Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2021 Aug 1.
Published in final edited form as: Exp Eye Res. 2020 May 4;197:108046. doi: 10.1016/j.exer.2020.108046

Isolation and Characterization of Primary Human Trabecular Meshwork Cells from Segmental Flow Regions: New Tools for Understanding Segmental Flow

JA Staverosky 1, K Dhamodaran 2, TS Acott 1, VK Raghunathan 2,3,4,*, JA Vranka 1,*
PMCID: PMC7483402  NIHMSID: NIHMS1601166  PMID: 32376472

Abstract

Segmental flow in the human trabecular meshwork is a well-documented phenomenon but in depth mechanistic investigations of high flow (HF) and low flow (LF) regions are restricted due to the small amount of tissue available from a single donor. To address this issue we have generated and characterized multiple paired HF and LF cell strains. Here paired HF and LF cell strains were generated from single donors. Cells were characterized for growth and proliferation, as well as gene and protein expression of potential segmental region markers. Cells isolated from HF and LF regions have similar growth and proliferation rates. Gene expression data reveals vascular cell adhesion protein 1 (VCAM1), thrombospondin 2 (THBS2), and tissue inhibitor of metalloproteinase 1 (TIMP1) are potential markers of LF cells in vitro. Protein expression of VCAM1, THBS2 and TIMP1 are complex and may reflect the dynamic nature of the TM. Initial protein expression levels of these genes is either similar between HF and LF cells (VCAM1, THBS2), or higher in HF compared to LF in some strains (TIMP1). However, after long term culture LF cells express higher levels of VCAM1, TIMP1 and THBS2 protein compared to HF cells. HF and LF cell strains are a powerful new tool that enable understanding segmental flow allowing for multiple experiments on the same genetic background.

Keywords: trabecular meshwork, aqueous humor outflow, anterior segment, segmental outflow, glaucoma

1. Introduction

Glaucoma is the primary cause of blindness worldwide yet its etiology is poorly understood(Quigley, 2011; 1996). An increase in intraocular pressure (IOP) is both a key diagnostic tool and the only treatable feature of glaucoma(Grant, 1963; Rohen et al., 1993). The trabecular meshwork (TM) is the principal tissue responsible for regulation of IOP(Bradley et al., 1998; Johnson et al., 1989; Tamm, 2009). Aqueous outflow through the TM is segmental in nature, with areas of high flow (HF), intermediary or mixed flow (MF) and low flow (LF). These different regions can be visualized in standard anterior segment perfusion culture, and a number of studies have highlighted differences in stiffness as well as gene and protein expression(Buller and Johnson, 1994; Carreon et al., 2017; J. Y. H. Chang et al., 2014; de Kater et al., 1989; Ethier and Chan, 2001; Hann et al., 2005; Keller et al., 2011; Overby et al., 2009; Swaminathan et al., 2014; Vranka and Acott, 2017; 2016; Vranka et al., 2015a; 2015b; 2018).

To date, our understanding of HF and LF regions and the potential role they play in overall IOP regulation has been limited to immunohistochemical, proteomic, and gene array studies with few mechanistic insights. The TM is a dense tissue, with a complex extracellular matrix (ECM) surrounding relatively few cells(Acott and Kelley, 2008; Keller and Acott, 2013). It is estimated that the TM of a single eye has approximately 300,000 cells and this progressively declines with age and disease(Acott and Wirtz, 1996; Alvarado et al., 1984; 1981; Lütjen-Drecoll, 1999; R. C. Tripathi and B. J. Tripathi, 1982). Considering the fact that only about a third of an eye is HF, LF or MF, obtaining enough material from either normal or glaucomatous tissues to do comprehensive molecular mechanistic investigations, which may include intervention in the form of siRNA or therapeutics, is challenging(Vranka et al., 2015a).

To overcome these limitations and to gain further insight into HF and LF differences we have established paired HF and LF cell strains generated from single pairs of donor eyes. Here we characterize their morphology, growth, proliferation, elastic moduli, and ECM gene expression levels. Additionally, we identify specific ECM proteins that show a dynamic pattern of temporal expression which may provide further insights into the underlying mechanisms of segmental flow.

2. Materials and Methods

2.1. Generation of HF and LF cell strains

Human donor eye tissue use was approved by the Oregon Health and Science University Institutional Review Board and all experiments were done in accordance with the tenets of the Declaration of Helsinki for the use of human tissue. Human donor eyes were obtained within 48 hours of death from either SavingSight (Kansas City, MO) or from Lion’s VisionGift (Portland, Oregon). Eyes from donors with a history of ocular disease were not used. Non-identifying information was supplied from the eye banks including age, race, sex and known ocular history (summarized in Table 1). Anterior segments were cultured in stationary organ culture for five days prior to perfusion to facilitate recovery from postmortem effects. Standard perfusion culture apparatus and media. (Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Paisley, UK) supplemented with 1% Penicillin/Streptomycin/Fungizone (Gibco, Grand Island, NY)) were utilized at a constant pressure of 8.8 mmHg for approximately 24 hours to establish normal flow conditions as described previously(Acott et al., 1988; Bradley et al., 1998; Keller et al., 2009; 2008). Fluorescently-labeled amine-modified 200nm FluoSpheres (ThermoFisher Scientific, Waltham, MA), or Cell-Mask Orange (ThermoFisher Scientific, Waltham, MA), and 200nm PolyStyrene Basic Black Uniform Dyed Microspheres (Banks Laboratories Inc, Fishers, IN) were diluted 1:500 (FluoSpheres and Cell Mask) or 1:100 (Microspheres) in phosphate buffered saline (PBS) and introduced to the media line via a 300μl bolus(Vranka et al., 2015a). The labeling was done for 1 hour or until 100μL had flowed, whichever came later. Accumulation of the black beads was visually obvious and allowed for separation of HF and LF regions under a laminar flow hood. Separation was confirmed and refined by fluorescent microscopy using an Olympus BX51 microscope or Leica MZ10. The HF regions from both eyes were combined, as were the LF regions. MF regions were not used in this experiment. From this point we followed the standard human TM (hTM) protocol as described in the recent consensus white paper(Keller et al., 2018). Briefly, TM tissue was dissected and cultures were either established using cytodex bead method, or digested overnight using 125μg/mL collagenase type IV (Gibco, Grand Island, NY) and 125μg/mL dispase (Gibco, Grand Island, NY). Cells were allowed to grow out of digested tissue into a 6cm plate in DMEM supplemented with 20% FBS (Sigma-Aldrich, St. Louis, MO) and 1% Penicillin/Streptomycin/Fungizone. Both methods yielded viable hTM cells. Upon establishment of primary cultures, cells were routinely cultured in DMEM with 10% FBS and 1% antibiotic/antimycotic supplements. Segmental cell strains were originally established from 14 pairs of human donor eyes; however, only 8 pair were included in these studies. Cell strains from 6 pairs of eyes were excluded based on the criteria outlined in the TM consensus paper (Keller et al 2018), as is described below.

