Integrating zeta potential with conventional sperm preparation: Comparative effects of swim-up–zeta and density gradient-zeta methods on motility and DNA integrity

Isarin Thanaboonyawat; Isaraporn Thanaratsiriworakul; Japarath Prechapanich; Pitak Laokirkkiat; Roungsin Choavaratana; Somsin Petyim

doi:10.5653/cerm.2025.08788

Clin Exp Reprod Med > Epub ahead of print

Thanaboonyawat, Thanaratsiriworakul, Prechapanich, Laokirkkiat, Choavaratana, and Petyim: Integrating zeta potential with conventional sperm preparation: Comparative effects of swim-up–zeta and density gradient-zeta methods on motility and DNA integrity

Original Article

Clinical and Experimental Reproductive Medicine 2026;cerm.2025.08788.

Published online: April 28, 2026

DOI: https://doi.org/10.5653/cerm.2025.08788

Integrating zeta potential with conventional sperm preparation: Comparative effects of swim-up–zeta and density gradient-zeta methods on motility and DNA integrity

Isarin Thanaboonyawat¹

, Isaraporn Thanaratsiriworakul¹, Japarath Prechapanich¹

, Pitak Laokirkkiat¹

, Roungsin Choavaratana¹, Somsin Petyim^1,²

¹Reproductive and Biology Unit, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand

²Center of Reproductive and Stem cell Biology, Department of Obstetrics and Gynecology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand

Corresponding author: Somsin Petyim Department of Obstetrics and Gynecology, Faculty of Medicine Siriraj Hospital, Mahidol University, 2 Prannok Road, Siriraj, Bangkoknoi, Bangkok 10700, Thailand
Tel: +66(0)-2419-4895-6 Fax: +66(0)-2418-2662 E-mail: somsin101@yahoo.com

^*The study was financially supported by Siriraj Grant for Research Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand (grant number R016133010).

Received October 10, 2025 Revised December 7, 2025 Accepted December 15, 2025

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Objective

Sperm preparation techniques, such as the swim-up method and density gradient centrifugation, are essential for selecting the highest-quality spermatozoa in the context of assisted reproductive technologies. The zeta method, in which sperm are selected based on their surface electrical charge, has recently emerged as a simple and cost-effective adjunct to improve sperm selection. This study aimed to compare sperm motility and DNA integrity between sperm prepared using a combination of the swim-up and zeta methods (S-Z) and sperm prepared with a combination of density gradient centrifugation and the zeta method (D-Z).

Methods

Thirty-nine normozoospermic semen samples were randomly divided into two aliquots. One aliquot was prepared using S-Z and the other using D-Z. Post-preparation sperm motility was assessed using computer-assisted sperm analysis, and DNA integrity was evaluated with acridine orange staining.

Results

Both preparation methods significantly reduced sperm concentration compared to neat semen (p<0.001). The total motility rate was significantly higher in the S-Z group (90%) than in the D-Z group (86%) (p<0.001). Normal sperm DNA integrity was comparable between the S-Z (95.0%) and D-Z (95.3%) groups (p=0.582).

Conclusion

Integrating the zeta method into conventional sperm preparation techniques may increase the efficiency of sperm selection. The S-Z approach appears particularly effective for recovering highly motile sperm with normal DNA integrity.

Keywords: Density gradient; Sperm DNA integrity; Sperm motility; Sperm preparation; Swim-up; Zeta method

Introduction

Infertility is a growing global concern, and effective sperm preparation is essential for optimizing assisted reproductive technologies (ARTs). In intrauterine insemination (IUI), prepared sperm must meet adequate quality and quantity standards [1], while in in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), selecting sperm with good motility and intact DNA remains critical for successful fertilization and embryo development [2]. Semen is a heterogeneous mixture containing normal and abnormal spermatozoa, immature germ cells, and cellular debris, making sperm preparation necessary to isolate functionally competent sperm [3].

Common preparation techniques include the swim-up method, density gradient centrifugation, and microfluidic sorting [4]. An ideal method should be simple, cost-effective, and capable of yielding motile sperm with minimal DNA damage. However, centrifugation-based methods may increase membrane disruption, DNA fragmentation, and reactive oxygen species generation. Swim-up relies on motile sperm migration, whereas density gradient centrifugation separates cells by sedimentation; both are widely used and generally produce comparable motility and morphology outcomes, although their effects on DNA integrity remain debated. Advanced techniques such as magnetic-activated cell sorting and microfluidics offer improved genomic selection but remain limited by high cost and specialized equipment requirements [5].

