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Oct 2021

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Preparation of a Single Cell Suspension from the Murine Iridocorneal Angle
从小鼠虹膜角膜角制备单细胞悬液   

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Abstract

Single cell RNA sequencing is a powerful tool that can be used to identify distinct cell types and transcriptomic differences within complex tissues. It has proven to be especially useful in tissues of the eye, where investigators have identified novel cell types within the retina, anterior chamber, and iridocorneal angle and explored transcriptomic contribution to disease phenotypes in age-related macular degeneration. However, to obtain high quality results, the technique requires isolation of healthy single cells from the tissue of interest, seeking complete tissue digestion while minimizing stress and transcriptomic changes in the isolated cells prior to library preparation. Here, we present a protocol developed in our laboratory for isolation of live single cells from the murine iridocorneal angle, which includes Schlemm’s canal and the trabecular meshwork, suitable for single cell RNA sequencing, flow cytometry, or other downstream analysis.


Graphical abstract:




Keywords: Single cell RNA sequencing (单细胞RNA测序), Schlemm’s canal (莱姆氏管), Eye (眼睛), Glaucoma (青光眼), Single cell ( 单细胞), Dissociation (离解), Iridocorneal angle (虹膜角膜角), Limbus (边缘), Mouse (小鼠)

Background

Glaucoma is a progressive neurodegenerative disease with no cure, affecting over 60 million individuals worldwide and leaving 8 million blind (Quigley and Broman, 2006). Current therapy is focused on reduction of intraocular pressure (IOP), the only treatable risk factor for disease progression. IOP is determined by the ratio between aqueous humor secretion by the ciliary body and drainage through tissues in the iridocorneal angle, including the trabecular meshwork and Schlemm’s canal, which comprise the conventional outflow pathway. Patients with glaucoma, including primary congenital glaucoma, exhibit defects in tissues of the conventional outflow pathway leading to decreased aqueous humor outflow and elevated IOP (Tektas and Lütjen-Drecoll, 2009; Braunger et al., 2015; Zagora et al., 2015). The importance of these tissues to glaucoma pathogenesis, and consequently as therapeutic targets, has focused intense research interest in understanding their physiology, molecular phenotypes, and the cell-cell interactions governing their function. However, due to the small sizes of the tissues involved and their location within the iridocorneal angle, relatively little is known about the transcriptomes or overall phenotypes of Schlemm’s canal or drainage channel endothelial cells, potential differences between inner and outer wall endothelial cells of Schlemm’s canal or cells of the trabecular meshwork.


Recently, single cell RNA sequencing has been used to identify cell types in the iridocorneal angle region of mice, humans, and other model species (van Zyl et al., 2020; Patel et al., 2020; Thomson et al., 2021). These studies have provided exciting hints about the cells making up the angle and identified potential new therapeutic targets, but the extreme rarity of Schlemm’s canal endothelial cells within published datasets [van Zyl et al. (2020) identified just 25 Schlemm’s canal endothelial cells after sequencing 13,833 cells (0.18%), while our group has identified 109 in a dataset of 19,232 (0.57%)] has emphasized the need for sequencing of more cells, in healthy and disease conditions, to better understand the full complexity of this important tissue.


In the present protocol, we optimized a straightforward method for dissecting and dissociating the murine iridocorneal angle to obtain a single cell suspension of healthy cells to be used for downstream applications such as single cell RNA sequencing or flow cytometry. Previous studies utilizing the full anterior segment have reported that corneal and ocular surface epithelial cells represent over 50% of total cells in the dataset (van Zyl et al., 2020). While glaucoma-relevant iridocorneal angle cell populations remain rare, by removing the majority of the cornea during dissection, our method substantially increases their yield, resulting in a ~200% increase in proportion of Schlemm’s canal endothelial cells and ~80% increase in trabecular meshwork cells within the sample. Depending on the tissue(s) of interest, this protocol could be combined with flow cytometry or antibody-conjugated bead-based methods to enrich the sample in relevant cell types prior to library preparation. We have also found that this procedure can be readily adapted to dissociate cells of the cornea or posterior eye with similar results and cell viability.

Materials and Reagents

  1. Pipet tip micro 40 μm cell strainers (Bel-Art Flomi, catalog number: 136800040)

  2. Standard 10 cm Petri dishes (one per sample)

  3. Standard 20 mm tissue culture dishes (one per sample)

  4. Standard 1.5 mL microcentrifuge tubes

  5. Standard 1 mL pipet tips (ThermoFisher ART 2079 or similar)

  6. Wide orifice 1 mL pipet tips (ThermoFisher ART 2079GPK or similar)

  7. DMEM, dye free (ThermoFisher, Gibco, catalog number: 21063029)

  8. Fetal bovine serum (ThermoFisher, Gibco, catalog number: 26140079 or similar)

  9. 0.25% Trypsin EDTA (ThermoFisher, Gibco, catalog number: 25200056)

  10. 10 μm Rock inhibitor Y-27632 (ATCC, catalog number: ACS-3030)

  11. DNase 1 (Roche, catalog number: 04716728001)

  12. Collagenase A (Roche, catalog number: 10103578001)

  13. HBSS (ThermoFisher, Gibco, catalog number: 14175095)

  14. Bovine Serum Albumen (Sigma-Aldrich, catalog number: 03117332001)

  15. Acridine orange/propidium iodide live/dead staining kit compatible with your automated cell counter (i.e., Nexcelom CS2-0106)

  16. Compatible disposable slides if needed for your automated cell counter (i.e., Nexcelom CHT4-PD100)

  17. Digestion mix (see Recipes)

  18. Resuspension solution (see Recipes)

  19. 25 mg/mL Collagenase A stock solution (see Recipes)

  20. 10 mM Y27632 stock solution (see Recipes)

    Note: All reagents stored per manufacturer’s recommendations and used before labeled expiry dates.

