Abstract
Problem:
A variety of methods have been used to process cervical cytobrush and genital tissue for flow cytometric evaluation of immune cell populations. We sought to optimize genital tract specimen processing and to determine if blood could be used as a model for assessment of tissue processing methods.
Method of Study:
Cervical cytobrushes, PBMCs, and genital tissue samples (cervical and endometrial biopsies) were subjected to varying processing conditions to characterize the effects on cell yields, lymphocyte viability and surface receptors. We exposed PBMC and tissue specimens to varied collagenase types, concentrations, and exposure durations and cytobrushes to immediate vs delayed processing with/without vortexing.
Results:
PBMCs and tissues exposed to varying enzymatic digestion conditions demonstrated stability of some cell surface receptors, including CD3+, CD4+, and CD8+, while others, including CCR6+, were cleaved when exposed to any concentration of collagenase B, or ≥0.25mg/mL of collagenase D. We observed increased CD69 expression (marker of cell activation) after exposure to collagenase B. Neither a 2-hour delay in cytobrush processing nor vortexing at a setting of 50% for 30 seconds had significant impacts on viability or quantities of genital immune cells of interest.
Conclusions:
Although tissue digestion with collagenase D was sufficient to recover and analyze cells from endometrial biopsy specimens, cervical biopsy specimens required a limited exposure to collagenase B at 1mg/mL to optimize cell yield and viability for cytometric analysis. PBMCs can be used as a model to assess the impact of tissue processing on co-receptor expression and to optimize methods prior to study implementation.
Keywords: Cytobrush, Tissue, Biopsy, Flow Cytometry, Collagenases, Lymphocytes
1. Introduction
Flow cytometry is a useful tool designed to characterize cellular populations based on assessment and quantification of individual cellular features of interest. Flow cytometry has been widely employed in clinical and research applications to evaluate blood cells. Since single-cell suspensions are necessary for flow cytometric evaluation, application of this tool to resident cells in tissues or cells collected using cytobrushes requires processing to yield single cell suspensions. Protocols for processing tissues are referenced in the literature, including the Worthington Tissue Dissociation Guide1, which provides citations and examples of collagenase digestion for almost all tissue types. Many protocols for tissue digestion recommend long exposures to relatively high concentrations of collagenase.1 To date there have not been published assessments of the impacts of tissue processing on important cellular markers and receptors of interest in the female genital tract. Cell surface receptors may be disrupted or altered by tissue digestion procedures, or cells may express new markers of activation or stress in response to tissue processing. While there have been some head-to-head comparisons of specific digestion conditions2–4, currently there are no published comparisons evaluating the impacts of varying digestion and processing conditions on specific immune cell populations from female genital tract tissues.
We sought to evaluate the effects of sample processing and enzymatic digestion on immune cell populations resident in female genital tract tissue and mucosa collected by biopsy and cytobrush. We also sought to determine if PBMCs could be used as a model to quantify cellular changes in response to tissue processing exposures. We compared enzymatic digests using collagenase B (col B) (Millipore Sigma, St. Louis, MO) and collagenase D (col D) (Millipore Sigma) at varying concentrations (0.25–1.0mg/mL) and for varying exposure times (20–60 minutes). For this study, CD4+CCR5+ was selected because it is the co-receptor for HIV on B and T lymphocytes.5 CCR6+ was chosen because it is a marker for Th17 lineage polarization and HIV permissiveness in memory CD4+ T cells.6 CD69+ was selected because T cells expressing this activation marker are also considered to be more susceptible to HIV.7
2. Material and Methods
2.1. Study population
Samples were collected from non-pregnant, healthy women at the Center for Family Planning Research at UPMC Magee-Womens Hospital, Pittsburgh, PA. The Institutional Review Board at The University of Pittsburgh approved the collection of samples and all participants signed informed consent prior to performing any study procedures. Blood and genital samples were collected from participants during any day of their menstrual cycle with no active bleeding or spotting and were eligible regardless of birth control method used. Women were excluded if they were post-menopausal, pregnant or within 60 days of their last pregnancy, at risk of pregnancy due to heterosexual activity since last menses without contraception, reported exposure to a male sexual partner with symptoms of urethritis, or were positive for active infection (HIV by rapid test (OraQuick, OraSure Technologies, Inc., Bethlehem, PA); Trichomonas vaginalis by OSOM rapid test (Sekisui Diagnostics, LLC, Lexington, MA); Neisseria gonorrhoeae or Chlamydia trachomatis by nucleic acid amplification tests (Gen-Probe, Hologic, San Diego, CA); herpes simplex virus lesions or genital warts by visual examination; symptomatic candidiasis by examination and microscopy; bacterial vaginosis by Amsel’s criteria; suspected urinary tract infection by urinalysis; syphilis by RPR). Women were also excluded if they had used a systemic or vaginal antimicrobial agent in the last 30 days, prior hysterectomy, prior malignancy of the genital tract, or were immunosuppressed.