Compromised sperm DNA integrity is strongly associated with abnormal fertilization, impaired embryo development, implantation failure, and an increased risk of miscarriage [6,7]. In ART, sperm quality is assessed using parameters such as sperm concentration, motility, morphology, and DNA integrity, and both pre- and post-preparation evaluations are essential for optimal clinical outcomes [1,8]. Among commonly used preparation methods, the swim-up method and density gradient centrifugation both reduce apoptotic and necrotic sperm and improve viability [9]. Swim-up typically yields a higher proportion of viable and progressively motile sperm but lower sperm concentrations, whereas density gradient centrifugation often results in better overall motility and reduced DNA damage [8,10].

The zeta method, which leverages the negative surface charge of mature spermatozoa, is a simple, low-cost technique that can be combined with conventional preparation methods [11]. Although it is not widely standardized and is generally not effective as a standalone approach, combining the zeta method with density gradient centrifugation has been shown to improve morphology and progressive motility through a synergistic effect [12,13].

In prior research, sperm DNA integrity was evaluated using acridine orange (AO) staining, a rapid and cost-effective assay that differentiates intact double-stranded DNA (green fluorescence) from denatured single-stranded DNA (red fluorescence) [14,15]. An increased proportion of single-stranded DNA detected by AO has been associated with reduced fertilization rates, delayed embryo development, and a higher risk of miscarriage in IVF cycles [2].

The objective of this study was to compare sperm motility and the proportion of sperm with normal DNA integrity between samples prepared using a combination of the swim-up and zeta methods (S-Z) and those prepared with a combination of density gradient centrifugation and the zeta method (D-Z).

Methods

1. Study design

This experimental study was conducted from March 2015 to December 2015 at the Infertility Unit, Siriraj Hospital, Mahidol University, Bangkok, Thailand, in accordance with the Declaration of Helsinki. The Siriraj Institutional Review Board approved the protocol (SIRB 757/2557) before enrollment.

2. Participants

Male patients attending the Infertility Unit, Department of Obstetrics and Gynecology, Faculty of Medicine Siriraj Hospital, were enrolled in the study. All participants received comprehensive counseling and provided written informed consent. A total of 39 semen samples with normozoospermic parameters were included to evaluate the effectiveness of the sperm preparation techniques.

All participants were instructed to maintain 3–5 days of ejaculatory abstinence before sample collection. Semen specimens were obtained by masturbation directly into sterile plastic containers in a designated private room adjacent to the laboratory. Eligible participants were men older than 18 years with normal semen parameters according to the World Health Organization (WHO) 2010 criteria [16]. Exclusion criteria included abnormal semen analysis based on WHO 2010 guidelines, active sexually transmitted infections (human immunodeficiency virus, syphilis, or hepatitis B), genital tract infections (e.g., active prostatitis or urethritis), and semen obtained through surgical sperm retrieval methods.

3. Semen analysis

After liquefaction, semen processing and analysis were performed in accordance with the WHO guidelines [16]. Seminal volume was measured in a graduated tube, and sperm concentration was determined using computer-assisted sperm analysis (Hamilton Thorne) and expressed in millions per milliliter. Sperm motility was recorded as the percentage of motile sperm (grade A, rapid progressive; grade B, slow progressive; grade C, non-progressive; and grade D, immotile). Eosin-nigrosin staining (Sigma-Aldrich Inc.) and Diff-Quik staining (Clinag Co. Ltd.) were performed to assess sperm vitality and sperm morphology, respectively, under microscopic visualization. All processes were performed by experienced technicians. Sperm morphology was reported as the percentage of sperm with normal morphology. Sperm DNA integrity was assessed using AO staining and expressed as the percentage of sperm with intact DNA.

