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Feb 2019

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3D Organoid Formation from the Murine Salivary Gland Cell Line SIMS
小鼠唾液腺SIMS细胞系的三维类器官形成   

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Abstract

Salivary glands consist of multiple phenotypically and functionally unique cell populations, such as the acinar, ductal, and myoepithelial cells that help produce, modify, and secrete saliva (Lombaert et al., 2011). Identification of mechanisms and factors that regulate these populations has been of key interest, as salivary gland-related diseases have detrimental effects on these cell populations. A variety of approaches have been used to understand the roles different signaling mechanisms and transcription factors play in regulating salivary gland development and homeostasis. Differentiation assays have been performed with primary salivary cells in the past (Maimets et al., 2016), however this approach may sometimes be limiting due to tissue availability, labor intensity of processing the tissue samples, and/or inability to long-term passage the cells. Here we describe in detail a 3D differentiation assay to analyze the differentiation potential of a salivary gland cell line, SIMS, which was immortalized from an adult mouse submandibular salivary gland (Laoide et al., 1996). SIMS cells express cytokeratin 7 and 19, which is characteristic for a ductal cell type. Although adult acinar and myoepithelial cells were found in vivo to preserve their own cell population through self-duplication (Aure et al., 2015; Song et al. 2018), in some cases duct cells can differentiate into acinar cells in vivo, such as after radiation injury (Lombaert et al., 2008; Weng et al., 2018). Thus, utilization of SIMS cells allows us to target and analyze the self-renewal and differentiation effects of ductal cells under specific in vitro controlled conditions.

Keywords: Salivary gland (唾液腺), Epithelial (上皮), SIMS (SIMS), Differentiation assay (分化测定), Mouse cell line (小鼠细胞系)

Background

In vitro differentiation assays are a remarkable tool that enables us to analyze the potency of cells without relying on in vivo models. Cells in these assays can be manipulated for gene expression to, for instance, analyze their differentiation ability. While previous protocols have described the differentiation potential of primary salivary gland cells in vitro, our protocol is optimized for the use of the SIMS cell line (Laoide et al., 1996; Maimets et al., 2016). SIMS cells express cytokeratin 7 and 19, which is characteristic for a ductal cell type. Although adult acinar and myoepithelial cells were found in vivo to preserve their own cell population through self-duplication (Aure et al., 2015; Song et al. 2018), in some cases duct cells can differentiate into acinar cells in vivo, such as after radiation injury (Lombaert et al., 2008; Weng et al., 2018). Thus, utilization of SIMS cells allows us to target and analyze the self-renewal and differentiation effects of ductal cells under specific in vitro controlled conditions. Our recent work shows that SIMS cells have the ability to differentiate into unique populations of acinar, myoepithelial, and duct cells in 3D differentiation conditions, that normally produce, modify and secrete saliva in vivo (Lombaert et al., 2011; Athwal et al., 2019). This protocol describes the workflow necessary for the generation and analysis of 3D differentiated SIMS cells. SIMS cells are cultured in a 3D matrigel/collagen matrix over a period of 7-9 days. Matrigel is a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma, which is rich in several growth factors and extracellular matrix proteins, such as laminin-1, collagen IV, and heparin sulfate proteoglycans (Patel et al., 2016). Matrigel has been shown to induce differentiation of stem/progenitor cells and outgrowth of already differentiated cell types by causing polarization of cells embedded in or on top of it. For SIMS grown in this 3D matrix, organoid formation post single cell culture is apparent around day 5 of culture. These organoids are also stained either via cryosectioning or in 3D to analyze for differentiation markers and cell populations. Alterations, such as cell transfections, transductions and/or media changes, to this condition can easily be made to address specific research questions (Athwal et al., 2019). The media components were adjusted using literature from both existing salivary and mammary gland differentiation protocols, which deemed necessary to induce differentiation of the SIMS cells in vitro. Here we show the robust use of this cell line to address specific research questions without having to rely on primary salivary gland cells.