2.2.1. PBMC sample collection, processing, and conditions tested
PBMC exposure to col B vs. col D at specific concentrations and exposure times were evaluated to determine the effects on viability and lymphocyte populations of interest. Blood was collected using BD Vacutainer Cell Preparation Tubes (CPT) (Becton Dickinson and Co, Franklin Lakes, NJ). CPTs were processed within one hour of collection following the manufacturer’s instructions. Briefly, tubes were subjected to centrifugation at 1300x g for 30 minutes with brake off, cell monolayers were transferred to 15mL conical tubes, DPBS was added to bring to 15mL total volume, and then centrifuged at 300x g for 15 minutes. After decanting the supernatant, cellular pellets were washed with 10mL DPBS, centrifuged (10 minutes at 300x g), and the resulting pellets were resuspended in 2mL DPBS. Lymphocytes were quantified using trypan blue exclusion criteria5, and DPBS added to adjust lymphocyte concentration to 2×106 per mL. We utilized 1×106/mL PBMC lymphocytes per digest condition, including exposure to varying collagenase types (col B vs. col D at 0.25 mg/mL for 20 minutes), varying collagenase concentrations (col D at 0.25 mg/ml, 0.5 mg/mL and 1.0 mg/mL for 20 minutes), and/or varying exposure time (col D at 0.25 mg/mL for 20, 40, and 60 minutes). All PBMC exposures were paired from the same individual for each of the conditions tested and compared to control PBMCs from the same individual not exposed to any collagenase. PBMCs from individual participants were exposed to collagenase and agitated at 750–800 rpm at 37°C for the specified time in a total volume of 20mL. After digestion, cells were washed in the digest buffer by centrifugation (400x g, 10 min), and the cellular pellet resuspended in 1mL DPBS before staining.
2.2.2. Endometrial sample collection, processing, and conditions tested
Endometrial and cervical tissue biopsy samples were used to evaluate digestion methods that were found to be least impactful on PBMCs to assess whether these methods would adequately digest tissue stroma and recover cells of interest.
For direct comparison, multiple endometrial biopsies collected at a single visit were subjected to various digestion conditions. Use of paired endometrial samples collected from an individual additionally controlled for cellular variation due to menstrual cycle day. Endometrial biopsies were collected using endometrial samplers (GynoSampler®, McKesson, San Francisco, CA) and the tissues were transported to the laboratory in the biopsy curettes. Processing began within one hour of tissue collection. Each biopsy was weighed to standardize all calculations as events per gram of tissue and divided such that equal volumes of endometrial tissue were exposed to the varying digest conditions. Endometrial tissue was digested for 20 minutes with: no enzyme (control), 0.25 mg/mL col D, and ≤0.25 mg/mL col B in 20 mL and agitated at 750–800 rpm at 37°C. After digestion exposure, samples were filtered through a 70μm cell sieve to yield single cell suspensions. Any visible tissue on the sieve surface was pushed through using the rubber-coated plunger from a syringe while the sieve was rinsed with tRPMI and resultant single-cell endometrial suspensions were washed and counted as described above.
2.2.3. Cervical biopsy collection, processing, and conditions tested
Two ectocervical biopsies were collected using Tischler forceps and transported to the laboratory in 3 mL of tRPMI within one hour of collection. Upon receipt in the laboratory, each biopsy was cut into two pieces of approximately equal size, and half of the first biopsy was placed with half of the second biopsy, constituting a cervical tissue sample, to minimize the potential impact of heterogeneous cell distribution in tissue. Cervical tissue samples were weighed to standardize all calculations as events per gram of tissue, then scissor minced in 500μl of the test digest media, and transferred to 50cc conical tubes for digestion. Samples from seven individual participants were used for head-to-head digestion comparisons, including varying collagenase types (col B vs. col D at 0.25–1.0 mg/mL for 20 minutes), samples from 16 individual participants were used for varying collagenase concentrations (col B at 0.5 mg/mL and 1.0 mg/mL for 20 minutes), and samples from 13 participants were used to test varying exposure time (col B at 0.25–1.0 mg/mL for 5–60 minutes) in 20 mL and agitated at 750–800 rpm at 37°C. After each digestion exposure, cellular suspensions were filtered, washed, resuspended and counted as described above.