Following the initial assessment, each semen sample was divided into two equal aliquots for processing according to two different sperm preparation protocols (Figure 1). The first protocol combined the swim-up technique with the zeta method, while the second combined density gradient centrifugation with the zeta method. For both protocols, standard laboratory procedures were followed to ensure consistency and minimize variability. After preparation, the processed samples were reanalyzed for sperm concentration, motility, and DNA integrity using the same analytical techniques described above.

4. Sperm preparation

Following routine semen analysis, each sample was processed using the following methods.

5. Swim-up method

An aliquot of 1 mL of whole semen was gently mixed with 2 mL of sperm preparation medium (Sperm Washing Medium; Irvine Scientific) in a test tube. The mixture was centrifuged at 350 ×g for 10 minutes. The supernatant was discarded, and the pellet was washed again with the same medium at 350 ×g for 10 minutes. Subsequently, 0.5 mL of sperm preparation medium was carefully added to the pellet. The tube was inclined at a 45° angle and incubated for 1 hour at 37 °C in a 5% CO₂ incubator.

6. Density gradient method

The sperm washing procedure involved layering 1 mL of semen over a discontinuous two-layer colloid gradient consisting of 1 mL of 80% colloid solution at the bottom and 1 mL of 40% colloid solution on top (Percoll Plus; GE Healthcare) in a 15-mL polystyrene centrifuge tube. The tube was centrifuged at 350 ×g for 20 minutes. The resulting sperm pellet was then resuspended in 2 mL of sperm preparation medium (Sperm Washing Medium; Irvine Scientific). The diluted sperm suspension was centrifuged again at 250 ×g for 10 minutes, and the final pellet was resuspended in 0.5 mL of the same medium.

7. Zeta method

The zeta method was performed as described in a previous publication [11]. Washed sperm (0.1 mL) were transferred to a new tube and diluted with 5 mL of serum-free 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered human tubal fluid (HTF) medium (Sperm Washing Medium; Irvine Scientific). To induce a positive surface charge, the tube was inserted into a latex glove up to the cap; while holding the cap, the tube was rotated two to three times and then quickly removed. The tube was then held by the cap at room temperature for 1 minute to allow negatively charged mature sperm (−16 to −20 mV) to adhere to the tube wall. Care was taken to avoid grounding the tube by holding it only at the cap. After incubation, the tube was centrifuged at 300 ×g for 5 minutes. The supernatant and pellet were discarded to remove non-adhering sperm and other cells. The tube wall was then rinsed with 0.2 mL of serum-supplemented HEPES-buffered HTF medium (Sperm Washing Medium; Irvine Scientific) to neutralize the charge and detach the adhering sperm. The rinsate was collected and used to wash the tube wall several times to maximize sperm recovery. The recovered sperm fractions were then assessed for concentration, motility, viability, and DNA integrity (Figure 1).

8. DNA integrity testing

A small aliquot (10 µL) of sperm suspension was spread evenly on a glass slide and allowed to air-dry for 20 minutes. The smeared slides were fixed overnight in freshly prepared Carnoy solution (glacial acetic acid and absolute methanol at a 1:3 ratio), then air-dried and stained with AO (Sigma-Aldrich Inc.) at pH 2.5 for 5 minutes at room temperature in the dark. Stained slides were evaluated on the same day using a fluorescence microscope equipped with a 490-nm excitation filter and a 530-nm barrier filter (BX51; Olympus). For each smear, 200 spermatozoa were examined. Normal DNA content was characterized by green fluorescence over the sperm head, whereas abnormal DNA integrity was indicated by fluorescence ranging from yellow to red.

9. Statistical analysis

Statistical analyses were performed using SPSS ver. 18 (SPSS Inc.). Data are presented as mean±standard deviation. The normality of each dataset was assessed using the Kolmogorov–Smirnov test. Paired t-tests were used to compare outcomes between groups. For multiple comparisons, repeated measures analysis of variance with Bonferroni post hoc testing was applied. A p-value of less than 0.05 was considered to indicate statistical significance. The sample size was calculated to achieve 80% power at a two-tailed significance level of 0.05.

Results

A total of 39 semen samples were included in this study. The mean age of the male partners in the infertile couples was 36.4±3.8 years. Baseline semen characteristics are summarized in Table 1. The mean sperm concentration in neat semen samples was 55.4 million/mL. Sperm parameters after preparation using S-Z and D-Z are detailed in Table 2.