Equipment

  1. Shaking hot block (e.g., Eppendorf Thermomixer 5384000020)

  2. Dissecting Microscope (we prefer a microscope with 6.3×–40× variable zoom optics for this method)

  3. Refrigerated microcentrifuge capable of 350 × g (e.g., Eppendorf 5425R)

  4. Automated cell counter (Nexcelom Cellometer Auto2000 or similar with compatible fluorescent live/dead staining kit)

  5. Vannas scissors, curved (Fine Science Tools, catalog number: 15019-10 or similar)

  6. Fine Bonn Scissors, straight or straight bladed Vannas scissors (Fine Science Tools, catalog number: 14084-08 or similar).

  7. Fine forceps (Dumont #1, #5 or similar)

Procedure



Figure 1. Dissection of corneal rim/iridocorneal angle/limbal region for dissociation.

(A–D) Working in a 10 cm dish containing ice-cold HBSS, freshly harvested eyes are cleaned of connective tissue, bisected, and the lens, retina, iris, and ciliary body are removed. (E) Using curved Vannas scissors, the cornea is removed immediately anterior to the limbus. (F) A second cut is then made posterior to the ciliary body to isolate the corneal rim/iridocorneal angle/limbal region. (G–H) Isolated corneal rim/iridocorneal angle/limbal region samples are immediately transferred to ice-cold dye-free DMEM and minced using fine scissors. (I) Minced tissue is transferred to a microcentrifuge tube for subsequent dissociation.


  1. Dissection of iridocorneal angle region

    Once begun, dissection should be performed as quickly as practical to limit cell mortalities. In our experience, the full dissection procedure takes a practiced surgeon about 5 min per eye, with a few minutes added if the same person is performing both euthanasia/enucleation and microdissection.

    1. Sacrifice mice according to the protocol approved by your local animal care and use committee. In our laboratory, mice are first anesthetized by intraperitoneal injection of ketamine/xylazine cocktail followed by cervical dislocation for euthanasia.

    2. Immediately after euthanasia, enucleate eye globes by cutting on the lateral side of the eye socket using scissors to cut through the conjunctiva and surrounding tissue. Take care not to puncture the eye at this stage. After making this incision, enucleate the eye by pulling back the tissue surrounding the eye socket and firmly grasping the optic nerve with forceps. The eye can then be cleanly pulled away and placed in ice-cold HBSS.

    3. To dissect the iridocorneal angle/limbal region, first use straight scissors to cleanly trim as much conjunctival and connective tissue from globe as possible (Figure 1A). Take care not to puncture the globe itself, as deflation of the eye can make this process more difficult. Next, cut the globe in half in either the sagittal or transverse direction so that the limbus and ciliary body bisect each section (Figure 1B–1C). Remove the lens and discard. Remove the retina using forceps and then, using curved Vannas scissors, carefully trim away the iris and ciliary body (Figure 1D). Take care not to damage the angle region, and err on the side of leaving some ciliary body or iris tissue. Next, while grasping the sclera near the hole left by the optic nerve, use the curved scissors to remove the cornea, cutting as close to the limbus as possible without damaging the angle region (Figure 1D–1E). Remove the cornea, and then make a second smooth cut immediately below the root of the ciliary body to free the limbal/iridocorneal angle region (Figure 1E–1F). Working in albino mice, this piece of tissue will look similar to a soft fingernail clipping. Immediately transfer this tissue to a 20 mm cell culture dish containing ice-cold DMEM with 10% FBS and continue isolating the limbal regions from the other half of the eye and the opposite globe. After dissection, you will have 4 slivers of limbal/iridocorneal angle tissue from each mouse.

    4. The tissues are then minced before digestion. Working submerged in the small dish of ice-cold DMEM + 10% FBS to prevent small tissue fragments from sticking to pipet tips, grasp each sliver of tissue by one end and, working from the other end with the fine scissors, reduce the tissue to fine slices by making a series of cuts perpendicular to the length of the tissue (Figure 1G–1I). Repeat until all tissues have been minced into the ice-cold media and proceed immediately with tissue digestion.


  2. Tissue dissociation

    1. Using a 1 mL pipet equipped with a wide bore tip, collect the minced tissues and transfer them into 2 mL microcentrifuge tubes for tissue dissociation (Figure 2). If necessary, allow tissues to settle (1–3 min), or centrifuge 350 × g for 30 s and remove supernatant to make space for additional volume until all tissue is collected.



      Figure 2. Flow chart showing an overview of the dissociation procedure.


    2. Allow samples to settle on ice or centrifuge 350 × g for 30 s before carefully aspirating media without disturbing tissues.