2.2.4. Endocervical cytobrush sample collection, processing, and conditions tested
Cytobrush processing conditions evaluated included testing the effects of vortexing and of processing delays on cellular viability and recovery. Two cytobrushes were collected per participant by consecutively inserting each cytobrush into the cervical os, rotating 360° and placing the cytobrush in 3 mL of total RPMI [tRPMI:RPMI 1640, 1X with L-glutamine and 25 mM HEPES (Corning, New York, NY) with 10%HI-FBS (Life Technologies, Carlsbad, CA)] on ice and transporting to the laboratory. To test the impact of vortexing on cytobrush cell recovery there were paired samples tested from 10 participants, one of the paired cytobrushes was vortexed at 50% of the maximum setting for 30 seconds while the other was not subjected to vortexing. Sample processing began within 30 minutes of collection and the order of the cytobrush collected for vortexing was alternated to minimize systematic bias since more cells may be collected with the first brush. To assess the impact of transport time cytobrushes were collected from seven participants, one cytobrush was processed within 30 minutes of collection and the other was stored at 4°C, then processed two-hours after collection. No vortexing was utilized in studies evaluating time to processing.
Mucus and cellular material were recovered from cytobrushes by washing with tRPMI and scraping along the inside edges of 15mL conical tubes. Once all visible material was removed, mucus was disassociated in tRPMI using transfer pipettes to create a visually uniform suspension before filtering through a 70μm sieve (Corning) using the rubber-coated plunger from a syringe while washing with tRPMI. Filtered cell suspensions were washed by centrifugation at 400x g for 10 minutes, the supernatant discarded, and cellular pellets re-suspended in 1ml Dulbecco’s Phosphate Buffered Saline (DPBS) (Mediatech, Inc., Manassas, VA). Lymphocytes were counted using trypan blue exclusion criteria.8
2.3. Flow cytometry
Samples were stained for viability9 (LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit; Invitrogen, Carlsbad, CA) per manufacturer’s instructions, including room temperature incubation for 25 minutes in the dark. Samples were then washed with 1mL FACS buffer (eBioscience®, San Diego, CA) and centrifuged (400x g, 5 minutes). Supernatant was decanted, and the following stains from BD Biosciences (San Jose, CA) were added to the cellular pellet: CD3 (PerCP, SK7), CD4 (FITC, RPA-T4), CD8 (AmCyan, SK1), CD69 (PE, FN50), CCR5/CD195 (PE-Cy7™, 2D7) and CCR6/CD196 (Alexa Flour® 647, 11A9). Cells were incubated for 25 minutes at room temperature in the dark, washed with 1mL FACS, centrifuged (400x g, 5 minutes), the supernatant was decanted, and 2mL RBC Lysis Buffer (eBioscience®) was added and mixed by pipetting. Samples were incubated for two minutes, 2 mL cold DPBS was added, samples were centrifuged (400x g, 5 minutes), and the cellular pellet was resuspended in 200μL FACS buffer. Samples were analyzed with a LSRII FACS (BD Biosciences) and FlowJo software (FlowJo, LLC, Ashland, OR). The flow cytometric gating strategy utilized for this study is shown in Figure 1.
Figure 1. Gating strategy for cervical cytobrush, PBMC, endometrial tissue, and cervical tissue.
For all sample types, live single cells were identified from the lymphocyte population. CD4+ and CD8+ T cells were identified from CD3+ lymphocyte populations; CCR5+, CD69+, and CCR6+ cells were identified from both CD4+ and CD8+ cell populations.
2.4. Statistical analysis
Wilcoxon signed-rank tests were used to evaluate the effect of vortexing on immune cell population recovery from paired endocervical cytobrush samples. Differences in cell recovery from matched PBMC samples treated with collagenases B and D and untreated samples from 11 participants were evaluated using paired Student’s t-tests. Mixed effects generalized linear models were used to estimate the effects of collagenase type, concentration, and exposure time on immune cell populations (number of cells and percentage of parent population) in PBMC, cervical tissue, and endocervical cytobrushes. Generalized estimating equations were used to analyze the endometrial tissue data due to the small number of samples. For the endometrial tissue, analyses were performed on all samples presented below and restricted to paired samples. Estimates of mean difference in cell counts and percent of parent population of the treatment conditions from the referent condition in each model are shown along with 95% confidence intervals and corresponding p-values. A 2-sided P-value <0.05 was considered statistically significant.