Table 1:

Demographic summary of donors.

Donor Age Sex Race HF LF Myocilin upreg Growth Proliferation AFM Stiffness qRT-PCR passage qRT-PCR serum WB ICC
2018–0426 49 M C ✓ ✓ Yes ✓ ✓ ✓ ✓ ✓ ✓
2018–1189 65 M C ✓ ✓ Yes ✓ ✓ ✓ ✓ ✓ ✓
2018–1235 66 F C ✓ ✓ Yes ✓ ✓ ✓ ✓ ✓
2018–1244 69 M C ✓ ✓ Yes ✓ ✓ ✓ ✓ ✓ ✓ ✓
2018–1524 51 M ND ✓ ✓ Yes ✓ ✓ ✓ ✓ ✓ ✓
2018–1723 50 M C ✓ ✓ Yes ✓ ✓ ✓ ✓ ✓
UHCO-2289 67 M C ✓ ✓ Yes ✓ ✓
UHCO-2328 53 M C ✓ ✓ Yes ✓ ✓
Open in a new tab

C -Caucasian, ND - not disclosed, HF - High Flow, LF - Low Flow, WB - Western Blots, ICC - Immunocytochemistry.

2.2. Myocilin expression in response to dexamethasone

Cells were plated in duplicate at 1.5×105 cells per well in 6-well plates. Cells were treated with 100nM dexamethasone (dex) (Sigma-Aldrich, St. Louis, MO) or DMSO, as vehicle control, with media changes every three days for two weeks. Three days before the end of the experiment treatment cells were washed twice with PBS and final treatment was done in serum free media (SFM). Secreted proteins were isolated from 1mL of the media by Trichloroacetic Acid (TCA) precipitation followed by Western blotting for myocilin (MAB3446, R&D Systems, Minneapolis, MN).

2.3. MTT growth assay

To determine growth we utilized the Vybrant MTT Cell Proliferation Assay Kit (Invitrogen, Eugene OR). The assay was done in accordance with the recommendations of the manufacturer with the following modifications. Cells were plated at 1×104 cells per well in 96 well plates. Cells were incubated with MTT at 37° for 3 hours at which point formazan was solubilized and incubated at 37° for another 2 hours followed by absorbance reading at 595nm using a Victor3 plate reader (PerkinElmer, Richmond, CA). HF and LF values were normalized to the same starting point, where necessary (2018–1524, UHOC-2289 and USOC-2328).

2.4. Proliferation assay

To determine the percent proliferation we utilized Click-iT EdU Alexa Fluor 488 Imaging Kit (Invitrogen, Eugene, OR). The assay was done in accordance with the recommendations of the manufacturer. Cells were plated on coverslips as described in the cell count growth assay and coverslips were transferred to 12 well plates to grow for 3 days after plating. Cells were incubated with 10μM 5-ethynyl-2’-deoxyuridine (EdU) for 20 hours. Imaging was done on an inverted microscope (epifluorescent Leica DMi8 microscope located at the UHCO lab, or on a confocal Olympus FV1000 microscope located at the OHSU lab) and a minimum of 1000 cells were counted for each data point.

2.5. Cellular biomechanics

Elastic moduli of single TM cells were determined by atomic force microscopy as describe previously(Y.-R. Chang et al., 2014; McKee et al., 2011b). Briefly, TM cells from HF or LF regions were plated (2,500 cells/cm2) on glass bottom petri dishes for a minimum of 36 hours in complete medium. Force-distance curves were obtained in contact mode (Bruker BioScope Resolve, Santa Barbara, CA) using PNP-TR cantilevers (nominal spring constant 0.32 N/m; Nano and More, USA) with a pyramidal tip. Prior to measurements, all cells were thermally equilibrated in Dulbecco’s PBS for 30 min. For all samples, force curves were obtained with AFM tips placed over the nucleus from at least 15–20 cells with 5 force curves for each cell. Elastic modulus was determined using Sneddon model; details of the equation for each are described elsewhere(McKee et al., 2011a).

2.6. Gene Expression

Changes in cellular phenotype or in gene expression levels in primary cell cultures may be due to changes in replicative differentiation or senescence, and may occur over extended periods of time. Here we investigated changes in gene expression levels over multiple passages and under different cell culture media conditions. To characterize effects of passage number, cells were seeded in 60mm plates and allowed to grow to 90% confluence in DMEM supplemented with 10% FBS and 1% Penicillin/Streptomycin/Fungizone, before trypsinizing and replating at low density in fresh flasks. To test different concentrations of serum, cells were plated in 6-well plates at 1×105 cells per well. The day after plating cells were washed twice with PBS, SFM, 0.5% or 10% FBS media was added, and cells were incubated for 24 hours. RNA was isolated using Direct-zol (Zymo Research, Seattle, WA) or Gene-Jet (Thermo Fisher, CA) RNA MiniPrep Kit. The SuperScript IV First-Strand Synthesis System (ThermoFisher Scientific, Waltham, MA) was used to make cDNA using 500ng of total RNA. Quantitative PCR was done on a QuantStudio3 using TaqMan Fast Advanced Master Mix (Applied Biosystems, Austin, TX). TaqMan Gene Expresssion Assays (Supplemental Table S1)(ThermoFisher Scientific, Waltham, MA) were used to determine gene expression levels of 17 target ECM genes.