    3. Add 1 mL of pre-warmed digestion mix (see Recipes) containing 10% FBS, 1 mg/mL Collagenase A and 1 μM of Y27362. Rotate gently to mix and then place on a pre-warmed 37°C hot block shaking at 450 rpm.

    4. Incubate tissues for 2 h. At 30 min intervals, gently mix by inverting the tube 3–4 times to ensure tissue remains in suspension.

    5. After 2 h incubation, collect tissue fragments and dissociated cells by centrifugation at 350 × g for 5 min at 20°C. Aspirate supernatant and resuspend gently in 1 mL of DPBS to wash.

    6. Repeat centrifugation step, and then resuspend tissues in 1mL of pre-warmed 0.25% Trypsin EDTA containing 100 units of RNAse free DNase 1.

    7. Return samples to shaking 37°C block for 10 min.

    8. After 10 min, triturate samples using a 1 mL pipet equipped with a wide bore tip. Gently resuspend tissues by slowly aspirating 1 mL and returning it to the tube. Make 10 strokes, taking care not to introduce bubbles or damage cells by aggressive mixing.

    9. Return to 37°C block for an additional 10 min, then use a normal bore P1000 tip to make 10 additional mixing strokes. Incubate for 5 additional mins and then mix using 10 additional strokes with the standard bore tip. On the final stroke, transfer the cell suspension from the 2 mL to a 1.5 mL microcentrifuge tube.

    10. Collect dissociated cells by centrifugation at 350 × g for 5 min at 20°C. Gently aspirate supernatant and resuspend cell pellet in 1 mL of DMEM + 10% FBS to neutralize the Trypsin EDTA.

    11. Gently aspirate the full sample using a 1 mL pipet equipped with a standard bore P1000 tip, place a Flowmi micro 40 μm filter over the aperture of the pipet tip and slowly filter the cells into a new 1.5 mL microcentrifuge tube to remove debris. Take care not to damage the cells by subjecting them to high pressure during filtration.

    12. Repeat centrifugation step and resuspend in 1 mL of HBSS containing 1% BSA.

    13. Repeat centrifugation step and aspirate supernatant. Gently resuspend cells in 200 μL (see Notes) of additional HBSS containing 1% BSA.

    14. Filter cells through an additional 40 μm Flowmi filter, transferring to a new 1.5 mL microcentrifuge tube.

    15. Place cells on ice and use immediately for downstream applications.

Data analysis

Prior to library preparation for single cell RNA sequencing or other applications, assess cell concentration and viability using an automated cell counter equipped with live/dead cell detection (see note). Typically, we obtain 75,000–100,000 cells from dissociation of six eyes and 150–200,000 cells from dissociation of eight eyes with viability of between 80–95%. Smaller samples can be prepared using a similar protocol, but will result in disproportionally fewer cells as per-sample losses due to filtration and sample handling appear similar regardless of final cell number. Final sample concentration should be optimized based on downstream application and abundance of target cell population.

In one typical experiment, following sequencing of 19,232 cells from 12 wild-type mouse eyes in two independent preps, cells were clustered using Seurat, and distributions of cell types were estimated (Figure 3). While the majority of cells obtained mapped to uveal, scleral, corneal tissues, and melanocytes, Schlemm’s canal endothelial cells made up <1% of total cells present in the sample (109/19232), and trabecular meshwork cells made up almost 4% (701/19232). The extreme rarity of these cell types within the tissue is a critical consideration during experimental design and optimization.



Figure 3. Distribution of cell types obtained after single cell RNA sequencing on the 10x genomics platform.

Following sequencing of 19,232 cells from 12 wild-type mouse eyes in two independent preps, cells were clustered using Seurat, and distributions of cell types were estimated. SC: Schlemm’s canal endothelial cells, Conj: Conjunctival epithelial cells, RPE: Retina pigment epithelia, B/T: B and T cells, vSMC: Vascular smooth muscle cells, BEC: Blood vascular endothelial cells, TM: Trabecular meshwork, SMC: Smooth muscle cells.

Notes

Mouse strain selection: We have found that dissociation of pigmented eyes leads to aggregation of pigment material that is difficult to remove through washing and filtration and makes subsequent viability testing or single cell RNA sequencing library preparation difficult. Using eyes from albino mice eliminates this issue, but limits model selection and necessitates extensive backcrossing for use with genetic models present on a pigmented background. When using pigmented eyes, additional flow cytometry or filtration steps may be required to eliminate pigment aggregate from the sample prior to use in downstream applications.


Dissection: We typically perform this stage of the protocol using two people, so that one person can be responsible for humane euthanasia of mice and enucleation of the globes while their colleague works under the dissecting microscope on dissection of the iridocorneal angle region. It is possible for a single investigator to handle both tasks, but in that case, we typically collect all eyes in ice-cold HBSS before beginning the fine dissections.


Tissue digestion: Each sample requires 1 mL of pre-warmed DMEM + 10% FBS, and before beginning dissections, we prepare 1 mL aliquots in 1.5 mL microcentrifuge tubes. These can be placed on the 37°C block to pre-warm prior to beginning the protocol.