3. Results and Discussion
Enzymatic digestion is a step intended to enhanced disaggregation of tissues into single cell suspensions. PBMCs, which are more readily available than tissue biopsy samples, were exposed to various tissue digestion conditions to screen for off-target effects on cellular markers of interest. We found no significant processing-induced alterations to PBMCs exposed for 20 minutes to 0.25mg/ml col D compared to PBMCs exposed to buffer alone (Table 1). Conversely, we found exposure of PBMCs to 0.25mg/ml col B for 20 minutes significantly induced cellular changes, including higher mean viability; lower proportion of CD3+CD4+; lower proportion of CD3+CD4+CCR6+; and higher numbers and proportion of CD3+CD8+ T-cell populations compared to buffer-only controls (Table 1). Compared to controls, which were not subjected to digestion, exposure to either col D or col B digestion resulted in the recovery of higher numbers of CD3+CD8+CCR5+ cells.
Table 1.
Comparison of 0.25 mg/mL of collagenase D, collagenase B used to digest PBMC for 20 minutes compared to buffer-only (no enzyme) exposure
| Cell Populations | No Digest (n=11) Mean (‡SD) | Col D 0.25mg/mL 20 min (n=11) Mean (‡SD) | Mean difference (§95% CI) | †P-value | Col B 0.25mg/mL 20 min (n=11) Mean (‡SD) | Mean difference (§95% CI) | †P-value |
|---|---|---|---|---|---|---|---|
| %Viability | 98.7 (0.9) | 99.1 (0.4) | 0.4 (−0.1, 0.9) | 0.12 | 99.4 (0.3) | 0.7 (0.1, 1.2) | 0.03 |
| #CD3+ | 93,395 (15,298) | 93,987 (14,509) | 592 (−1276, 2461) | 0.50 | 94,335 (14,274) | 939 (−2143, 4021) | 0.51 |
| #CD3+ CD4+ | 58,507 (13,543) | 59,121 (13,025) | 615 (−539, 1768) | 0.26 | 58,354 (12,441) | −152 (−2076, 1771) | 0.86 |
| %CD3+CD4+ | 64.2 (8.1) | 63.9 (7.5) | −0.2 (−1.4, 1.0) | 0.70 | 63.0 (7.7) | −1.1 (−2.1, −0.2) | 0.03 |
| #CD3+CD4+CCR5+ | 5,089 (2,261) | 5,329 (1,999) | 240 (−735, 1215) | 0.60 | 5,178 (1,772) | 90 (−846, 1025) | 0.84 |
| %CD3+CD4+CCR5+ | 9.6 (6.2) | 10.1 (6.8) | 0.5 (−1.1, 2.1) | 0.48 | 9.9 (6.5) | 0.4 (−1.2, 1.9) | 0.61 |
| #CD3+CD4+CD69+ | 3,371 (1,536) | 3,030 (1,619) | −341 (−1691, 1009) | 0.59 | 4,334 (2,544) | 964 (−795, 2722) | 0.25 |
| %CD3+CD4+CD69+ | 5.7 (2.5) | 5.3 (3.1) | −0.4 (−2.5, 1.7) | 0.66 | 7.4 (4.5) | 1.7 (−1.4, 4.8) | 0.25 |
| #CD3+CD4+CCR6+ | 8,824 (9,087) | 6,368 (2,121) | −2457 (−7964, 3051) | 0.34 | 3,812 (2,830) | −5012 (−10614, 589) | 0.07 |
| %CD3+CD4+CCR6+ | 14.9 (12.8) | 11.1 (3.8) | −3.7 (−11.9, 4.4) | 0.33 | 6.3 (4.5) | −8.6 (−16.8, −0.4) | 0.04 |
| #CD3+CD8+ | 28,421 (7,747) | 29,611 (7,549) | 1191 (−330, 2711) | 0.11 | 30,265 (7,815) | 1844 (350, 3337) | 0.02 |
| %CD3+CD8+ | 31.7 (7.6) | 32.4 (7.2) | 0.8 (−0.4, 1.9) | 0.16 | 33.1 (7.3) | 1.4 (0.5, 2.2) | 0.005 |
| #CD3+CD8+CCR5+ | 5,661 (2,288) | 6,916 (2,881) | 1255 (215, 2295) | 0.02 | 6,995 (2,991) | 1334 (−10, 2678) | 0.05 |