2.7. Protein expression by Western blot

To investigate protein expression levels cells were plated at 3×105 cells per well in 6 well plates. The next day media was changed to either 1mL of SFM or 1mL of DMEM + 10%FBS. After two days cells grown in normal conditions were lysed. Lysates were normalized based on BSA assay and used to determine VCAM1 expression. SFM was collected after 5 days and used to determine THBS2 and TIMP1 expression. Equal volumes of SFM for each sample were loaded on SDS-PAGE gels. GAPDH was used as a loading control. The following primary antibodies were utilized for Western blot: anti-VCAM1 (sc-20070), anti-THBS2 (sc-136238) and anti-TIMP1 (sc-365905) purchased from Santa Cruz Biotechnology (Dallas, TX) and anti-GAPDH (MA5–15738) purchased from ThermoFisher (Rockford, IL).

2.8. Immunocytochemistry of cultured cell line

Cells were seeded in plastic 4-well chamber slides at 40,000 cells per well and grown to full confluence for 3 weeks in DMEM supplemented with 10% FBS and 1% Penicillin/Streptomycin/Fungizone. Cells were rinsed once in PBS prior to fixation in 4% paraformaldehyde for 30 minutes at room temperature. Cells were next incubated in CAS-Block, a universal blocking reagent to saturate the non-specific binding sites (Invitrogen, Grand Island, NY), for 1 hour at room temperature, and then incubated overnight at 4°C with one or more of the following antibodies: anti-VCAM1 mouse monoclonal antibody (Cat.#1.G11B1) purchased from ThermoFisher Scientific, (Rockford, IL), anti-thrombospondin-1 mouse monoclonal antibody (#39–9300) purchased from Invitrogen, (Camarillo, CA), anti-thromobospondin-2 rabbit polyclonal antibody (LS-C312578) purchased from LSBio (Seattle, WA), anti-collagen IV mouse monoclonal antibody (M3F7) purchased from Developmental Studies Hybridoma Bank (Iowa City, IA), anti-collagen VI rabbit polyclonal antibody (AB6588) purchased from AbCam (Cambridge, MA), or anti-TIMP-1 rabbit polyclonal antibody (AAJ47032) purchased from Antibody Verify (Las Vegas, NV). Primary antibodies were detected with Alexa-fluor 488-conjugated anti-mouse or 594-conjugated anti- rabbit secondary antibodies (Invitrogen, Grand Island, NY). Secondary antibodies were previously tested for non-specific immune reactivity using no-primary antibody controls. Immuno-stained cells were mounted using Slowfade Gold antifade reagent with DAPI (Invitrogen), and imaged by confocal microscopy using an Olympus FV1000 microscope. Optical sections were acquired using sequential scanning in separate laser channels. Image acquisition settings and number of optical sections in a stack were kept constant when comparing images.

2.9. Statistical Analyses of Growth Data

Data were analyzed using Graph-Pad Prism 6 software for statistical significance. The exponential growth equation was used to determine the growth rates and doubling times. R square values for all fits were greater than 0.7 indicating reasonable goodness of fit to the growth data. Growth rate data was analyzed for statistical significance using a 2-way ANOVA for each HF vs. LF comparison for each cell strain (N = 3 technical replicates) at each time point. Two tailed paired t-test analyses were used to determine significance between HF and LF mean proliferation rates (N = 6 biological replicates), where P<0.05 was considered statistically significant.

2.10. Statistical Analysis of qRT-PCR data

Data were analyzed using the online program SATQPCR (http://satqpcr.sophia.inra.fr/cgi/home.cgi) (Rancurel et al., 2019). Each cell strain pair was treated as a biological replicate and data was normalized to 18s and ITGB1 (the latter being chosen by the program). Expression was rescaled across biological replicates. In addition to t-tests we also performed 1-way and 2-way ANOVA with factors of type (HF/LF), and either condition (0, 0.5, 10% FBS), or passage number (p2, p4, p5). Analysis of passage number expression contained 4 biological replicates and 3 technical replicates. Analysis of condition expression contained 5 biological replicates and 4 technical replicates.

3. Results

3.1. Establishment and growth of HF/LF cell strains

We initially attempted HF and LF cell strain generation from 14 pairs of eyes. Of these, 8 paired strains were created and utilized for further characterization. Demographic data is summarized in Table 1. We excluded cell strains that did not grow well, lacked a paired strain, or did not meet the pre-determined exclusion/inclusion criteria (TM morphology and dex-responsiveness)(Keller et al., 2018).

3.2. Confirmation of hTM by dexamethasone induced myocilin expression and morphology

A defining feature of primary cultured hTM cells is their increased myocilin expression in response to treatment with dexamethasone(Keller et al., 2018). In this study, HF and LF cell strains were plated and treated with 100nM dexamethasone for 14 days. Myocilin expression levels were quantified for each of the cell strains either by qRT-PCR or by Western blotting, and cell strains that did not respond appropriately were excluded from this study. Both methods are reliable, as is documented in the recent white paper on TM cell generation (Keller et al., 2018). Two paired cell lines, 2018–1718 and 2018–1515 were excluded from further study based on their lack of response to dex treatment. All other cell strains characterized in this paper showed increased myocilin levels in response to dex treatment (Figure 1). There was no trend observed in the ability of segmental cells to upregulate myocilin differentially.

Figure 1: HF and LF cells are hTM. Dexmethasone-induced myocilin expression.

Figure 1:

Open in a new tab

Cells were plated at 1.5×105 per well in 6 well plates and treated +/− dex every 3 days for 2 weeks. Cells were incubated in SFM for 3 days prior to the end of the experiment. (A) Proteins were concentrated by TCA precipitation followed by Western blot analysis to determine myocilin expression. (B) RNA was collected and qRT-PCR was utilized to determine myocilin expression.