Final suspension volume: For a prep using six adult eyes, we typically perform final resuspension in a volume of 200 μL. Following filtration, this results in a final sample volume of ~150 μL and a cell concentration of ~650 cells per μL. For preparations using fewer eyes, a smaller volume should be used as needed to optimize sample concentration for downstream applications.


Cell counting and live-dead staining: In our laboratory, cell counting and live-dead staining is performed using a Nexcelom Cellometer Auto2000 automatic cell counter and compatible Nexcelom-branded Acridine orange/propidium iodide live/dead staining reagents.

Recipes

  1. Digestion mix

    Make 1 mL per sample immediately before beginning the digestion step of the protocol.

    Typically, we pre-warm 1 mL aliquots of DMEM to 37°C and add other reagents immediately (i.e., <5 min) before use.

    Reagent Final concentration Amount
    DMEM containing 10% FBS - 1 mL
    Collagenase A, 25 mg/mL stock 1 mg/mL 40 μL
    Y27362, 10 mM stock 10 μM 1 μL
    n/a

  2. Resuspension solution

    Filter sterilize before use and store at 4°C

    Reagent Final concentration Amount
    HBSS 10 mL
    BSA 1% W/V 0.1 g
    Total 10 mL

  3. 25 mg/mL Collagenase A stock solution

    Store at -20°C in 50 μL of aliquots

    Reagent Final concentration Amount
    Collagenase A (Lyophilized) 25 mg/mL 100 mg
    ddH2O n/a 4 mL
    Total n/a 4 mL

  4. 10 mM Y27632 stock solution

    Store at -20°C in 50 μL of aliquots. Once thawed, aliquots can be kept at 4°C for two weeks.

    Reagent Final concentration Amount
    Y27632 10 mM 10 mg
    ddH2O n/a 3 mL
    Total n/a 3 mL

Acknowledgments

We are grateful to Drs. Robert Lavker and Nihal Kaplan (Northwestern University Feinberg School of Medicine) for helpful discussions and for sharing their protocol for cell dissociation (described in Kaplan et al., 2019), upon which this protocol was based. Automated cell counting and quality control was performed by Dr. Jennifer Chin Man Wai of the Center for Genetic Medicine of the Feinberg School of Medicine. Work described here was funded by R01 EY025799 (to SEQ.), R01 EY032609 (to BRT) and a Brightfocus foundation new investigator grant in macular degeneration research to BRT.

Competing interests

Benjamin Thomson receives research funding from Bayer. Susan Quaggin owns stock in Mannin Research Inc. and receives consulting fees from AstraZeneca, Janssen, the Lowy Medical Research Foundation, Novartis, Pfizer, Janssen, and Roche/Genentech.

Ethics

Animal experiments were approved by the Animal Care and Use Committee at Northwestern University (Evanston IL, USA, animal protocol number IS000150015, validity period 2020/4/30-2023/4/29) and comply with ARVO guidelines for care and use of vertebrate research subjects in Ophthalmology research.

References

  1. Braunger, B. M., Fuchshofer, R. and Tamm, E. R. (2015). The aqueous humor outflow pathways in glaucoma: A unifying concept of disease mechanisms and causative treatment. Eur J Pharm Biopharm 95(Pt B): 173-181.
  2. Kaplan, N., Wang, J., Wray, B., Patel, P., Yang, W., Peng, H. and Lavker, R. M. (2019). Single-Cell RNA Transcriptome Helps Define the Limbal/Corneal Epithelial Stem/Early Transit Amplifying Cells and How Autophagy Affects This Population. Invest Ophthalmol Vis Sci 60(10): 3570-3583.
  3. Patel, G., Fury, W., Yang, H., Gomez-Caraballo, M., Bai, Y., Yang, T., Adler, C., Wei, Y., Ni, M., Schmitt, H., et al. (2020). Molecular taxonomy of human ocular outflow tissues defined by single-cell transcriptomics. Proc Natl Acad Sci U S A 117(23): 12856-12867.
  4. Quigley, H. A. and Broman, A. T. (2006). The number of people with glaucoma worldwide in 2010 and 2020. Br J Ophthalmol 90(3): 262-267.
  5. Tektas, O. Y. and Lutjen-Drecoll, E. (2009). Structural changes of the trabecular meshwork in different kinds of glaucoma. Exp Eye Res 88(4): 769-775.
  6. Thomson, B., Liu, P., Onay, T., Du, J., Tompson, S., Misener, S., Purohit, R., Young, T., Jin, J. and Quaggin, S. (2021). Cellular crosstalk regulates the aqueous humor outflow pathway and provides new targets for glaucoma therapies. Nat Commun 12(1): 6072.
  7. van Zyl, T., Yan, W., McAdams, A., Peng, Y. R., Shekhar, K., Regev, A., Juric, D. and Sanes, J. R. (2020). Cell atlas of aqueous humor outflow pathways in eyes of humans and four model species provides insight into glaucoma pathogenesis. Proc Natl Acad Sci U S A 117(19): 10339-10349.
  8. Zagora, S. L., Funnell, C. L., Martin, F. J., Smith, J. E., Hing, S., Billson, F. A., Veillard, A. S., Jamieson, R. V. and Grigg, J. R. (2015). Primary congenital glaucoma outcomes: lessons from 23 years of follow-up. Am J Ophthalmol 159(4): 788-796.

