outcome. To remedy that, Single-cell analysis was proposed. Current approaches operating based on single-cell analysis include the FACS, MACS, LCM, micro uidics, and limiting dilution. Each of the aforementioned strategies possesses its own merits and downsides: choosing them is majorly a matter of personal preference. Nonetheless, the limiting factors involving in selecting an approach is straightforwardness, cost-effectiveness, being high-throughput, and less time-consuming. The serial dilution approach as a variant of the Limiting dilution strategy has demonstrated to be satisfactory. The method possess considerable advantages, it is easy to perform and does not require high-tech facilities.
Methods that currently exist to separate single cell-clones are FACS, MACS, LCM, micro uidics, and limiting dilution (8,18). Researchers choose which technology is most appropriate, based on the purpose of isolation (i.e. therapeutic or research); and their advantages and disadvantages, (19). Researchers tend to use methods with adequate throughput, simple, fast, and inexpensive properties (20). Among these techniques, serial dilutions that is a kind of limiting dilution provides relatively desired conditions for researchers. Although the Limiting dilution is inherently easy to perform, the downstream processes may be demanding, as handling a single cell comes with its own challenges, to this end, techniques such as automatic microscopic imaging have come to facilitate the process.
Unlike the FACS and LCM strategies, the probability that cells would be contaminated during the performance of serial dilution is quite low. Furthermore, the method holds a promising future due to costeffectiveness and unlike other methods does not impose any stress that would compromise cellular viability (21). Here, we endeavored to isolate single cells from 10 6 primary populations. We report a 41% e ciency, which could be further improved by e cient pipetting. Considering that the approach is economically favorable, the overall results are satisfactory.

Figure 1
An overview of preparing serial dilutions and expanding single-cell colonies. A. Transferring 100 µl of the previously prepared cell suspension to a subsequent falcon. B. The diluted cells are transferred to 96 wells. C. Theoretically, wells ought to receive a single cell, however, practically some wells may be devoid of any cells while others may receive more than one, particularly in situations that the cell suspension is not thoroughly pipetted and evenly distributed among wells. D. Propagation of cells takes place within 7 to 10 days. Reaching higher con uency, cells are transferred to a 48-well, then a 24-well, and nally a 6well plate in a step-wise manner. If high yield DNA/RNA is required for downstream applications, a T-25 or T-75 ask is suitable for subsequent passages.

Figure 2
Monitoring the process of single-cell colony formation. The gure presents a clear demonstration of a single (left) and a dual (right) colony formation within a well after 7 days of cultivation. Those containing two colonies should be identi ed and excluded from further examination.

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