HTM cells are also known to have a distinct morphology(Keller et al., 2018). While there was some variation across cell strains, all strains exhibited a cobblestone-like morphology with some overlapping structures. The cell strains presented here were checked regularly to ensure they maintained characteristic hTM morphology.

3.3. Growth and proliferation

Once segmental cell strains were established and myocilin expression was confirmed, cell growth rates were measured. MTT growth assay data demonstrated that differences between the HF and LF cell strain growth rates were not statistically significant (8 paired cell strains were tested) (Figure 2). To determine whether HF and LF strains had different proliferation rates we used an EdU assay that directly quantitates newly synthesized DNA. Cells were imaged and both EdU and Hoechst stained nuclei were counted to determine the percent proliferation (Figure 3). HF strain proliferation was 53.85% ±6.528 and LF strain proliferation was 48.95% ±5.016, and the difference between them was not statistically significant (paired t-test, P value = 0.565)

Figure 2: HF and LF hTM cell growth.

Figure 2:

Open in a new tab

MTT assay: Cells were plated in triplicate in 96 well plates, one plate for each day. On the experimental day cells were incubated with MTT for 3 hours and then formazan was solubilized. Absorbance was measured at 595 nm.

Figure 3: Percent proliferation of HF and LF strains.

Figure 3:

Open in a new tab

Proliferation was determined by EdU incorporation assay. Cells were plated on coverslips and allowed to grow for 2 days. Cells were incubated with EdU for 20 hours, fixed and EdU was visualized using Click-iT imaging kit. Total nuclei were determined using Hoescht. Data represents a minimum of 1000 cells per cell line. HF strain proliferation mean was 46.8 ±6.79 and LF strain proliferation mean was 45.24 ±6.43 (paired two-tailed t-test, P value = 0.565). Difference between HF and LF means was not significant (N = 6 biological replicate cell strains.)

3.4. Stiffness of cells

Elastic moduli were determined by curve fitting the Hertz equation in the region of linear elasticity. Two cell strains from each testing site (OHSU vs UHCO) were chosen randomly for this experiment. The region of linear elasticity was determined from Elastic moduli vs indentation curves after determining the contact point. Three of the four strains did not show any significant difference in moduli. Similarly, when values from all 4 strains were pooled (HF strains: 3.59 +/− 1.57 kPa; LF strains: 3.32 +/− 1.21 kPa), no statistical significance was observed (data shown in Figure 4 and Table 2) from all four donor HF strains where moduli were 3.59 +/− 1.57 kPa vs 3.32 +/− 1.21 kPa for all LF donor cells. Moduli of individual segmental pair of cell strains can be found in Table 2.

Figure 4: Elastic moduli of TM cells from segmental regions.

Figure 4:

Open in a new tab

Mean Elastic modulus (± standard deviation) of LF and HF cell strains from four biological donors. Stiffness difference between LF and HF strains is not significant (paired t-test).

Table 2:

Elastic moduli of TM cells from segmental regions.

hTM cell strain LF (kPa) HF (kPa) p-value (t-test)
HTM2289 3.5570 +/− 1.49 3.3630 +/− 1.15 0.262
HTM2328 3.0050 +/− 1.45 3.3360 +/− 1.07 0.440
HTM1524 3.0240 +/− 0.97 4.1710 +/− 0.87 <0.001
HTM1244 3.7600 +/− 0.67 3.9040 +/− 0.82 0.320
Open in a new tab

Data presented are mean ± standard deviation from 15–20 single cells, with 5 force vs indentation curves per cell, for each cell strain cultured routinely in complete media. AFM measurements were performed by equilibrating cells in Dulbecco’s PBS.

3.5. HF and LF cell strains grown in complete media show similar gene expression patterns

Previous studies have investigated protein and gene expression differences between HF and LF regions, with a specific emphasis on ECM molecules(Keller et al., 2011; Vranka et al., 2015a; Vranka and Acott, 2017). From these prior data we selected 17 genes (primarily ECM) as potential markers to distinguish HF cell strains from LF cell strains consistently across passage number and under full growth conditions.. Primary cultured hTM cells are known to dedifferentiate after approximately 5 passages. Here, mRNA was harvested from normally growing cells at passages 2, 4 and 5 and gene expression levels were compared between HF and LF cells. This was done with 4 paired cell strains. Under these conditions, none of the genes assayed here were consistent markers of HF or LF cells comparing all of the cell strains from multiple donors. For all genes investigated t-tests were performed to determine statistical significance for HF versus LF comparisons and resulted in p-values greater than 0.05 (Supplemental Figure 1).

3.6. Under limited serum growth conditions, THBS2, TIMP1 and VCAM1 gene expression is elevated in LF compared to HF cell strains

Since qRT-PCR on normally growing cells did not reveal common markers we refined the conditions under which we looked at gene expression. To do this we standardized the number of cells plated, length of growth time and varied the serum concentration testing serum free (SFM), 0.5% and 10% serum. We tested these three conditions on five sets of paired HF/LF strains (2018–0426, 2018–1189, 2018–1235, 2018–1524 and 2018–1723) for a total of ten strains.

Initially, the most promising markers of LF cells appear to be VCAM1, TIMP1 and THBS2 (Figure 5). LF cells express VCAM1 at higher levels than their HF counterparts (t-test HF versus LF; SFM p=0.255, 0.5% serum p=0.0475, 10% serum p=0.0663; 1-way ANOVA type p=0.0438, 2-way ANOVA type: condition p=0.31, n=5). TIMP1 is overexpressed in LF compared to HF (t-test HF versus LF; SFM p=0.0895, 0.5% serum p=0.0423, 10% serum p=0.358; 1-way ANOVA type p=0.0077, 2-way ANOVA type: condition p=0.62, n=5). Finally, THBS2 is elevated in LF compared to HF (t-test HF versus LF; SFM p=0.279, 0.5% serum p=0.0204, 10% serum p=0.0482; 1-way ANOVA type p=0.0343, 2-way ANOVA type: condition p=0.457). Expression of other genes can be found in Supplemental Figure 2.