简介

[摘要]单细胞 RNA测序是一种强大的工具,可用于识别复杂组织内不同的细胞类型和转录组差异。它已被证明在眼睛组织中特别有用,在这些组织中,研究人员已经在视网膜、前房和虹膜角膜角内发现了新的细胞类型,并探索了转录组对年龄相关性黄斑变性疾病表型的贡献。然而,为了获得高质量的结果,该技术需要从感兴趣的组织中分离出健康的单细胞,寻求完全的组织消化,同时在文库制备之前尽量减少分离细胞中的压力和转录组变化。在这里, 我们提出了一个在我们的实验室开发的协议, 用于从小鼠虹膜角膜角度分离活单细胞, 其中包括 Schlemm 的运河和小梁网, 适用于单细胞 RNA 测序、流式细胞术或其他下游分析。

图形概要:


[背景] 青光眼是一种无法治愈的进行性神经退行性疾病,在全世界影响超过 6000 万人并导致 800 万人失明(Quigley 和 Broman,2006) 。目前的治疗集中在降低眼压(IOP),这是疾病进展的唯一可治疗风险因素。眼压由睫状体的房水分泌与通过虹膜角膜角组织(包括小梁网和施累姆管)的引流之间的比率决定,这些组织构成了传统的流出通路。青光眼患者,包括原发性先天性青光眼,在常规流出途径的组织中表现出缺陷,导致房水流出减少和眼压升高(Tektas 和 Lütjen-Drecoll,2009;Braunger等人,2015;Zagora等人,2015) .这些组织对青光眼发病机制的重要性以及因此作为治疗靶点的重要性,使得研究兴趣集中在了解它们的生理学、分子表型和控制其功能的细胞-细胞相互作用上。然而,由于所涉及的组织尺寸较小且它们位于虹膜角膜角内,因此对施累姆管或引流通道内皮细胞的转录组或整体表型、施累姆管内壁和外壁内皮细胞之间的潜在差异知之甚少。或小梁网的细胞。
最近,单细胞 RNA 测序已被用于鉴定小鼠、人类和其他模型物种的虹膜角膜角区域的细胞类型(van Zyl等人,2020;Patel等人,2020;Thomson等人,2021) .这些研究为构成角的细胞提供了令人兴奋的提示,并确定了潜在的新治疗靶点,但在已发表的数据集中,施莱姆氏管内皮细胞极为罕见 [van Zyl等人。 (2020 年)在对 13,833 个细胞(0.18%)进行测序后,仅鉴定了 25 个施累姆管内皮细胞(0.18%),而我们的小组在 19,232 个(0.57%)的数据集中鉴定了 109 个)]强调需要在健康和疾病条件下对更多细胞进行测序,以更好地了解这一重要组织的全部复杂性。
在本协议中,我们优化了一种简单的方法,用于解剖和分离小鼠虹膜角膜角,以获得健康细胞的单细胞悬浮液,用于下游应用,如单细胞 RNA 测序或流式细胞术。先前利用完整眼前节的研究报告称,角膜和眼表上皮细胞占数据集中总细胞的 50% 以上(van Zyl等人,2020) 。虽然与青光眼相关的虹膜角膜角细胞群仍然很少见,但通过在解剖过程中去除大部分角膜,我们的方法大大提高了它们的产量,导致施累姆管内皮细胞的比例增加约 200%,小梁网增加约 80%样品中的细胞。根据感兴趣的组织,该协议可以与流式细胞术或基于抗体结合珠的方法相结合,以在库制备之前丰富相关细胞类型的样品。我们还发现,该程序可以很容易地适应分离角膜或后眼的细胞,具有相似的结果和细胞活力。

关键字:单细胞RNA测序, 莱姆氏管, 眼睛, 青光眼, 单细胞, 离解, 虹膜角膜角, 边缘, 小鼠



材料和试剂


1.移液器尖端微型 40 μ m 细胞过滤器(Bel-Art Flomi,目录号: 136800040)
2.标准 10 cm 培养皿(每个样品一个)
3.标准 20 mm 组织培养皿(每个样品一个)
4.标准1.5 mL 微量离心管
5.标准 1 mL 移液器吸头(ThermoFisher ART 2079 或类似产品)
6.宽孔 1 mL 移液器吸头(ThermoFisher ART 2079GPK 或类似产品)
7.DMEM,无染料(ThermoFisher,Gibco,目录号:21063029)
8.胎牛血清(ThermoFisher,Gibco,目录号:26140079或类似)
9.0.25% 胰蛋白酶 EDTA(ThermoFisher,Gibco,目录号: 25200056)
10.10μm岩石抑制剂Y-27632(ATCC,目录号:ACS-3030 )
11.DNase 1(Roche,目录号:04716728001)
12.胶原酶A(Roche,目录号:10103578001)
13.HBSS(ThermoFisher,Gibco,目录号: 14175095)
14.牛血清蛋白(Sigma-Aldrich,目录号: 03117332001)
15.与您的自动细胞计数器兼容的吖啶橙/碘化丙啶活/死染色试剂盒(即Nexcelom CS2-0106)
16.如果您的自动细胞计数器需要兼容的一次性玻片(即Nexcelom CHT4-PD100)
17.消化混合物(见食谱)
18.重悬溶液(见配方)
19.25 mg/mL 胶原酶 A 原液(参见配方)
20.10 mM Y27632 原液(参见配方)
注意:所有试剂均按照制造商的建议储存并在标记的有效期之前使用。