| %CD3+CD8+CCR5+ | 20.0 (6.8) | 23.0 (7.1) | 3.1 (0.5, 5.6) | 0.02 | 22.8 (6.6) | 2.8 (−0.9, 6.5) | 0.13 |
| #CD3+CD8+CD69+ | 2,427 (957) | 2,368 (1,314) | −59 (−833, 714) | 0.87 | 2,707 (987) | 280 (−302, 862) | 0.31 |
| %CD3+CD8+CD69+ | 8.0 (3.1) | 7.8 (3.3) | −0.2 (−2.4, 1.9) | 0.80 | 9.0 (3.3) | 1.0 (−1.3, 3.3) | 0.37 |
| #CD3+CD8+CCR6+ | 3,854 (2,997) | 3,806 (2,450) | −48 (−2172, 2076) | 0.96 | 2,834 (2,106) | −1020 (−3194, 1155) | 0.32 |
| %CD3+CD8+CCR6+ | 14.3 (12.1) | 12.7 (7.9) | −1.6 (−9.4, 6.2) | 0.66 | 8.9 (5.9) | −5.3 (−13.3, 2.7) | 0.17 |
P-values from paired Student’s t-test
95% CI refers to a 95% Confidence Interval
SD refers to the Standard Deviation
Data considered statistically significant if P≤0.05
After varying the concentration of col D in the digestion mixture, there were significant concentration-dependent decreases in number and proportion of CD3+CD4+CCR6+ cells (Figure 2) and increases in number and proportion of CD3+CD8+CCR5+ cells, (mean difference/0.25mg col D of 421 (183, 659) and 0.8 (0.4, 1.2) respectively, both P≤0.001 (data not shown)). There were no significant differences in cellular viability or any other cell populations of interest with increasing col D concentration up to 1mg/mL.
Figure 2. Impact on CD3+CD4+CCR6+ cells from PBMCs exposed to increasing concentrations of collagenase D.
PBMCs were exposed to increasing concentrations of collagenase D (col D) and CD3+CD4+CCR6+ cells were measured using flow cytometry. Compared to exposure of PBMC to a no digest control of buffer only, there was a concentration dependent decrease in (a) number and (b) proportion of CD3+CD4+CCR6+ cells recovered (P=0.002 and P<0.001 respectively for control compared to exposure to 1mg/mL col D). These PBMC screening results suggest trial of lowest col D concentration tissue digestion for analysis of CD3+CD4+CCR6+ cells.
Exposure time-dependent shifts in cell populations were also observed. When col D exposure times were increased, the proportions of CD3+CD4+ significantly decreased and CD3+CD8+ cells significantly increased, with mean difference/minute −0.02 (−0.04, −0.002), P=0.032 and 0.03 (0.01, 0.04), P=0.008 respectively (data not shown). There were no significant differences in cellular viability or any of the other cell populations of interest with increasing col D exposure time up to 60 minutes.
Our PBMC model found fewer processing-associated changes using col D compared to col B digestion. For endometrial tissues, more lymphocytes (CD3+) were recovered when tissues were digested with col D or col B compared to no digest controls with a mean difference of 566,767 cells (35,090, 1,098,445) P=0.04 for col D compared to control (Table 2). Similar to our PBMC model, we found no significant changes in cell viability (P=0.35), number or proportion of CD3+CD4+CD69+ cells (P=0.08 and P=0.91 respectively) for col D exposed tissue compared to control.
Table 2.