Both sperm preparation methods produced significantly lower sperm concentrations than neat semen samples (p<0.001). Notably, the D-Z group had a significantly higher sperm concentration than the S-Z group (p<0.001). For total sperm motility, both S-Z and D-Z groups displayed a significantly higher proportion of highly motile spermatozoa than neat semen (p<0.001). Total motility was significantly higher in the S-Z group than in the D-Z group (p<0.001) (Figure 2).

Sperm viability was also significantly higher after S-Z and D-Z preparation than in neat semen (p<0.001). The difference in viability between the two preparation methods was statistically significant, with the S-Z method displaying higher viability (p<0.001) (Table 2). The percentage of sperm with normal DNA integrity in neat semen was significantly lower than that in the S-Z group (87.6%±5.9% vs. 95.0%±3.3%, p<0.001) (Figure 3).

Similarly, the percentage of sperm with normal DNA integrity was significantly lower in neat semen than in the D-Z group (87.6%±5.9% vs. 95.3%±3.2%, p<0.001). The proportion of sperm with normal DNA integrity in the S-Z group (95.0%±3.3%) was slightly lower than in the D-Z group (95.3%±3.2%); however, the difference was not statistically significant (p=0.582) (Figure 4).

Discussion

Sperm preparation is a critical step in selecting high-quality sperm based on their physical properties to optimize outcomes in ART [1]. A variety of sperm preparation techniques are currently in use, including sperm washing, swim-up, density gradient centrifugation, and microfluidic methods [5,9]. Conventional techniques—such as sperm washing, swim-up, and density gradient centrifugation—remain widely used because they are simple, cost-effective, and do not require expensive disposable sperm chips [9]. Notably, the zeta method offers a simple and inexpensive approach for selecting sperm with intact DNA, potentially improving fertilization and pregnancy rates in ICSI procedures. This study aimed to integrate the zeta method with conventional techniques—the swim-up method and density gradient centrifugation—to evaluate their comparative effectiveness. These findings suggest the potential value of incorporating these combined methods into routine sperm preparation protocols in ART.

The results showed a significant reduction in sperm concentration after preparation in both groups. This is expected, as sperm preparation selectively separates and discards abnormal spermatozoa, retaining only morphologically normal and motile sperm. These findings align with previous studies [8-10]. Interestingly, the D-Z method appeared to recover a greater number of spermatozoa than the S-Z method, suggesting that D-Z may be more suitable for patients with sperm concentrations in the low-normal range. Moreover, sperm motility after preparation in both the S-Z and D-Z groups was higher than in neat semen, indicating that these methods effectively enrich motile sperm for use in ART. Previous studies have reported that the zeta method improves sperm parameters related to DNA integrity and morphology, but not total motility, consistent with the lack of association between zeta potential and motility [11]. The swim-up technique relies on the inherent motility of sperm, allowing those that migrate to the upper layer to be collected, which likely explains the higher motility observed in the S-Z group than in the D-Z group [9]. In contrast, the D-Z method recovered a greater number of moderately motile spermatozoa, thereby increasing the total motile sperm count in samples with abnormal semen parameters. These observations align with studies demonstrating that swim-up and density gradient centrifugation provide complementary advantages [8,9]. In addition, both S-Z and D-Z improved the proportion of normal motile sperm in each sample, as primarily progressive motile sperm were obtained in both groups (Figure 2), with the S-Z group yielding a higher proportion than the D-Z group. Notably, incorporating the zeta method did not appear to substantially improve the performance of either technique in terms of sperm concentration or total motile sperm yield, and the zeta potential may trap more abnormal sperm on the tube surface, contributing to a reduction in total sperm number and motility. Indeed, prior work has shown that using the zeta method alone does not increase total sperm motility [11].

The present study demonstrated that sperm viability in both groups was higher than in neat semen. This is unsurprising, as sperm preparation inherently selects viable sperm. However, sperm viability in the S-Z group was significantly higher than in the D-Z group. This may be explained by the fact that the swim-up method is particularly effective at selecting viable sperm because it relies on motility, whereas the D-Z method may allow some non-viable sperm to remain within the separated fraction. Similar findings have been reported in previous studies [9,17]. In addition, sperm preparation using the zeta method, which involves an attachment/detachment process, has been shown to activate sperm metabolic activity without inducing premature acrosome reaction [18]. However, when combined with conventional sperm preparation methods, the zeta potential may fail to trap non-viable spermatozoa if they have lost their surface charge due to degenerative changes. This could explain the observed differences in sperm viability between the two groups.