设备


1.摇动热块(例如,Eppendorf Thermomixer 5384000020)
2.解剖显微镜(对于这种方法,我们更喜欢具有 6.3 × – 40 ×可变变焦光学元件的显微镜)
3.冷冻微量离心机 350 × g (例如,Eppendorf 5425R)
4.自动细胞计数器(Nexcelom Cellometer Auto2000 或类似的兼容荧光活/死染色试剂盒)
5.Vannas剪刀,弯曲的(Fine Science Tools,目录号:15019-10或类似)
6.Fine Bonn Scissors,直或直刃 Vannas 剪刀(Fine Science Tools,目录号:14084-08 或类似)。
7.细镊子(Dumont #1、#5 或类似产品)


程序


 
图 1. 分离角膜缘/虹膜角膜角/边缘区域。
(A - D)在一个 10 厘米的盘子中工作,其中包含冰冷的 HBSS,新鲜收获的眼睛被清除结缔组织,一分为二,晶状体、视网膜、虹膜和睫状体被移除。 (E) 使用弯曲的 Vannas 剪刀,在角膜缘前立即去除角膜。 (F) 然后在睫状体后方进行第二次切割,以隔离角膜缘/虹膜角膜角/角膜缘区域。 (G - H) 分离的角膜缘/虹膜角膜角/边缘区域样品立即转移到冰冷的无染料 DMEM 中,并用细剪刀切碎。 (I) 将切碎的组织转移到微量离心管中进行后续分离。


A.虹膜角膜角区解剖
一旦开始,应尽可能快地进行解剖以限制细胞死亡率。根据我们的经验,完整的解剖过程需要一名经验丰富的外科医生每只眼睛大约 5 分钟,如果同一个人同时执行安乐死/摘除和显微解剖,则需要增加几分钟。
1.根据当地动物护理和使用委员会批准的协议牺牲小鼠。在我们的实验室中,首先通过腹膜内注射氯胺酮/甲苯噻嗪混合物对小鼠进行麻醉,然后进行颈椎脱位以实施安乐死。
2.安乐死后,立即用剪刀剪开眼窝的外侧,切开结膜和周围组织,从而摘除眼球。在这个阶段注意不要刺破眼睛。做这个切口后,通过拉回眼窝周围的组织并用镊子牢牢抓住视神经来摘除眼睛。然后可以将眼睛干净地拉开并置于冰冷的 HBSS 中。
3.要解剖虹膜角膜角/角膜缘区域,首先使用直剪刀从地球上干净地修剪尽可能多的结膜和结缔组织(图 1A)。注意不要刺破地球本身,因为眼睛的通缩会使这个过程更加困难。接下来,在矢状或横向方向将地球切成两半,使边缘和睫状体将每个部分一分为二(图 1B - 1C)。取下镜头并丢弃。使用镊子取出视网膜,然后使用弯曲的 Vannas 剪刀小心地修剪虹膜和睫状体(图 1D)。注意不要损坏角度区域,并在留下一些睫状体或虹膜组织的一侧犯错。接下来,在抓住视神经留下的孔附近的巩膜时,使用弯曲的剪刀去除角膜,在不损坏角度区域的情况下尽可能靠近角膜缘切割(图 1D - 1E)。取出角膜,然后在睫状体根部正下方进行第二次平滑切割,以释放角膜缘/虹膜角膜角区域(图 1E - 1F)。在白化病小鼠中工作,这块组织看起来类似于柔软的指甲剪。立即将此组织转移到含有 10% FBS 的冰冷 DMEM 的 20 mm 细胞培养皿中,并继续将角膜缘区域与眼睛的另一半和对面的地球隔离开来。解剖后,您将获得每只小鼠的 4 片角膜缘/虹膜角膜角组织。
4.然后在消化前将组织切碎。将工作浸入冰冷的 DMEM + 10% FBS 的小盘中,以防止小组织碎片粘在移液管尖端上,从一端抓住每一片组织,用细剪刀从另一端工作,将组织减少到通过垂直于组织长度的一系列切口进行精细切片(图 1G - 1I)。重复直到所有组织都被切碎到冰冷的培养基中,并立即进行组织消化。


B.组织解离
1.使用配备宽孔尖端的 1 mL 移液器,收集切碎的组织并将其转移到 2 mL 微离心管中以进行组织分离(图 2)。如有必要,让组织沉降( 1-3分钟),或以 350 × g离心30 秒并去除上清液,为额外的体积腾出空间,直到收集到所有组织。