Materials and Reagents

  1. 500 ml filter units with 0.22 μm filter (Fisher, catalog number: 974102)
  2. 24-well tissue culture plates (Thermo Fisher, catalog number: 142475)
  3. T75 tissue culture flasks (Thermo Fisher, catalog number: 156499)
  4. 2 ml pipettes (Fisher, catalog number: 13-678-25C)
  5. 10 cm dishes (Thermo Fisher, catalog number: 150464)
  6. 15 ml Falcon tubes (Corning, catalog number: 35296) 
  7. 1 ml pipettes (Fisher, catalog number: 13-678-25B)
  8. Forceps (Fisher Scientific, catalog number: XX6200006P)
  9. Razor blade (Fisher Scientific, catalog number: 12-640)
  10. Glass slides (VWR, catalog number: 89500-466)
  11. SIMS cells (available per request) 
  12. Sterile DMEM medium + 4.5g/L high glucose + L-glutamine without sodium pyruvate (Invitrogen, catalog number: 11965-092)
  13. Sterile 1x PBS-Ca+2-Mg+2 free (Thermo Fisher, catalog number: 110010031) or 1x HBSS-Ca+2-Mg+2 free (Thermo Fisher, catalog number: 14175079)
  14. Penicillin-Streptomycin (Thermo Fisher, catalog number: 15140122)
  15. Sterile USDA-approved FBS (Sigma-Aldrich, catalog number: F2442-500ML)
  16. 0.25% Trypsin-EDTA (Sigma-Aldrich, catalog number: T4049-100ML)
  17. Epidermal growth factor (EGF) (Sigma-Aldrich)
  18. Glutamax (Gibco/ThermoFisher, catalog number: 35050061)
  19. Fibroblast growth factor-2 (FGF2) (R&D, catalog number: 3139FB025)
  20. N2 (Thermo Fisher, catalog number: 17502048)
  21. Dexamethasone (VWR, catalog number: 80056-298)
  22. Triiodothyronine (Sigma-Aldrich, catalog number: 709719)
  23. Retinoic Acid (Thermo Fisher, catalog number: AA4454077)
  24. Hydrocortisone (Sigma-Aldrich, catalog number: H0888)
  25. Cholera toxin (Sigma-Aldrich, catalog number: C8052)
  26. Calcium, 99%, Granular (Fisher, catalog number: AC201180050)
  27. Growth factor reduced Matrigel (BD Biosciences, catalog number: 354230)
  28. Collagen Type I, Rat Tail (Millipore, catalog number: 2790888)
  29. 4% PFA (VWR, catalog number: AAJ61899-AK)
  30. Trypan blue 0.4% (Thermo Fisher, catalog number: 15250061)
  31. O.C.T. Compound (Fisher scientific, catalog number: 23-730-571)
  32. SIMS subculture media (see Recipes)
  33. Differentiation media (see Recipes)

Equipment

  1. Water bath (Thermo Scientific, catalog number: TSGP02)
  2. Vacuum pump aspirator (standard)
  3. CO2 incubator (standard)
  4. Cryotome (standard)
  5. Brightfield microscope (standard)
  6. Confocal laser scanning microscope (standard)

Software

  1. ImageJ software (http://imagej.net, available online for download)

Procedure

  1. SIMS subculture
    1. Pre-warm cell medium, 1x PBS-Ca+2-Mg+2 free or 1x HBSS- Ca+2-Mg+2 free and 0.25% trypsin-EDTA in a water bath at 37 °C. 
    2. Aspirate off old medium with vacuum aspirator or use 10 ml pipettes to manually aspirate from the T75 cell culture flask. 
    3. Wash twice with 10 ml of sterile 1x PBS/HBSS-Ca+2-Mg+, aspirate off completely-either manually with a 2 ml pipette or with an aspirator and sterile fresh 2 ml glass pipette. 
    4. Add 3 ml of pre-warmed 0.25% Trypsin-EDTA per T75 flask. 
    5. Incubate plate with cells at 37 °C for 5-7 min. Tap and gently shake the plate to dislodge cells.
    6. Neutralize trypsin by adding fresh ~3-5 ml culture medium using a 10 ml pipette. Pipette gently up and down several times to re-suspend cells and transfer to a 15 ml Falcon tube.
    7. Centrifuge at 100 x g for 3 min, wash 2 times with 1x PBS-Ca+2-Mg+2 free, and take up the cells in desired amount of culture medium.
    8. Plate up to 1 million cells to get 95% confluency in 4-5 days in a T75 tissue culture flask.
      Note: Cell viability (~90-95%) is determined by trypan blue cell count.