Figure 5: Expression of VCAM1, TIMP1 and THBS2 with varying serum concentration.

Figure 5:

Open in a new tab

Cells were plated, switched to SFM, 0.5% or 10% FBS the next day followed by a 24 hour incubation. Relative expression levels were determined using the ΔΔCt method. Each data point includes rescaled expression data from the following paired strains 2018–0426, 2018–1189, 2018–1235, 2018–1524 and 2018–1723. Error bars are SEM.

3.7. Short term protein expression reveals similar expression of VCAM1, THBS2 and TIMP1 in HF compared to LF

To further understand the potential of VCAM1, THBS2 and TIMP1 to distinguish between HF and LF cells we performed western blots on cell lysates (VCAM1) or on SFM from HF and LF cell strains (THBS2 and TIMP1). Unlike the gene expression data, we did not see obvious differences in protein levels between HF and LF cells when cultured under full-serum conditions (Figure 6A), or when cultured under serum-free conditions (Figure 6B). THBS2 protein expression is very similar in HF and LF cells of all strains tested (Figure 6C). Finally, TIMP1 relative protein expression levels appear to vary somewhat with cell strains where it is similar in HF versus LFin strains 2018–1189, 2018–1235 and 2018–1244, but higher in HF compared to LF for strains 2018–0426, 2018–1524 and 2018–1723 (Figure 6C). TIMP1 data is representative of two independent experiments.

Figure 6: Protein expression of VCAM1, TIMP1 and THBS2.

Figure 6:

Open in a new tab

Cells were plated in 6 well plates, the next day media was changed to 10% FBS (A) or SFM (B and C). Cell lysates (A and B) were collected after 3 days in culture or (C) SFM was collected after 5 days of incubation with cells. Lysates were normalized using the BCA assay. Equal amounts of SFM were added to equal numbers of cells and the same volume was run for each sample. TIMP1 expression is representative of two independent experiments.

3.7. Immunocytochemistry shows elevated VCAM1, TIMP1 and THBS2 in LF cells compared to HF when cells are cultured long enough to lay down ECM

Immunostaining on confluent HF and LF cell cultures grown for 3 weeks shows the presence of a dense ECM network for HF and LF cells (Figure 7). Antibodies to VCAM1 and TIMP1 were used to determine relative expression levels of each protein in the ECM of the cultured cells. Representative confocal images show a strong extracellular staining in LF cell cultures compared with that in HF cells (Figure 7A, HF in upper panel, LF in lower panel). This is in agreement with our gene expression data for these genes. HF and LF cell cultures were also immunostained using antibodies to THBS1 and THBS2. Representative confocal images show relatively more THBS2 in LF cell cultures compared with the HF cell cultures (Figure 7B), which is in agreement with our qRT PCR data. In contrast, protein levels of THBS1 are higher in HF cells compared to LF cells, suggesting a differential expression pattern from THBS2… This data suggests that ECM protein deposition of HF and LF cell cultures reflects similar expression level differences as were observed at the RNA level. Images are representative of multiple cell strains showing a similar pattern of staining. We also performed immunostaining for types IV and VI collagen (Figure 7C) to verify that there was robust ECM matrix laid down by both HF and LF cells. The proteins were expressed at similar levels in both HF and LF cells.

Figure 7: Immunocytochemistry on HF and LF TM cells cultures using select ECM antibodies.

Figure 7:

Open in a new tab

HF and LF TM cell cultures were grown on chamber slides for 3 weeks in DMEM and 10%FBS prior to fixation and immunostaining. Alexa-fluor secondary antibodies were used to detect the primary antibodies and fluorescent images were captured on an Olympus FV1000 confocal microscope. Panel A shows HF (upper panels) and LF (lower panels) cells where VCAM1 is in green and TIMP1 is in red and all colors are merged in the far right panels (DAPI is blue nuclear stain). VCAM1 is expressed at higher levels in LF cells than in HF cells, as is TIMP1. In panel B THBS1 (green) is expressed at relatively similar levels in HF (upper panels) and LF (lower panels) cells, whereas THBS2 (in red) is expressed more in LF cells than HF. All colors are merged in the far right panels (DAPI is blue nuclear stain). Panel C shows robust staining for type IV collagen (green) and type IV collagen (red) and expression levels are similar in HF (upper panels) and LF (lower panels) cells. All colors are merged in the far right panels (DAPI is blue nuclear stain). Images are representative of a minimum of n = 3 different cell strains tested. Scale bar = 100μm.

4. Discussion

Previous studies on the segmental nature of aqueous humor outflow have been limited by the availability of donor eye tissues, as well as the relatively small number of TM cells that can be grown from a single donor eye. Here we demonstrate that HF and LF cell strains can be generated and grow robustly in primary cultures. Furthermore, no significant differences were found in the morphology, growth, or proliferation rates of HF and LF cell strains, suggesting that differences between the segmental flow regions in situ are not due to differences in cell growth or proliferation. We also measured the dex responsiveness of each of the established cell strains through their upregulation of myocilin. HF and LF cell strains did not show differences in their ability to upregulate myocilin in response to dex. These studies provide baseline measures of growth and expression of the cell strains allowing for a direct comparison of segmental hTM cells on the same genetic background.

In this study we have measured ECM gene expression levels across several passage numbers, and when the HF and LF cells are grown under varying serum concentrations, since we hypothesize that the underlying segmental phenotype in hTM cells is likely retained over multiple cell divisions. Here we measured expression levels of ECM genes of interest based on our previous studies, and determined the effect of cell growth under different conditions. It is of note that the most consistent differences in gene expression levels were seen when cells were cultured in 0.5% FBS. These conditions may be more physiologically relevant since aqueous humor is devoid of many of the proteins found in plasma(Goel et al., 2010; Reiss et al., 1986). Standard perfusion culture of anterior segments is done using SFM. Likewise, we find that hTM cells can be maintained, though not plated, in SFM for extended amounts of time with little observable cell death (personal observations). This is unsurprising, as TM cells perpetually reside in a serum-starved environment in situ. Serum starvation of many other cell types has long been known to induce a stress response that may or may not be beneficial. Here we did not determine stress responses in our cells under serum-starved conditions. It is feasible to assume that processes such as metabolism, signaling, and gene and protein synthesis in hTM cells favor a medium mimicking in vivo conditions of less than 1% serum(Freddo, 2013).