 
图 2 。流程图显示了解离过程的概述。


2.让样品在冰上沉淀或以 350 × g离心30 秒,然后小心吸出培养基,而不会干扰组织。
3.μM 的 Y27362的1 mL 预热消化混合物(参见食谱) 。轻轻旋转以混合,然后放置在预热的37 ° C热块上,以 450 rpm 的速度摇动。
4.将组织孵育 2 小时。每隔 30 分钟,将试管倒置 3-4 次,轻轻混匀,以确保组织保持悬浮状态。
5.孵育 2 小时后,在 20°C 下以 350*G 离心 5 分钟收集组织碎片和分离的细胞。吸出上清液并在 1 mL 的 DPBS 中轻轻重新悬浮以洗涤。
6.重复离心步骤,然后将组织重悬于 1mL 预热的 0.25% 胰蛋白酶 EDTA 中,其中含有 100 单位的 RNAse free DNase 1。
7.将样品放回 37°C 块中振荡 10 分钟。
8.10 分钟后,使用配备宽孔尖端的 1 mL 移液器研磨样品。通过缓慢吸出 1 mL 并将其返回管中,轻轻地重新悬浮组织。进行 10 次冲程,注意不要通过剧烈混合引入气泡或损坏细胞。
9.回到 37°C 再加热 10 分钟,然后使用普通口径的 P1000 吸头进行 10 次额外的混合冲程。再孵育 5 分钟,然后使用标准孔尖端使用 10 次额外冲程混合。在最后一次冲程中,将细胞悬浮液从 2 mL 转移到 1.5 mL 微量离心管中。
10.在 20°C 下以 350 × g离心 5 分钟收集分离的细胞。在 1 mL 的 DMEM + 10% FBS 中轻轻吸出上清液并重新悬浮细胞颗粒,以中和胰蛋白酶 EDTA。
11.使用配备标准孔 P1000 吸头的 1 mL 吸管轻轻吸出全部样品,将 Flowmi 微型 40 μm 过滤器放在吸管吸头的孔径上,然后将细胞缓慢过滤到新的 1.5 mL 微量离心管中以去除碎屑。注意不要在过滤过程中使细胞承受高压而损坏细胞。
12.重复离心步骤并重新悬浮在含有 1% BSA 的 1 mL HBSS 中。
13.重复离心步骤并吸出上清液。在含有 1% BSA 的 200 μL(见注释)中轻轻重悬细胞。
14.通过额外的 40 μm Flowmi 过滤器过滤细胞,转移到新的 1.5 mL 微量离心管中。
15.将细胞置于冰上并立即用于下游应用。


数据分析


在为单细胞 RNA 测序或其他应用准备文库之前,使用配备活/死细胞检测功能的自动细胞计数器评估细胞浓度和活力(见注)。通常,我们从六只眼睛的解离中获得 75,000 – 100,000 个细胞,从八只眼睛的解离中获得150 – 200,000 个细胞,存活率在 80 – 95% 之间。较小的样品可以使用类似的方案制备,但会导致细胞数不成比例地减少,因为过滤和样品处理导致的每个样品损失看起来相似,而与最终细胞数无关。最终样品浓度应根据下游应用和靶细胞群的丰度进行优化。
在一项典型实验中,在两次独立的准备中对来自 12 只野生型小鼠眼睛的 19,232 个细胞进行测序后,使用 Seurat 对细胞进行聚类,并估计细胞类型的分布(图 3 )。虽然获得的大多数细胞映射到葡萄膜、巩膜、角膜组织和黑素细胞,但施累姆管内皮细胞占样本中总细胞的比例不到 1% (109/19232),而小梁网细胞几乎占 4%。 701/19232)。组织中这些细胞类型的极端稀有性是实验设计和优化过程中的一个关键考虑因素。


 
图 3. 在 10x 基因组学平台上单细胞 RNA 测序后获得的细胞类型分布。
在两次独立准备中对来自 12 只野生型小鼠眼睛的 19,232 个细胞进行测序后,使用 Seurat 对细胞进行聚类,并估计细胞类型的分布。 SC:施累姆管内皮细胞,Conj:结膜上皮细胞,RPE:视网膜色素上皮细胞,B/T:B 和 T 细胞,vSMC:血管平滑肌细胞,BEC:血管内皮细胞,TM:小梁网,SMC:平滑肌肌肉细胞。


笔记


小鼠品系选择:我们发现色素眼睛的解离导致色素物质的聚集,这些物质难以通过洗涤和过滤去除,并使得后续的活力测试或单细胞 RNA 测序文库的制备变得困难。使用白化病小鼠的眼睛可以消除这个问题,但会限制模型选择,并且需要进行广泛的回交,以便与色素背景上存在的遗传模型一起使用。当使用有色眼睛时,可能需要额外的流式细胞术或过滤步骤以在用于下游应用之前从样品中去除色素聚集体。


解剖: 我们通常使用两个人执行该协议的这个阶段,这样一个人就可以负责小鼠的人道安乐死和球体的摘除,而他们的同事在解剖显微镜下对虹膜角膜角区域进行解剖。一个调查员可以同时处理这两项任务,但在这种情况下,我们通常会在开始精细解剖之前将所有眼睛收集在冰冷的 HBSS 中。


组织消化: 每个样品需要 1 mL 的预热 DMEM + 10% FBS,在开始解剖之前,我们在 1.5 mL 微量离心管中准备 1 mL 等分试样。这些可以放在 37°C 块上以在开始协议之前预热。


最终悬浮体积:对于使用六只成人眼睛的准备,我们通常以 200 μL 的体积进行最终再悬浮。过滤后,最终样品体积约为 150 μL,细胞浓度约为每 μL 650 个细胞。对于使用较少眼睛的制备,应根据需要使用较小的体积来优化下游应用的样品浓度。