  2. Organoid differentiation culture 
    1. Resuspend SIMS cells in SIMS medium at 0.4 x 106 cells/ml.
    2. Thaw out growth factor reduced matrigel matrix on ice for 10 min before culturing cells. Both collagen and matrigel need to remain on ice until they are resuspended with cells for plating.
    3. Add 50 μl of cell solution (10,000 cells) to 100 µl of 60:40 ratio of Type I rat tail collagen to growth factor reduced Matrigel on ice. Mix gently while slowly pipetting up and down using a 200 μl pipette.
      Notes:
      1. Aliquot collagen first then Matrigel. Do not mix with pipette until you add cells to avoid bubbles. 
      2. Avoid fast aspiration or suspension of aliquoted matrix mix to avoid bubble formation. 
    4. Deposit cell/collagen/matrigel mix in the center of 24-well tissue culture plate on ice and incubate for 20 min at 37 °C. 
    5. Add up to 1 ml differentiation medium. 
    6. Change medium every 2-3 days.
    7. Cultures are maintained up to 14 days as the cells reach maximum confluency within the matrix, and the matrix starts breaking down. 
    8. Organoid formation can be seen starting Day 5 of culture (Figure 1).


      Figure 1. Collagen-matrigel embedded SIMS cells expand and form organoids by Day 5 (A) and 7 (B). Scale bars, 200 μm (4x magnification).

  3. Organoid fixation 
    1. Wash matrix with ice cold 1x PBS-Ca+2-Mg+2 free gently up to 3 times. 
    2. Cells can be fixed at any timepoint using 2% PFA in 1x PBS-Ca+2-Mg+2 free for 20 min on ice or at 4 °C. 
    3. Rinse the matrix with ice cold 1x PBS-Ca+2-Mg+2 free 3 times. 
    4. Incubate matrix in 1x PBS-Ca+2-Mg+2 free for 10 min. Repeat this 3 times for a total of 30 minutes.
    5. Using forceps, gently remove the matrix from the tissue culture plate.
      Note: You can cut the matrix into 2-3 pieces using a fine blade if you want multiple cryo blocks.
    6. Embed with OCT (a full description of embedding can be found in Campbell et al., 2011).
      Note: Matrix will likely curl up in OCT if it is poured too quickly. Gently pour, and fix any curling using your forceps. 
    7. Optimal cutting thickness is between 12 and 16 μm. 
    8. Sections can be post fixed with 4% PFA, ice cold acetone, or acetone/methanol for staining optimization.

  4. 3D matrix staining
    To conserve the 3D morphology, staining is performed on 1 mm3 pieces of the organoids embedded in matrix.
    1. Post fixation, small pieces of matrix including organoids are cut using a sharp scalpel blade and pointed tweezers. Treat each chunk separately in a plastic dish or 24-well plate. 
    2. Matrix can be treated again with 2-4% PFA, acetone/methanol, or methanol treatments if necessary. 
    3. Matrix is treated like tissue slides for staining. An example is described in Athwal et al., 2019. 
    4. Mount each sample on glass slides with 1-2 spacers depending on the size of the matrix.
    5. Capture images using a confocal microscope (Figure 2). 
    6. This staining can also be performed on cryosections of OCT embedded organoid matrix (Athwal et al., 2019). A complete description of the primary and secondary antibodies used in Figure 2 can be found in above-mentioned manuscript.


      Figure 2. 1 mm3 collagen/matrigel matrix with 3D grown SIMS organoids were stained with E-cadherin post-fixation with 4% PFA. Scale bar, 10 μm.

Data analysis

Brightfield images are taken starting Day 3 until the end of the experiment. Organoid diameter can be quantified using ImageJ. Each experiment is repeated 3 times with n = 5 images per day for quantification. Details on statistical analysis can be found in our previous manuscript (Athwal et al., 2019).