We have long hypothesized that cell- and ECM-context is important for normal TM cell function. HTM cells in segmental flow regions in tissues represent a complex and dynamic interplay of cells and their matrix, where the resident cells sense and respond to the changing stiffness of their immediate environment by secreting ECM and ECM modifying proteins. Prior studies of TM tissues perfused at normal or physiologic pressure have shown few marked protein level differences between HF and LF regions(Vranka et al., 2015a; Vranka and Acott, 2017). Our current study in which HF and LF cell cultures were grown under normal conditions showed few protein level differences as well, corroborating the prior tissue studies. In prior studies larger protein level differences were observed between segmental flow regions when tissues were subjected to a continuously-elevated pressure challenge for an extended period of time. Thus future studies will investigate ECM gene and protein expression levels in HF and LF cells in response to stretch and varying substratum stiffness. VCAM1, THBS2 and TIMP1 may continue to be interesting genes in these studies and highlight the dynamic nature of segmental regions at the cellular level.

The data presented here show that HF and LF cell strain generation is possible and that these cell strains can be utilized to further understand the features of segmental flow. This initial study focused on the feasibility of isolating and culturing TM cells from segmental outflow regions, establishing their normal growth and proliferative behavior in cell culture, their biomechanical properties, and in investigating a limited number of potential ECM gene markers under a variety of growth conditions. Further studies are underway to not only expand the number of potential markers unique to HF and LF cells, but also to determine the influence of the ECM on the behavior of the HF and LF cell strains in culture. We anticipate that HF and LF cell strains may respond differently to their environment, with ECM gene expression differences in hTM cells in response to stretch(Vranka and Acott, 2017). Although recent findings demonstrate that LF tissue is stiffer than HF tissue, we did not observe significant differences in the elastic moduli of HF compared to LF cells cultured from these segmental regions. However, it is important to note that the HF and LF cell strains established in these studies were cultured on rigid plastic or glass surfaces not native to their microenvironment in vivo. Recent studies have determined the importance of TM tissue compliance in health and disease(Vranka et al., 2018; Wang et al., 2017). Other studies have demonstrated that cells may have a mechanical memory and that their functions may be altered by this memory of past matrix stiffness(Nasrollahi et al., 2017). With these HF and LF cell cultures established and characterized, it is now possible to study the effects of substrate composition and compliance on downstream gene and protein expression levels. Combined, these studies will help to improve the understanding of the molecular mechanisms underlying segmental outflow regions of the TM, and will provide important mechanistic insights into cell-ECM interactions in the context of segmental outflow and TM pathophysiology.

Supplementary Material

1
4
5

Supplemental Figure 1: Expression of potential differentially expressed genes at passage 2, 4 and 5. Cells were plated in 60mm dishes and RNA was collected at 90% confluency for p2, p4 and p5. Paired cell strains 2018–0426, 2018–1189, 2018–1235 and 2018–1244 were utilized in this experiment. Relative expression levels were determined using the ΔΔCt method. Expression was rescaled across biological replicates. Error bars are SEM.

6

Supplemental Figure 2: Expression of potential differentially expressed genes at in different concentrations of serum. Gene expression was investigated 24 hours after incubation with SFM, 0.5% or 10% FBS. Paired cell strains 2018–0426, 2018–1189, 2018–1235, 2018–1524 and 2018–1723 were utilized in this experiment. Relative expression levels were determined using the ΔΔCt method. Expression was rescaled across biological replicates. Error bars are SEM.

Highlights.

  • Here we generate novel cell strains from segmental regions of human TM tissues.

  • High flow and low flow cell strains have similar growth and proliferation rates.

  • THBS2, VCAM1, and TIMP1 are potential low flow cell markers.

  • Low flow cell markers may be useful for developing targeted therapeutics.

Acknowledgments

The authors would like to thank their funding sources: NIH/NEI grants EY026048-01A1 (JAV, VKR), EY030238, EY008247, EY025721 (TSA), P30 EY010572, and by an unrestricted grant to the Casey Eye Institute from Research to Prevent Blindness, New York, NY. We would also like to thank the Lions VisionGift and SavingSight (Kansas City, MO) for procuring all human donor eyes used in this work. Most importantly, we would like to thank the families of the organ donors without whose consent these experiments would be impossible. We also would like to acknowledge the late John Bradley for his thoughtful comments and discussions over the years. His enthusiasm will be sorely missed.