Comparison of 0.25 mg/mL of collagenase D, ≤ 0.25 mg/mL collagenase B used to digest endometrial tissue compared to buffer-only (no enzyme) exposure
| Cell Populations | No Digest (n=5) Mean (‡SD) | Col D 0.25mg/mL 20 min (n=9) Mean (‡SD) | Mean Difference (§95% CI) | †P-value | Col B ≤ 0.25mg/mL 20 min (n=11) Mean (‡SD) | Mean difference (§95% CI) | †P-value |
|---|---|---|---|---|---|---|---|
| %Viability | 61.8 (29.3) | 67.2 (37.6) | 6.0 (−6.7, 18.7) | 0.35 | 80.6 (6.9) | 7.3 (−7.0, 21.6) | 0.32 |
| #CD3+ | 264,724 (302,560) | 809,420 (721,385) | 566767 (35090, 1098445) | 0.04 | 650,104 (517,953) | 109568 (−77716, 296851) | 0.25 |
| #CD3+CD4+ | 58,632 (71,287) | 203,758 (257,916) | 151439 (−17609, 320487) | 0.08 | 166,440 (122,833) | 109568 (−77716, 296851) | 0.25 |
| %CD3+CD4+ | 30.2 (7.2) | 32.6 (11.6) | 1.6 (−6.3, 9.6) | 0.69 | 30.6 (8.6) | 2.0 (−6.9, 10.9) | 0.66 |
| #CD3+CD4+CCR5+ | 33,995 (36,724) | 116,709 (150,430) | 86858 (−8360, 182075) | 0.07 | 85,741 (62,032) | 50653 (−54961, 156267) | 0.35 |
| %CD3+CD4+CCR5+ | 65.9 (13.2) | 56.3 (14.8) | −9.5 (−23.1, 4.1) | 0.17 | 57.4 (10.8) | −8.4 (−23.2, 6.3) | 0.26 |
| #CD3+CD4+CD69+ | 34,298 (36,367) | 133,455 (181,199) | 104588 (−10427, 219603) | 0.08 | 87,190 (65,621) | 51322 (−76082, 178,725) | 0.43 |
| %CD3+CD4+CD69+ | 64.9 (16.0) | 65.0 (19.6) | 1.1 (−17.9, 20.1) | 0.91 | 59.0 (14.0) | −9.1 (−28.6, 10.5) | 0.36 |
| #CD3+CD4+CCR6+ | 6,309 (9,164) | 17,854 (27,642) | 11408 (−7559, 30374) | 0.24 | 2,875 (2,942) | −2775 (−23542, 17992) | 0.79 |
| %CD3+CD4+CCR6+ | 11.1 (8.9) | 9.4 (5.7) | −2.0 (−7.6, 3.7) | 0.49 | 1.9 (1.5) | −8.5 (−14.6, −2.3) | 0.007 |
| #CD3+CD8+ | 150,583 (231,945) | 318,752 (402,282) | 196111 (−100021, 492244) | 0.19 | 395,385 (380,463) | 215861 (−113747, 545468) | 0.20 |
| %CD3+CD8+ | 64.7 (4.9) | 55.0 (13.8) | −9.7 (−18.1, −1.3) | 0.02 | 61.1 (10.1) | −8.1 (−17.5, 1.3) | 0.09 |
| #CD3+CD8+CCR5+ | 105,833 (166,633) | 183,417 (213,821) | 91427 (−75367, 258222) | 0.28 | 251,448 (251,506) | 114345 (−71732, 300422) | 0.23 |
| %CD3+CD8+CCR5+ | 71.9 (8.7) | 62.5 (15.8) | −11.3 (−20.3, −2.3) | 0.01 | 67.9 (11.9) | −8.8 (−18.9, 1.3) | 0.09 |
| #CD3+CD8+CD69+ | 113,811 (165,898) | 251,202 (330,959) | 158641 (−76845, 394127) | 0.18 | 283,016 (264,982) | 114734 (−114003, 409471) | 0.27 |
| %CD3+CD8+CD69+ | 81.2 (5.8) | 78.5 (13.3) | −3.0 (−14.4, 8.4) | 0.60 | 75.1 (7.8) | −6.6 (−18.4, 5.2) | 0.27 |
| #CD3+CD8+CCR6+ | 2,699 (4,931) | 5,258 (10,227) | 2311 (−4379, 9002) | 0.87 | 873 (794) | −903 (−8302, 6496) | 0.81 |
| %CD3+CD8+CCR6+ | 2.8 (3.9) | 1.9 (1.7) | −1.1 (−3.0, 0.8) | 0.75 | 0.3 (0.3) | −2.0 (−4.0, 0.1) | 0.06 |
P-values and estimates of mean difference from mixed effects generalized linear models
95% CI refers to a 95% Confidence Interval
SD refers to the Standard Deviation
Data considered statistically significant if P≤0.05
The proportion of CD3+CD4+CCR6+ cells significantly decreased when endometrial tissue was digested with col B (mean difference −8.5 (−14.6, −2.3) P=0.007) and was not impacted by col D digestion relative to control (Figure 3), similar to what was observed with the PBMC model. However, the PBMC model did not predict the significant decreases in the proportions of CD3+CD8+ lymphocytes and CD3+CD8+CCR5+ cell with col D digestion compared to control with mean differences −9.7 (−18.1, −1.3) P=0.02 and −11.3 (−20.3, −2.3) P=0.01) respectively. There were no other significant processing-induced alterations in lymphocyte populations when col D or col B were used to digest endometrial tissue. All data from endometrial experiments can be seen in Table 2. Analyses restricted to paired samples yielded similar results although some of the above associations lost statistical significance due to small sample sizes (n=5 for Col D and n=3 for Col B).