Notes

  1. Cell death is noticed by Day 14 as cells become confluent within the matrix. 
  2. Serial passage of cells dissociated from organoids has not been performed with SIMS cell organoids. 
  3. Increase the volume of collagen/matrigel if cell number is increased. 
  4. Transduced or transfected SIMS cells are selected prior to the 3D culture assay.

Recipes

  1. SIMS subculture medium (Table 1)

    Table 1. SIMS subculture medium


  2. Differentiation medium

    Table 2. SIMS differentiation medium

    Notes:
    1. Make 100 μl aliquots of growth factor reduced matrigel and store at -20 °C.
    2. Make 100 μl aliquots of rat tail collagen and store at 4 °C. 

Acknowledgments

This work was supported by the National Institutes of Health (NIH)/National Institute of Dental and Craniofacial Research (NIDCR) grants DE027034 and DE022557. This protocol was adapted from Maimets et al., 2016. Timeline of organoid formation using SIMS cells and changes in media recipe are described in our protocol.

Competing interests

There are no conflicts of interest.

References

  1. Athwal, H. K., Murphy, G., 3rd, Tibbs, E., Cornett, A., Hill, E., Yeoh, K., Berenstein, E., Hoffman, M. P. and Lombaert, I. M. A. (2019). Sox10 regulates plasticity of epithelial progenitors toward secretory units of exocrine glands. Stem Cell Reports 12(2): 366-380.
  2. Aure, M. H., Konieczny, S. F. and Ovitt, C. E. (2015). Salivary gland homeostasis is maintained through acinar cell self-duplication. Dev Cell 33(2): 231-237.
  3. Campbell, J. J., Davidenko, N., Caffarel, M. M., Cameron, R. E. and Watson, C. J. (2011). A multifunctional 3D co-culture system for studies of mammary tissue morphogenesis and stem cell biology. PLoS One 6(9): e25661.
  4. Laoide, B. M., Courty, Y., Gastinne, I., Thibaut, C., Kellermann, O. and Rougeon, F. (1996). Immortalised mouse submandibular epithelial cell lines retain polarised structural and functional properties. J Cell Sci 109 (Pt 12): 2789-2800.
  5. Lombaert, I. M., Brunsting, J. F., Wierenga, P. K., Faber, H., Stokman, M. A., Kok, T., Visser, W. H., Kampinga, H. H., de Haan, G. and Coppes, R. P. (2008). Rescue of salivary gland function after stem cell transplantation in irradiated glands. PLoS One 3(4): e2063.
  6. Lombaert, I. M., Knox, S. M. and Hoffman, M. P. (2011). Salivary gland progenitor cell biology provides a rationale for therapeutic salivary gland regeneration. Oral Dis 17(5): 445-449.
  7. Maimets, M., Rocchi, C., Bron, R., Pringle, S., Kuipers, J., Giepmans, B. N., Vries, R. G., Clevers, H., de Haan, G., van Os, R. and Coppes, R. P. (2016). Long-term in vitro expansion of salivary gland stem cells driven by Wnt signals. Stem Cell Reports 6(1): 150-162.
  8. Patel, R. and Alahmad, A. J. (2016). Growth-factor reduced Matrigel source influences stem cell derived brain microvascular endothelial cell barrier properties. Fluids Barriers CNS 13: 6.
  9. Song, E. C., Min, S., Oyelakin, A., Smalley, K., Bard, J. E., Liao, L., Xu, J. and Romano, R. A. (2018). Genetic and scRNA-seq analysis reveals distinct cell populations that contribute to salivary gland development and maintenance. Sci Rep 8(1): 14043.
  10. Weng, P. L., Aure, M. H., Maruyama, T. and Ovitt, C. E. (2018). Limited regeneration of adult salivary glands after severe injury involves cellular plasticity. Cell Rep 24(6): 1464-1470 e3.