Regarding DNA integrity, this study confirmed that both the D-Z and S-Z methods can select a high proportion of sperm with normal DNA integrity, with no significant difference between the two techniques. Previous reports have suggested that DNA integrity following density gradient centrifugation is sometimes lower than that obtained with swim-up, possibly because mechanical stress during centrifugation causes micro-injury to the sperm membrane and subsequent DNA damage [17,19]. Interestingly, the zeta method may enhance the selection of sperm with optimal DNA integrity, while motility-based selection in swim-up also favors sperm with intact DNA. Our findings support previous reports indicating that the zeta method alone can select a sperm population with normal DNA integrity but is not effective in improving sperm motility [11-13]. When combined with conventional sperm preparation techniques, the zeta method may offer a synergistic ‘double selection’ effect, increasing the likelihood of retrieving spermatozoa with normal DNA integrity.

In the butterfly diagram in our study, both sperm selection methods demonstrated homogeneous results, showing higher percentages of sperm motility, viability, and DNA integrity than neat semen. These findings confirm the efficacy of both approaches in selecting functionally competent sperm. A key advantage of the zeta method is the ability to prepare sperm without exposure to high-voltage electricity, expensive electrophoresis equipment, extreme pH conditions, or ultraviolet irradiation [20]. However, a major limitation of the zeta method is its low sperm recovery rate, which restricts its use in cases of oligozoospermia. In addition, it cannot be applied to testicular sperm extraction because sperm obtained using this technique have insufficient net electrical charge on the membrane surface.

This study has several methodological limitations. First, we did not perform direct comparisons between each sperm preparation technique with and without the zeta method. This limits our ability to assess the isolated effect and true efficacy of the zeta method; however, our findings still provide useful insights by comparing outcomes between the S-Z and D-Z groups. The primary constraint was the limited semen volume available from each sample. Recruiting samples with a larger semen volume in future studies would allow comprehensive comparisons among all four groups (swim-up, density gradient, S-Z, and D-Z). Although comparative studies between swim-up and density gradient methods have been reported elsewhere [8,21], our study was restricted to normozoospermic samples. The potential applicability of this sperm preparation strategy should be explored in abnormal semen profiles, such as oligozoospermia and asthenozoospermia.

Additionally, the zeta method alone remains a topic of debate due to several limitations, including low sperm recovery, a lack of direct correlation with motility, time-sensitive surface charge properties, and the absence of standardized protocols. These factors likely explain why the zeta method is rarely used as a standalone sperm selection technique in ART. Finally, this study was conducted in 2015 under an approved protocol, and semen analysis followed the 5th edition of the WHO criteria, which differ slightly from current reference ranges. In the 6th edition of the WHO criteria, the reference ranges for normal semen parameters are slightly lower than in the previous version; this difference does not significantly affect the interpretation of our results, supporting the reliability of the study.

Our results suggest a promising role for the simple zeta method in enhancing sperm selection outcomes when combined with conventional methods. Some evidence suggests that centrifugation may negatively affect sperm quality, even when the zeta method is added to either swim-up or density gradient methods [22,23]. Nevertheless, further research is warranted, particularly in semen samples with different abnormalities and in exploring combinations of selection techniques to optimize sperm recovery. This approach may be applicable in ART for couples with low sperm counts; in such cases, D-Z may be the preferred option for IVF or IUI.

In conclusion, this study demonstrates the potential of integrating the zeta method into conventional sperm preparation techniques to enhance the efficiency of sperm selection. This approach is simple, low-cost, and easily adaptable to routine ART laboratory practice, offering a practical strategy for improving the selection of high-quality spermatozoa.

Conflict of interest

No potential conflict of interest relevant to this article was reported.