Figure 3. Impact on CD3+CD4+CCR6+ cells from endometrial tissue digested with collagenase D or collagenase B.
Endometrial tissue was digested with 0.25mg/mL collagenase D (col D) or ≤0.25mg/mL collagenase B (col B) for 20 minutes. Compared to a no digest control, (a) a non-significant trend toward more CD3+CD4+CCR6+ cells recovered from the col D digest compared to no digest and (b) a significantly decreased proportion of CD3+CD4+CCR6+ cells were recovered after col B digest compared to no digest controls (P=0.49 and P=0.007 respectively). CCR6 surface receptor is particularly vulnerable to destruction by collagenase, particularly col B.
For optimal processing of endometrial tissue, we found a brief exposure to low concentration col D minimized cellular changes and increased viable CD3+ recovery compared to either longer exposures or exposures to higher col D concentrations. In contrast, we found 89% mean cell viability from cervical tissue digested with high concentration (1mg/mL) col B for ≥30 minutes compared to 60% for matched col D exposure (P=0.01). There was a non-significant, concentration-dependent trend towards increased number of viable CD3+ cells with col B digestion (Figure 4a), a significant increase in number of CD3+CD4+CD69+ cells (P=0.03) with 1mg/mL col B digestion for ≥30 minutes, and a significant decrease in the proportion of CD3+CD4+CD69+ cells (P=0.05) with 0.5mg/mL col B digestion compared to matched col D exposures. With a short (<30 minute) exposure to col B, there was an increase in number of CD3+CD8+ cells (P=0.04) and CD3+CD8+CD69+ cells compared to matched col D exposure (Figure 4d). As shown in Figure 4, col B compared to matched col D cervical tissue digestion significantly impacted CCR6 receptors with decreased number and proportion of CD3+CD4+CCR6+ cells (Figures 4b and 4c; P=0.004 at 0.5 mg/mL) and decreased proportion of CD3+CD8+CCR6+ cells (Figure 4e; P=0.005 at 1mg/ml for < 30 minutes). Thus, col B appears to cleave CCR6 cell surface receptors and to induce activation of cells as measured by CD69 expression. These observed col B effects on CCR6+ and CD69+ receptors were identified in our PBMC model and confirmed in tissue digestions (Table 1). Figure 5 also demonstrates this effect on CCR6+ and CD69+ receptors using paired samples of PBMCs exposed to col B for 20 minutes.
Figure 4. Impact of varying cervical tissue digestion methods on cell populations of interest.
Cervical tissue was digested with 1mg/mL collagenase D (col D) and 0.5mg/mL collagenase B (col B), 1mg/mL col B for less than 30 minutes, and 1mg/mL col B for time periods of longer than 30 minutes. Compared to col D, (a) there was a non-significant in the number of CD3+ cells, there was a significant decrease in the number of CD3+CD4+CCR6+ cells when digesting with 0.5 mg/mL col B, 1 mg/mL for < 30 minutes and 1 mg/mL for > 30 minutes (P=0.027, P=0.011, and P=0.039 respectively), (c) and significant decrease in the proportion of CD3+CD4+CCR6+ cells when digest with col D was compared to digest with 0.5 mg/mL col B, 1 mg/mL for < 30 minutes and 1 mg/mL for > 30 minutes (P≤ 0.001 for all three digests), (d) there was a non-significant increase in CD3+CD8+CD69+ when digest with col D was compared to digest with 0.5 mg/mL col B and 1 mg/mL col B for > 30 minutes, and a significant increase when digest with col D was compared to digest with 1 mg/mL col B for < 30 minutes, (e) and a non-significant decrease in the proportion of CD3+CD8+CCR6+ cells when digest with col D was compared to digest with 0.5 mg/mL col B and a significant decrease in the proportion of CD3+CD8+CCR6+ cells when digest with col D was compared to digest with 1 mg/mL col B for < 30 minutes and 1 mg/mL > 30 minutes (P=0.014 and P=0.031 respectively). Although a higher number of CD3+ cells were recovered with col B there is a possibility of inadvertently effecting the number or proportion of surface receptors when this enzyme is used for digest.