简介

唾液腺由多个表型和功能独特的细胞群组成,如腺泡、导管和肌上皮细胞,帮助产生、修饰和分泌唾液(LoBault et al.,2011)。由于唾液腺相关疾病对这些细胞群有不利影响,因此确定调节这些细胞群的机制和因素一直是人们关注的重点。各种方法已经被用来了解不同的信号机制和转录因子在调节唾液腺发育和稳态中的作用。过去曾用唾液腺细胞进行分化试验(Mimet等2016),但这种方法有时由于组织的可用性、处理组织样本的劳动强度和/或不能长期通过细胞而受到限制。在这里,我们详细描述了一个3D分化试验分析唾液腺细胞系,SIMS,这是永生从成年小鼠颌下腺唾液腺(LAOIDE等1996)的分化潜力。sims细胞表达细胞角蛋白7和19,这是导管细胞类型的特征。虽然成人腺泡和肌上皮细胞在活体< 中发现通过自身复制保存自身的细胞群(Aure 等人, >,2015;宋等.2018),但有些情况下,管细胞可分化为腺泡细胞<体内[],如放射线损伤后(Lombaert 等.,2008;文格 ET。2018年)。因此,利用SIMS细胞可以在特定的体外>控制条件下,靶向和分析导管细胞的自我更新和分化效应。
【背景】在体外E/EM>分化检测是一种非常有效的工具,使我们能够不依赖于体内>模型来分析细胞的潜能。这些分析中的细胞可以通过操纵基因表达来分析其分化能力。虽然以前的协议描述了唾液腺细胞的分化潜能在体外>,但我们的方案是优化SIMS细胞系的使用(LAOIDE等>1996,Maimet等。2016)。sims细胞表达细胞角蛋白7和19,这是导管细胞类型的特征。虽然成人腺泡和肌上皮细胞在活体< 中发现通过自身复制保存自身的细胞群(Aure 等人, >,2015;宋等.2018),但有些情况下,管细胞可分化为腺泡细胞<体内[],如放射线损伤后(Lombaert 等.,2008;文格 ET。2018年)。因此,利用SIMS细胞可以在特定的体外>控制条件下,靶向和分析导管细胞的自我更新和分化效应。我们最近的研究表明,SIMS细胞有能力在3D分化条件下分化为腺泡、肌上皮和导管细胞的独特群体,通常在唾液中产生、修饰和分泌唾液<(LoBault 等, >,2011;AthWale等,>,2019)。该协议描述了生成和分析3d分化sims细胞所需的工作流程。SIMS细胞在3-基质胶/胶原基质中培养7-9天。MatRelEL是从Engelbreth Holm Swarm(EHS)小鼠肉瘤中提取的一种可溶解的基底膜制剂,富含多种生长因子和细胞外基质蛋白,如层粘连蛋白-1、Ⅳ型胶原和硫酸肝素蛋白多糖(帕特尔等。>,2016)。Matrigel已经被证明诱导干/祖细胞分化和已经分化的细胞类型的生长,通过使细胞的极化嵌入或嵌入细胞顶部。对于生长在这种3d基质中的sims,在培养的第5天左右,单细胞培养后的器官形成是明显的。这些有机物也被染色通过冷冻或3D分析分化标记和细胞群体。为了解决特定的研究问题(Athwal等人,2019年),可以很容易地对这种情况进行改变,如细胞转染、转基因和/或介质改变。利用现有唾液腺和乳腺分化方案的文献,对培养基成分进行了调整,认为这是体外诱导SIMS细胞分化的必要条件。在这里,我们展示了该细胞系的稳健使用,以解决特定的研究问题,而不必依赖于原唾液腺细胞。