Grant information: The authors would like to thank their funding sources - NIH/NEI grants EY026048-01A1 (JAV, VKR), EY030238, EY008247, EY025721 (TSA), P30 EY010572, and by an unrestricted grant to the Casey Eye Institute from Research to Prevent Blindness, New York, NY.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  1. Acott TS, Kelley MJ, 2008. Extracellular matrix in the trabecular meshwork. Exp. Eye Res 86, 543–561. doi: 10.1016/j.exer.2008.01.013 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Acott TS, Kingsley PD, Samples JR, Van Buskirk EM, 1988. Human trabecular meshwork organ culture: morphology and glycosaminoglycan synthesis. Invest. Ophthalmol. Vis. Sci 29, 90–100. [PubMed] [Google Scholar]
  3. Acott TS, Wirtz M, 1996. Biochemistry of Aqueous Outflow. The Glaucomas. [Google Scholar]
  4. Alvarado J, Murphy C, Juster R, 1984. Trabecular meshwork cellularity in primary open-angle glaucoma and nonglaucomatous normals. Ophthalmology 91, 564–579. doi: 10.1016/s0161-6420(84)34248-8 [DOI] [PubMed] [Google Scholar]
  5. Alvarado J, Murphy C, Polansky J, Juster R, 1981. Age-related changes in trabecular meshwork cellularity. Invest. Ophthalmol. Vis. Sci 21, 714–727. [PubMed] [Google Scholar]
  6. Bradley JM, Vranka J, Colvis CM, Conger DM, Alexander JP, Fisk AS, Samples JR, Acott TS, 1998. Effect of matrix metalloproteinases activity on outflow in perfused human organ culture. Invest. Ophthalmol. Vis. Sci 39, 2649–2658. [PubMed] [Google Scholar]
  7. Buller C, Johnson D, 1994. Segmental variability of the trabecular meshwork in normal and glaucomatous eyes. Invest. Ophthalmol. Vis. Sci 35, 3841–3851. [PubMed] [Google Scholar]
  8. Carreon TA, Edwards G, Wang H, Bhattacharya SK, 2017. Segmental outflow of aqueous humor in mouse and human. Exp. Eye Res 158, 59–66. doi: 10.1016/j.exer.2016.08.001 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chang JYH, Folz SJ, Laryea SN, Overby DR, 2014. Multi-scale analysis of segmental outflow patterns in human trabecular meshwork with changing intraocular pressure. J Ocul Pharmacol Ther 30, 213–223. doi: 10.1089/jop.2013.0182 [DOI] [PubMed] [Google Scholar]
  10. Chang Y-R, Raghunathan VK, Garland SP, Morgan JT, Russell P, Murphy CJ, 2014. Automated AFM force curve analysis for determining elastic modulusof biomaterials and biological samples. J Mech Behav Biomed Mater 37, 209–218. doi: 10.1016/j.jmbbm.2014.05.027 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. de Kater AW, Melamed S, Epstein DL, 1989. Patterns of aqueous humor outflow in glaucomatous and nonglaucomatous human eyes. A tracer study using cationized ferritin. Arch. Ophthalmol 107, 572–576. doi: 10.1001/archopht.1989.01070010586035 [DOI] [PubMed] [Google Scholar]
  12. Ethier CR, Chan DW, 2001. Cationic ferritin changes outflow facility in human eyes whereas anionic ferritin does not. Invest. Ophthalmol. Vis. Sci 42, 1795–1802. [PubMed] [Google Scholar]
  13. Freddo TF, 2013. A contemporary concept of the blood-aqueous barrier. Prog Retin Eye Res 32, 181–195. doi: 10.1016/j.preteyeres.2012.10.004 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Goel M, Picciani RG, Lee RK, Bhattacharya SK, 2010. Aqueous humor dynamics: a review. Open Ophthalmol J 4, 52–59. doi: 10.2174/1874364101004010052 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Grant WM, 1963. Open-Angle Glaucoma Associated with Vitreous Filling the Anterior Chamber. Trans Am Ophthalmol Soc 61, 196–218. [PMC free article] [PubMed] [Google Scholar]
  16. Hann CR, Bahler CK, Johnson DH, 2005. Cationic ferritin and segmental flow through the trabecular meshwork. Invest. Ophthalmol. Vis. Sci 46, 1–7. doi: 10.1167/iovs.04-0800 [DOI] [PubMed] [Google Scholar]
  17. Johnson DH, Richardson TM, Epstein DL, 1989. Trabecular meshwork recovery after phagocytic challenge. Curr. Eye Res 8, 1121–1130. doi: 10.3109/02713688909000037 [DOI] [PubMed] [Google Scholar]
  18. Keller KE, Acott TS, 2013. The Juxtacanalicular Region of Ocular Trabecular Meshwork: A Tissue with a Unique Extracellular Matrix and Specialized Function. J Ocul Biol 1, 3. [PMC free article] [PubMed] [Google Scholar]
  19. Keller KE, Bhattacharya SK, Borrás T, Brunner TM, Chansangpetch S, Clark AF, Dismuke WM, Du Y, Elliott MH, Ethier CR, Faralli JA, Freddo TF, Fuchshofer R, Giovingo M, Gong H, Gonzalez P, Huang A, Johnstone MA, Kaufman PL, Kelley MJ, Knepper PA, Kopczynski CC, Kuchtey JG, Kuchtey RW, Kuehn MH, Lieberman RL, Lin SC, Liton P, Liu Y, Lütjen-Drecoll E, Mao W, Masis-Solano M, McDonnell F, McDowell CM, Overby DR, Pattabiraman PP, Raghunathan VK, Rao PV, Rhee DJ, Chowdhury UR, Russell P, Samples JR, Schwartz D, Stubbs EB, Tamm ER, Tan JC, Toris CB, Torrejon KY, Vranka JA, Wirtz MK, Yorio T, Zhang J, Zode GS, Fautsch MP, Peters DM, Acott TS, Stamer WD, 2018. Consensus recommendations for trabecular meshwork cell isolation, characterization and culture. Exp. Eye Res 171, 164–173. doi: 10.1016/j.exer.2018.03.001 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Keller KE, Bradley JM, Acott TS, 2009. Differential effects of ADAMTS-1, −4, and −5 in the trabecular meshwork. Invest. Ophthalmol. Vis. Sci 50, 5769–5777. doi: 10.1167/iovs.09-3673 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Keller KE, Bradley JM, Kelley MJ, Acott TS, 2008. Effects of modifiers of glycosaminoglycan biosynthesis on outflow facility in perfusion culture. Invest. Ophthalmol. Vis. Sci 49, 2495–2505. doi: 10.1167/iovs.07-0903 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Keller KE, Bradley JM, Vranka JA, Acott TS, 2011. Segmental versican expression in the trabecular meshwork and involvement in outflow facility. Invest. Ophthalmol. Vis. Sci 52, 5049–5057. doi: 10.1167/iovs.10-6948 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lütjen-Drecoll E, 1999. Functional morphology of the trabecular meshwork in primate eyes. Prog Retin Eye Res 18, 91–119. doi: 10.1016/s1350-9462(98)00011-1 [DOI] [PubMed] [Google Scholar]