Figure 5. Effects on CD3+CD4+CCR6+ and CD3+CD4+CD69+ cells isolated from PBMC exposed to collagenase B for 20 minutes.
Paired PBMC samples exposed to buffer control (a and c) or collagenase B (b and d) for 20 minutes demonstrating the reduction in CD3+CD4+CCR6+ cells when exposed to collagenase B (b) compared to controls (a) and the increase in cellular activation as measured by CD69+ from collagenase B exposure (d) compared to controls (c).
Collectively our data suggest that PBMCs can serve as a model for initial screening of tissue digestion methods and that pilot testing and refinement of digestion methods in the target tissue will often also be necessary. We confirmed suitable digestion of endometrial tissue biopsies with col D. However, we found that adequate recovery of cells from cervical tissue, which has far greater stromal density compared to endometrial tissue, required use of col B. For cervical tissue, we found that exposure to 1mg/mL of col B for 20 minutes, balanced adequate tissue digestion and obtained higher viable cell yield with limiting artifacts due to processing-related cellular changes.
There were no significant differences in cell yields or receptor expression comparing cytobrushes processed within 30 minutes vs 2 hours of collection, data not shown. Cell viability from cytobrushes subjected to vortexing were similar to control samples. Vortexing is commonly used when processing cytobrush specimens10–14 and is intended to increase cell recovery by dislodging cells trapped in the cytobrush. However, we found a non-significant reduction in the median number of cells recovered for all populations of interest from vortexed cytobrushes compared to non-vortexed cytobrushes (Figure 6). Thus, we found no benefit and a trend towards reduced cell recovery when cervical cytobrush specimens were vortexed.
Figure 6. Impact of vortexing on median number of cells recovered from cervical cytobrush.
Paired cervical cytobrushes were collected from 10 participants and processed for flow cytometry with or without use of vortexing. To minimize collection bias, we alternated vortexing the first-collected and the second-collected cytobrush since generally more cells are collected with the first cytobrush. There were non-significant reductions in the median number of cells for all populations of interest recovered from cytobrushes that were vortexed compared to those that were not vortexed (P=0.39 for overall recovery of viable lymphocytes).
4. Conclusions
We recommend optimizing tissue collection and processing techniques and understanding processing impacts prior to initiating any protocol that relies on enzymatic digestion for flow cytometric analysis. A strength of our evaluation was use of paired samples and techniques to minimize cellular heterogeneity, especially in small tissue biopsies. We were limited in the number of parameters that could be tested using a single sample due to the small size of biopsies and the number of biopsies that can reasonably be obtained from a participant at a given time.
Although our use of PBMCs to model impacts of tissue digestion exposures was not novel,15–18 we conducted a step-by-step evaluation of the impact of different enzymes, enzymatic concentrations, and digestion times on cell surface receptors using female genital tract tissues. We agree with previous studies that enzymatic and physical tissue processing methods can artifactually alter cells of interest.15–18 The most novel finding of this study is that collagenase treatment during processing can cleave certain cell surface markers and can increase cellular activation. For the specific markers of interest to us, both CCR6+ and CD69+ were clearly impacted by collagenase treatment. Collagenase treatment is routinely used for tissue processing for flow cytometry, and this work demonstrates that collagenase treatment can induce changes and result in data that do not reflect the cells in the native tissue. The present study also adds to the literature supporting the use of purified PBMCs to evaluate processing methods prior to their use with biopsy tissues.
Here we have outlined techniques for both optimizing tissue and cytobrush processing and for understanding possible processing related impacts, which may help standardize procedures, minimize artifacts, and promote data comparisons across studies.
Acknowledgements
This work was supported by Division of AIDS, US National Institute of Allergy and Infectious Diseases, US National Institutes of Health, Grant Number: 1R01AI102835.
Footnotes
Conflicts of Interest
SLH is a consultant for Merck, Pfizer, Curatek, Hologic and Daré Bioscience, and receives research funding from Becton-Dickinson and Curatek. SLA has served as a consultant to Merck Sharp & Dohme Corp and has received research grants from Mithra and Evofem. All other authors declare no competing interests.
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