关键字:唾液腺, 上皮, SIMS, 分化测定, 小鼠细胞系

材料和试剂

  1. 500毫升过滤装置,带0.22μm过滤器(费希尔,目录号:974102)
  2. 24孔组织培养板(Thermo Fisher,目录号:142475)
  3. T75组织培养瓶(Thermo Fisher,目录号:156499)
  4. 2毫升移液管(Fisher,目录号:13-678-25C)
  5. 10cm盘子(Thermo Fisher,目录号:150464)
  6. 15毫升Falcon试管(康宁,目录号:35296)
  7. 1毫升移液管(Fisher,目录号:13-678-25B)
  8. 镊子(Fisher Scientific,目录号:XX6200006P)
  9. 刀片(Fisher Scientific,目录号:12-640)
  10. 玻片(VWR,目录号:89500-466)
  11. 模拟人生单元(根据要求提供)
  12. 不含丙酮酸钠的无菌DMEM培养基+高糖培养基
  13. 无菌1X PBS CA+2-mg+2免费(赛默飞世尔,目录号:110010031)或1X HBSS CA+2-mg+2免费(赛默飞世尔,目录号:14175079)
  14. 青霉素链霉素(Thermo Fisher,目录号:15140122)
  15. 无菌美国农业部批准的FBS(Sigma-Aldrich,目录号:F2442-500ml)
  16. 0.25%胰蛋白酶EDTA(Sigma-Aldrich,目录号:T4049-100ml)
  17. 表皮生长因子(egf)(sigma-aldrich)
  18. 谷氨酰胺(Gibco/Thermofisher,目录号:35050061)
  19. 成纤维细胞生长因子-2(FGF2)(研发,目录号:3139FB025)
  20. N2(赛默飞世尔,目录号:17502048)
  21. 地塞米松(vwr,目录号:80056-298)
  22. 三碘甲状腺原氨酸(Sigma-Aldrich,目录号:709719)
  23. 维甲酸(Thermo Fisher,产品目录号:AA4454077)
  24. Hydrocortisone(西格玛奥德里奇,目录号:H088)
  25. 霍乱毒素(Sigma奥德里奇,目录号:C8052)
  26. 钙,99%,颗粒状(Fisher,目录号:AC201180050)
  27. 生长因子还原基质凝胶(bd biosciences,目录号:354230)
  28. Ⅰ型胶原,Rat Tail(MiLople,目录号:2790888)
  29. 4%全氟辛烷磺酸(VWRAAJ61899-AK)
  30. 台盼蓝0.4%(赛默飞世尔,目录号:15250061)
  31. O.C.T.化合物(Fisher Scientific,目录号:23-730-571)
  32. 模拟人生亚文化培养基(见配方)
  33. 差异化媒体(参见配方)

设备

  1. 水浴(Thermo Scientific,目录号:TSGP02)
  2. 真空泵抽吸器(标准)
  3. 培养箱(标准)
  4. 冰冻切片机(标准)
  5. Brightfield显微镜(标准)
  6. 共焦激光扫描显微镜(标准)