  24. McKee CT, Last JA, Russell P, Murphy CJ, 2011a. Indentation versus tensile measurements of Young’s modulus for soft biological tissues. Tissue Eng Part B Rev 17, 155–164. doi: 10.1089/ten.TEB.2010.0520 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. McKee CT, Raghunathan V, Russell P, Murphy C, 2011b. Probing effect of biophysical cues on cell behavior with atomic force microscopy. Microscopy and Analysis 25, 25–28. [Google Scholar]
  26. Nasrollahi S, Walter C, Loza AJ, Schimizzi GV, Longmore GD, Pathak A, 2017. Past matrix stiffness primes epithelial cells and regulates their future collective migration through a mechanical memory. Biomaterials 146, 146–155. doi: 10.1016/j.biomaterials.2017.09.012 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Overby DR, Stamer WD, Johnson M, 2009. The changing paradigm of outflow resistance generation: towards synergistic models of the JCT and inner wall endothelium. Exp. Eye Res 88, 656–670. doi: 10.1016/j.exer.2008.11.033 [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Quigley HA, 2011. Glaucoma. Lancet 377, 1367–1377. doi: 10.1016/S0140-6736(10)61423-7 [DOI] [PubMed] [Google Scholar]
  29. Quigley HA, 1996. Number of people with glaucoma worldwide. Br J Ophthalmol 80, 389–393. doi: 10.1136/bjo.80.5.389 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rancurel C, van Tran T, Elie C, Hilliou F, 2019. SATQPCR: Website for statistical analysis of real-time quantitative PCR data. Mol. Cell. Probes 46, 101418. doi: 10.1016/j.mcp.2019.07.001 [DOI] [PubMed] [Google Scholar]
  31. Reiss GR, Werness PG, Zollman PE, Brubaker RF, 1986. Ascorbic acid levels in the aqueous humor of nocturnal and diurnal mammals. Arch. Ophthalmol 104, 753–755. doi: 10.1001/archopht.1986.01050170143039 [DOI] [PubMed] [Google Scholar]
  32. Rohen JW, Lütjen-Drecoll E, Flügel C, Meyer M, Grierson I, 1993. Ultrastructure of the trabecular meshwork in untreated cases of primary open-angle glaucoma (POAG). Exp. Eye Res 56, 683–692. doi: 10.1006/exer.1993.1085 [DOI] [PubMed] [Google Scholar]
  33. Swaminathan SS, Oh D-J, Kang MH, Rhee DJ, 2014. Aqueous outflow: segmental and distal flow. J Cataract Refract Surg 40, 1263–1272. doi: 10.1016/j.jcrs.2014.06.020 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Tamm ER, 2009. The trabecular meshwork outflow pathways: structural and functional aspects. Exp. Eye Res 88, 648–655. doi: 10.1016/j.exer.2009.02.007 [DOI] [PubMed] [Google Scholar]
  35. Tripathi RC, Tripathi BJ, 1982. Human trabecular endothelium, corneal endothelium, keratocytes, and scleral fibroblasts in primary cell culture. A comparative study of growth characteristics, morphology, and phagocytic activity by light and scanning electron microscopy. Exp. Eye Res 35, 611–624. doi: 10.1016/s0014-4835(82)80074-2 [DOI] [PubMed] [Google Scholar]
  36. Vranka JA, Acott TS, 2017. Pressure-induced expression changes in segmental flow regions of the human trabecular meshwork. Exp. Eye Res 158, 67–72. doi: 10.1016/j.exer.2016.06.009 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Vranka JA, Acott TS, 2016. Molecular differences in segmental regions of the trabecular meshwork, in: Knepper, Samples JR (Eds.), Glaucoma Research and Clinical Advances . pp. 115–120. [Google Scholar]
  38. Vranka JA, Bradley JM, Yang Y-F, Keller KE, Acott TS, 2015a. Mapping molecular differences and extracellular matrix gene expression in segmental outflow pathways of the human ocular trabecular meshwork. PLoS ONE 10, e0122483. doi: 10.1371/journal.pone.0122483 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Vranka JA, Kelley MJ, Acott TS, Keller KE, 2015b. Extracellular matrix in the trabecular meshwork: intraocular pressure regulation and dysregulation in glaucoma. Exp. Eye Res 133, 112–125. doi: 10.1016/j.exer.2014.07.014 [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Vranka JA, Staverosky JA, Reddy AP, Wilmarth PA, David LL, Acott TS, Russell P, Raghunathan VK, 2018. Biomechanical Rigidity and Quantitative Proteomics Analysis of Segmental Regions of the Trabecular Meshwork at Physiologic and Elevated Pressures. Invest. Ophthalmol. Vis. Sci 59, 246–259. doi: 10.1167/iovs.17-22759 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wang K, Johnstone MA, Xin C, Song S, Padilla S, Vranka JA, Acott TS, Zhou K, Schwaner SA, Wang RK, Sulchek T, Ethier CR, 2017. Estimating Human Trabecular Meshwork Stiffness by Numerical Modeling and Advanced OCT Imaging. Invest. Ophthalmol. Vis. Sci 58, 4809–4817. doi: 10.1167/iovs.17-22175 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1601166-supplement-1.xml (245B, xml)
4
NIHMS1601166-supplement-4.docx (10.4KB, docx)
5

Supplemental Figure 1: Expression of potential differentially expressed genes at passage 2, 4 and 5. Cells were plated in 60mm dishes and RNA was collected at 90% confluency for p2, p4 and p5. Paired cell strains 2018–0426, 2018–1189, 2018–1235 and 2018–1244 were utilized in this experiment. Relative expression levels were determined using the ΔΔCt method. Expression was rescaled across biological replicates. Error bars are SEM.

NIHMS1601166-supplement-5.tif (197.5KB, tif)
6

Supplemental Figure 2: Expression of potential differentially expressed genes at in different concentrations of serum. Gene expression was investigated 24 hours after incubation with SFM, 0.5% or 10% FBS. Paired cell strains 2018–0426, 2018–1189, 2018–1235, 2018–1524 and 2018–1723 were utilized in this experiment. Relative expression levels were determined using the ΔΔCt method. Expression was rescaled across biological replicates. Error bars are SEM.

NIHMS1601166-supplement-6.tif (180.3KB, tif)

RESOURCES