Acknowledgments

We would like to thank all participants and all staff from the Infertility and Reproductive Biology Unit, Department of Obstetrics and Gynecology, Faculty of Medicine Siriraj Hospital, Mahidol University, as well as Miss Suvanna Sangchoo, Miss Benjawan Thokha and Miss Suphadtra Phornwilardsiri from the Infertility and Reproductive Biology Unit, for their assistance.

Author contributions

Conceptualization: IT (Isarin Thanaboonyawat), IT (Isaraporn Thanaratsiriworakul), SP. Methodology: IT (Isaraporn Thanaratsiriworakul), SP. Formal analysis: IT (Isaraporn Thanaratsiriworakul), SP. Data curation: IT (Isaraporn Thanaratsiriworakul), SP. Funding acquisition: SP. Project administration: IT (Isaraporn Thanaratsiriworakul). Visualization: IT (Isaraporn Thanaratsiriworakul), SP. Software: IT (Isaraporn Thanaratsiriworakul), SP. Validation: SP. Investigation: IT (Isarin Thanaboonyawat), IT (Isaraporn Thanaratsiriworakul), SP. Supervision: SP. Writing-original draft: IT (Isaraporn Thanaratsiriworakul), SP. Writing-review & editing: IT (Isarin Thanaboonyawat), JP, PL, RC, SP. Approval of final manuscript: IT (Isarin Thanaboonyawat), IT (Isaraporn Thanaratsiriworakul), JP, PL, RC, SP.

Figure 1.

Schematic flowchart of the sperm preparation protocols, divided into two groups: swim-up combined with the zeta method (S-Z) and density gradient centrifugation combined with the zeta method (D-Z). Pre- and post-preparation analyses included computer-assisted semen analysis (CASA) and assessment of DNA integrity using acridine orange (AO) staining.

Figure 2.

Percentage of total sperm motility in neat semen, semen prepared by swim-up with zeta (S-Z), and semen prepared by density gradient centrifugation with zeta (D-Z). (A) Both S-Z and D-Z significantly increased total sperm motility compared with neat semen. The S-Z group displayed significantly higher motility than the D-Z group (p<0.01). (B) Individual sample analysis demonstrated a consistent improvement in motility after preparation compared with neat semen in both the S-Z (left) and D-Z (right) groups. (C) Distribution of sperm motility patterns—rapid progressive (a), slow progressive (b), non-progressive (c), and immotile (d)—in neat semen compared with post-preparation by S-Z and D-Z.

Figure 3.

Percentage of sperm vitality in neat semen, semen prepared by swim-up with zeta (S-Z), and semen prepared by density gradient centrifugation with zeta (D-Z). (A) Sperm vitality was significantly greater after preparation with both methods compared with neat semen, with the S-Z group displaying significantly higher vitality than the D-Z group (p<0.01). (B) Butterfly diagram illustrating paired changes in sperm vitality for each semen sample, showing consistent improvement after S-Z (left) and D-Z (right).

Figure 4.

Percentage of sperm DNA integrity in neat semen, semen prepared by swim-up with zeta (S-Z), and semen prepared by density gradient centrifugation with zeta (D-Z). (A) Both S-Z and D-Z significantly improved sperm DNA integrity compared with neat semen. No significant difference was observed between the two preparation methods. (B) Butterfly diagram illustrating paired changes in sperm DNA integrity for each semen sample, confirming consistent improvement after preparation with S-Z (left) and D-Z (right).

Table 1.

Baseline semen parameters (n=39)

Parameter	Mean±SD	Range
Sperm concentration (×10⁶/mL)	55.4±26.7	20–140
Total motility (%)	53.4±8.1	40–68
Viability (%)	74.7±7.3	55–88
Normal DNA integrity (%)	87.6±5.9	73–98

Concentration is expressed as ×10⁶.

SD, standard deviation.

Table 2.

Effects of sperm preparation compared between the swim-up with zeta (S-Z) and density gradient centrifugation with zeta (D-Z) methods

Parameter	Swim-up with zeta method (S-Z)	DG with zeta method (D-Z)	p-value
Sperm concentration (×10⁶/mL)	19±14.7	25.5±16.7	<0.001
Total motility (%)	90±4.4	86±4.3	<0.001
Viability (%)	95±2.3	92±3.1	<0.001

Values are presented as mean±standard deviation. Concentration is expressed as ×10⁶.

DG, density gradient.

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