软件

  1. ImageJ软件(http://imagej.net/“target=”\u blank“>http://imagej.net,可在线下载)

程序

  1. 模拟人生亚文化
    1. 预温细胞培养基,1倍PBS Ca+2-mg+2游离或1倍HBSS-Ca+2-mg+2游离,37°C水浴中0.25%胰蛋白酶EDTA。
    2. 用真空吸管吸出旧培养基或用10毫升吸管从T75细胞培养瓶中手动吸出。
    3. 用10毫升无菌1X PBS/HBSS Ca+2-mg+清洗两次,用2毫升移液管或用吸液器和无菌新鲜2毫升玻璃移液管手动完全吸出。
    4. 每T75烧瓶加入3毫升预热的0.25%胰蛋白酶EDTA。
    5. 将培养板与细胞在37°C下培养5-7分钟。轻敲并轻轻摇动培养板以取出细胞。
    6. 通过添加新鲜的3-5毫升培养基,用10毫升移液管中和胰蛋白酶。移液管轻轻上下数次,重新悬浮细胞,并转移到15毫升猎鹰管。
    7. 离心机在100X g>3 min,用1X PBS Ca>2+/SUP> - Mg 2自由洗涤2次,取所需培养基中的细胞。
    8. 在T75组织培养瓶中培养100万个细胞,在4-5天内达到95%的融合率。
      注:细胞活力(~90-95%)由台盼蓝细胞计数决定。>
      < BR>
  2. 器官分化培养
    1. 在sims培养基中以0.4x 106细胞/ml的速度重新培养sims细胞。
    2. 解冻生长因子,在培养前10分钟,在冰上还原基质胶基质。胶原蛋白和MatMatell都需要留在冰上,直到它们再悬浮于细胞中进行电镀。
    3. 将50μL的细胞溶液(10000个细胞)加入到100μL的I型大鼠尾胶原的60:40比例中,在冰上生长因子还原MatMatel.用200μl吸管缓慢上下吸管,轻轻混合。< BR> 注:>
      1. 首先是胶原,然后是Matrigel。在添加细胞以避免气泡之前,不要与吸管混合。>
      2. 避免快速吸入或悬浮校准的基质混合物,以避免形成气泡。>
    4. 沉积细胞/胶原/基质胶混合在冰上24孔组织培养板的中央,在37°C孵育20分钟。
    5. 加至1毫升分化培养基。
    6. 每2-3天更换一次介质。
    7. 当细胞在基质内达到最大汇合,基质开始分解时,培养物维持长达14天。
    8. 从培养的第5天开始可以看到类器官的形成(图1)。
      < BR> < IMG类=“DoOtStWh”Src=“//附/图像/ 20190925/209925211547×7072 .jpg”宽度=“208”高度=“100”ALT=“//> BR/> 图1。胶原MatRIL嵌入的SIMS细胞在第5天(A)和7(B)下扩张并形成类器官。鳞条,200μm(4X放大)。< BR>
      < BR>
  3. 类器官固定
    1. 冰冷1X PBS Ca>2 - Mg 23次。
    2. 在1x PBS Ca+2 - Mg+2在ICE或4°C下,自由时间为20分钟时,细胞可以在任何时间点固定2%个PFA。
    3. 用冰冷1X PBS Ca>2 - Mg>SUP> 23次。
    4. 在1X PBS Ca~(2) - Mg + 2 孵育基质10分钟,重复3次,共30分钟。
    5. 用镊子轻轻地从组织培养板上取出基质。< BR> 注意:如果需要多个冷冻块,可以用细刀片将基质切成2-3片。>
    6. 嵌入oct(有关嵌入的完整说明,请参见Campbell等人,2011年)。 注:如果浇得太快,10月份基质可能会卷曲。轻轻地倒,并用镊子固定任何卷边。>
    7. 最佳切削厚度在12~16μm之间;
    8. 切片可以用4% PFA、冰冷丙酮或丙酮/甲醇固定后进行染色优化。< BR>
      < BR>
  4. 三维基质染色< BR> 为了保存三维形态,在基质中嵌入1 mm×SUP>3有机物。
    1. 固定后,使用锋利的解剖刀刀片和尖镊子切割包括有机体在内的小块矩阵。在塑料盘或24孔板中分别处理每一块。
    2. 如有必要,可用2-4%的pfa、丙酮/甲醇或甲醇处理基质。
    3. 基质像组织切片一样进行染色。Athwal等人于2019年介绍了一个例子。
    4. 根据矩阵的大小将每个样品安装在玻璃片上,间隔1-2个间隔。
    5. 使用共焦显微镜捕获图像(图2)。
    6. 这种染色也可在OCT嵌入的类器官基质的冷冻切片上进行(AthWal等。>2019)。图2中的一级抗体和二级抗体的完整描述可以在上述手稿中找到。 < BR> < IMG类=“DoOtStWh”Src=“//附/图像/ 20190925 /209925211617Y2424.jpg”宽度=“129”高度=“100”ALT=“”/> BR/> 图2。1 mm 3胶原/Matrix基质,用E-cadherin固定4%个PFA后,用E-cadherin染色,< <强>鳞条,10μm < BR>

数据分析

从第3天开始拍摄布莱特菲尔德的照片,直到实验结束。使用imagej可以量化器官直径。每个实验重复3次,每天N=5幅图像进行定量。统计分析的细节可以在我们以前的手稿中找到(AthWal[EM>等, >,2019)。

笔记

  1. 当细胞在基质中融合时,在第14天细胞死亡。
  2. 从类有机物中分离出来的细胞没有用sims细胞类有机物进行连续传代。
  3. 如果细胞数目增加,则增加胶原/基质胶的体积。
  4. 在进行三维培养分析之前,选择导入或转染的sims细胞。< BR>

食谱

  1. sims继代培养基(表1)
    < BR> 表1。sims亚培养基 < IMG类=“DoOtStWh”Src=“//附/图像/ 20190925/209925211958227.7.JPG”宽度=“600”高度=“188”ALT=“”/> BR/> < BR>
  2. 分化培养基
    < BR> 表2。sims分化培养基 < IMG类=“DoOtStWh”Src=“//附/图像/ 20190925/20992525212021201201.JPG”宽度=“600”高度=“350”ALT=“”/> BR/> 注意:>
    1. 制备100μl等份生长因子还原基质凝胶,并在-20°C下储存。>
    2. 大鼠尾胶原100μl等分试样,保存于4°C >