细胞计数和活死染色:在我们的实验室中,使用Nexcelom Cellometer Auto2000 自动细胞计数器和兼容的 Nexcelom 品牌吖啶橙/碘化丙啶活/死染色试剂进行细胞计数和活死染色。


食谱


1.消化混合物
在开始协议的消化步骤之前立即为每个样品制作 1 mL。
通常,我们将 1 mL 的 DMEM 等分试样预热至 37°C,并在使用前立即添加其他试剂(即<5 分钟)。
试剂最终浓度数量
含有 10% FBS 的 DMEM-1毫升
胶原酶 A,25 mg/mL 原液1毫克/毫升40微升
Y27362,10 毫米库存10微米_1微升
不适用


2.再悬浮液
使用前过滤除菌,4°C保存
试剂最终浓度数量
HBSS10 毫升
牛血清白蛋白1% W/V0.1克
全部的10 毫升


3.25 mg/mL 胶原酶 A 储备液
以 50 μ L 的等分试样在 -20 °C 下储存
试剂最终浓度数量
胶原酶 A(冻干)25毫克/毫升100 毫克
ddH 2 O不适用4 毫升
全部的不适用4 毫升


4.10 mM Y27632 原液
以 50 μ L 的等分试样在 -20 °C 下储存。解冻后,等分试样可在 4 °C下保存两周。
试剂最终浓度数量
Y2763210毫米10毫克
ddH 2 O不适用3 毫升
全部的不适用3 毫升


致谢


我们感谢 Drs。 Robert Lavker 和 Nihal Kaplan(西北大学 Feinberg 医学院)进行了有益的讨论并分享了他们的细胞解离方案(在Kaplan等人,2019 年描述),该方案是基于该方案的。自动细胞计数和质量控制由 Feinberg 医学院遗传医学中心的 Jennifer Chin Man Wai 博士进行。此处描述的工作由 R01 EY025799(至 SEQ.)、R01 EY032609(至 BRT)和 Brightfocus 基金会在黄斑变性研究中对 BRT 的新研究人员资助。


利益争夺


Benjamin Thomson 从拜耳获得研究经费。 Susan Quaggin 拥有 Mannin Research Inc. 的股票,并从 AstraZeneca、Janssen、Lowy Medical Research Foundation、Novartis、Pfizer、Janssen 和 Roche/Genentech 获得咨询费。


伦理


动物实验经西北大学动物护理和使用委员会(美国伊利诺伊州埃文斯顿,动物协议号 IS000150015,有效期 2020/4/30-2023/4/29)批准,并符合 ARVO 脊椎动物护理和使用指南眼科研究的研究对象。


参考


1.Braunger, BM, Fuchshofer, R. 和 Tamm, ER (2015)。青光眼的房水流出途径:疾病机制和病因治疗的统一概念。 Eur J Pharm Biopharm 95(Pt B):173-181。
2.Kaplan, N.、Wang, J.、Wray, B.、Patel, P.、Yang, W.、Peng, H. 和 Lavker, RM (2019)。单细胞 RNA 转录组有助于定义角膜缘/角膜上皮干细胞/早期转运扩增细胞以及自噬如何影响这一群体。 Invest Ophthalmol Vis Sci 60(10):3570-3583。
3.Patel, G., Fury, W., Yang, H., Gomez-Caraballo, M., Bai, Y., Yang, T., Adler, C., Wei, Y., Ni, M., Schmitt, H .,等人。 (2020 年)。由单细胞转录组学定义的人眼流出组织的分子分类。 Proc Natl Acad Sci USA 117(23): 12856-12867。
4.Quigley, HA 和 Broman, AT (2006)。 2010 年和 2020 年全球青光眼患者人数。 Br J Ophthalmol 90(3):262-267。
5.Tektas, OY 和 Lutjen-Drecoll, E. (2009)。不同类型青光眼小梁网的结构变化。 Exp Eye Res 88(4):769-775。
6.Thomson, B.、Liu, P.、Onay, T.、Du, J.、Tompson, S.、Misener, S.、Purohit, R.、Young, T.、Jin, J. 和 Quaggin, S.( 2021)。细胞串扰调节房水流出途径并为青光眼治疗提供新靶点。 国家通讯12(1):6072 。
7.van Zyl, T., Yan, W., McAdams, A., Peng, YR, Shekhar, K., Regev, A., Juric, D. 和 Sanes, JR (2020)。人类和四种模型物种眼房水流出通路的细胞图谱提供了对青光眼发病机制的深入了解。 Proc Natl Acad Sci USA 117(19):10339-10349。
8.Zagora, SL, Funnell, CL, Martin, FJ, Smith, JE, Hing, S., Billson, FA, Veillard, AS, Jamieson, RV 和 Grigg, JR (2015)。原发性先天性青光眼结局:23 年随访的经验教训。 Am J Ophthalmol 159(4):788-796。


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引用:Thomson, B. R. and Quaggin, S. E. (2022). Preparation of a Single Cell Suspension from the Murine Iridocorneal Angle. Bio-protocol 12(10): e4426. DOI: 10.21769/BioProtoc.4426.
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