致谢

这项工作得到了美国国立卫生研究院(NIH)/美国国立牙科和颅面研究院(NIDCR)的资助,资助项目为DE027034和DE022557。该协议适用于Maimet 等. >,2016。我们的方案描述了使用sims细胞形成有机物的时间线和培养基配方的变化。

竞争利益

没有利益冲突。

推荐信

  1. 阿斯瓦尔,H. K.,墨菲,G,第三,蒂布斯,E,CalNETT,A,Hill,E,Yeoh,K.,BelEn斯坦,E,霍夫曼,M. P.和LoBaBet,I. M. A.(2019)。SOX10调节上皮祖细胞向外分泌腺分泌单位的可塑性。干细胞报告>12(2):366-380。
  2. Aure,M.H.,Konieczny,S.F.和Ovitt,C.E.(2015年)。通过腺泡细胞自我复制维持唾液腺的稳态。dev cell>33(2):231-237。
  3. Campbell,J.J.,Davidenko,N.,Cafferel,M.M.,Cameron,R.E.和Watson,C.J.(2011)。< HREF=“HTTP//NCBI.NLM.NIH.GOV/PUMEDME/21982 437”目标=“空白”>一个多功能三维共培养系统用于乳腺组织形态发生和干细胞生物学研究。 EM:PLOS1>6(9):E25661。
  4. 劳德,B. M.,库蒂,Y,GASTIN,I,Sybut,C.,克勒曼,O.和Rueeon,F(1996)。< HeRF=“HTTP//NCBI.NLM.NIH.GOV/PUBMED/9013327”靶=“亚空白”>永生化小鼠颌下腺上皮细胞系保留极化结构和功能特性。 E.J细胞SCI>109(PT 12):27 89~28 00。
  5. 伦巴特,I. M.,Brunsting,J. F.,Wierenga,P. K.,FabER,H.,Stokman,M. A.,Ko.T,维瑟,Pig,Y,Ye,de Haon,Yi和CoppS,(2008)。HREF=“HTTP//NCBI.NLM.NIH.GOV/PUBMED/1844 6241”靶=“空白”>干细胞移植后唾液腺功能的损伤。 EM > PLOS1>3(4):E2063。
  6. 伦巴特,I. M.,诺克斯,S. M.和霍夫曼,M. P.(2011)。< HREF=“HtpF//www. NcB.NLM.NIH.GOV/PUBMED/21223 445”目标=“空白”>唾液腺祖细胞生物学为治疗性唾液腺再生提供理论依据。 EME>口腔DIS>17(5):44~499。
  7. Maimets,M.,Rocchi,C.,Bron,R.,Pringle,S.,Kuipers,J.,Giepmans,B.N.,Vries,R.G.,Clevers,H.,de Haan,G.,Van OS,R.和Coppes,R.P.(2016年)。体外长期由wnt信号驱动的唾液腺干细胞的扩增。干细胞报告>6(1):150-162。
  8. 帕特尔,R和Alahmad,A. J.(2016)。生长因子减少的基质凝胶来源影响干细胞衍生的脑微血管内皮细胞屏障特性。液体屏障cns>13:6。
  9. Song,E.C.,Min,S.,Oyelakin,A.,Smalley,K.,Bard,J.E.,Liao,L.,Xu,J.和Romano,R.A.(2018年)。基因和scrna序列分析揭示了有助于唾液腺发育和维持的不同细胞群。sci rep>8(1):14043。
  10. Weng,P.L.,Aure,M.H.,Maruyama,T.和Ovitt,C.E.(2018年)。一个HREF=“HTPF//NCBI.NLM.NIH.GOV/PUMEDME/300 89258”目标=“α空白”>严重损伤后成人唾液腺的有限再生包括细胞可塑性。 EME>细胞RP>24(6):1464-1470 E3。
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引用:Athwal, H. K. and Lombaert, I. M. A. (2019). 3D Organoid Formation from the Murine Salivary Gland Cell Line SIMS. Bio-protocol 9(19): e3386. DOI: 10.21769/BioProtoc.3386.
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