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Sep 2017

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Total RNA Isolation from Separately Established Monolayer and Hydrogel Cultures of Human Glioblastoma Cell Line
单层水凝胶培养人胶质母细胞瘤细胞系的全RNA分离   

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Abstract

Astrocytoma is an invasive carcinoma occurring in the nervous system and currently lacks effective treatment options. A deeper understanding of the mechanisms of tumorigenesis and tumor progression is needed in order to develop novel therapeutic strategies. Recent advances in in vitro culture systems have demonstrated that the use of three-dimensional (3D) culture models could be more relevant for this purpose as compared to monolayer or two-dimensional (2D) models due to their resemblance to in vivo cancer pathology. High-throughput techniques such as RNA sequencing, microarray analyses and cloning could provide useful insights into the relevance of these systems to the native tissue. Previous studies have reported RNA extraction protocols needed for such applications. We have modified these protocols to suit the isolation of total RNA from monolayer and hydrogel cultures of astrocytoma established using basement membrane matrix, GeltrexTM. We have used this method to demonstrate the differences in the expression of genes involved in autophagy, a process deregulated in many cancer types, in monolayer and hydrogel cultures using quantitative polymerase chain reaction (qPCR). This protocol can be adopted by the researchers who wish to understand the molecular basis of gene expression in hydrogel cultures of normal as well as cancer cell lines.

Keywords: RNA (核酸), Monolayer (单层), 3D (3D), Hydrogel (水凝胶), Glioblastoma (胶质母细胞瘤), Cancer (癌症), TRIzol (TRIzol), Cell recovery solution (细胞回收液), Astrocytoma (星形细胞瘤)

Background

Astrocytomas are central nervous system tumors of glial origin. Poor survival rates among patients within 5 years of diagnosis and almost inevitable relapse are the hallmarks of this life-threatening disease (Krex et al., 2007). Our understanding of disease pathogenesis is limited, resulting in the lack of effective intervention strategies. In vitro culture systems could prove useful for studying cell-cell interactions and intracellular signaling events in astrocytoma, and provide platforms for testing potential therapeutic compounds (Reardon et al., 2006; Baker and Chen, 2012). Three-dimensional (3D) in vitro culture platforms have especially gained popularity in recent years due to their biological relevance to in vivo tumors (Li et al., 2007; Whiteside, 2008; Tibbitt and Anseth, 2009; Xu et al., 2014; Jogalekar and Serrano, 2018). 3D cultures established using various tissue engineering scaffolds (e.g., hydrogels) allow for multi-layered growth of cells in a fashion similar to that of native tissue, unlike two-dimensional (2D) cultures, making them better predictors of responses to drug candidates.

Hydrogel culture systems could be exploited in parallel with monolayer cultures to further our understanding of cell-cell and cell-matrix interactions with high-throughput applications that analyze cellular gene expression, such as RNA-sequencing, microarray analysis and cloning. To accomplish this, a pure, intact, high quality RNA needs to be isolated in parallel from separately cultured monolayer and hydrogel cultures so that both cultures are processed under identical conditions and are therefore comparable. Several protocols have been reported for extracting RNA from 3D cultures established using a variety of commonly used tissue engineering scaffolds such as Matrigel and collagen (Lee et al., 2007; Mroue and Bissell, 2013). However, there is a paucity of information available with respect to RNA extraction from 3D cultures maintained in GeltrexTM, a relatively new basement membrane matrix derived from murine Engelbreth-Holm-Swarm tumors and manufactured by Invitrogen.

When we used existing protocols to carry out RNA isolation from multiple cell lines including astrocytoma, we found that our astrocytoma cell line (CCF-STTG1) was highly sensitive to the reagents that dissolve extracellular matrices used in 3D cultures and could not yield an intact RNA at the end of the procedure, although this procedure worked well with other mammalian and amphibian cell lines we used in our experiment. Therefore, we modified RNA isolation protocols available in the literature to extract total RNA from monolayer and hydrogel cultures of astrocytoma. We used this RNA to carry out gene expression analyses for key autophagy pathway genes, Beclin-1 (BECN1) and microtubule associated protein 1 light chain 3 beta (MAP1LC3B), using quantitative polymerase chain reaction (qPCR) (Jogalekar et al., 2017). Our findings indicate a modest upregulation of these genes in hydrogel cultures of astrocytoma and are consistent with previous reports suggesting their upregulation in correlation with poor prognosis in glioma patients (Pirtoli et al., 2009; Giatromanolaki et al., 2014).

Materials and Reagents

  1. 0.8 μm 150 ml filter unit (Thermo Scientific Nalgene, catalog number: 125-0080)
  2. NuncTM Cell Culture Treated Flasks with Filter Caps (NuncTM, catalog number: 136196)
  3. Falcon® Cell Scrapers, Sterile (Corning®, catalog number: 353085)
  4. Falcon® 50 ml High Clarity PP Centrifuge Tubes (Corning®, catalog number: 352098) 
  5. RNase-free Microfuge Tubes (1.5 ml) (InvitrogenTM, catalog number: AM12400)
  6. 5cc BD Luer-LokTM Disposable Syringe (BD, catalog number: 309603)
  7. 21 G 1¼ needle (BD, catalog number: 305166)
  8. 10 ml serological pipette, sterile (USA Scientific, catalog number: 1071-0810)
  9. FisherbrandTM SureOneTM Aerosol Barrier Pipette Tips (FisherbrandTM SureOneTM, catalog numbers: 02-707-404 [1,000 μl], 02-707-430 [200 μl], 02-707-432 [20 μl])
  10. CCF-STTG1 cell line (ATCC, catalog number: CRL-1718)
  11. RPMI-1640 Medium (ATCC, catalog number: 30-2001)
  12. Fetal Bovine Serum (FBS) (ATCC, catalog number: 30-2020)
  13. LDEV-Free Reduced Growth Factor Basement Membrane Matrix, GeltrexTM (Invitrogen, catalog number: A1413202)
  14. 70% Ethanol
  15. RNaseZapTM RNase Decontamination Solution (AmbionTM, catalog number: AM9780)
  16. TRIzolTM Plus RNA Purification Kit (AmbionTM, catalog number: 12183555)
  17. Cell Recovery Solution (Corning, catalog number: 354253)
  18. Phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: P3813) 
  19. Chloroform (Sigma-Aldrich, catalog number: C2432)
  20. Nuclease-Free Water (not DEPC-Treated) (AmbionTM, catalog number: AM9937)
  21. DNA-free DNA Removal Kit (AmbionTM, catalog number: AM1906)
  22. RNA 6000 Nano Kit (Agilent, catalog number: 5067-1511)
  23. Ethyl alcohol, Pure (Sigma-Aldrich, catalog number: E7023)
  24. RPMI-1640 complete medium (see Recipes)
  25. Sterile 1x PBS (see Recipes)

Equipment

  1. GilsonTM PIPETMAN ClassicTM Pipettes (Gilson, catalog numbers: F123600 [P20], F123601 [P200], F123602 [P1000], F144802 [P10], F144801 [P2])
  2. 2100 bioanalyzer instrument (Agilent, catalog number: G2939BA)
  3. Drummond Pipet-Aid, Plain, 110V (Drummond, catalog number: DP-110)
  4. Biosafety cabinet (Nuaire, catalog number: NU-425-600)
  5. 4 °C centrifuge (Beckman Coulter, catalog number: AllegraTM 21R) 
  6. Centrifuge rotors
  7. Water bath at 37 °C (Sheldon Manufacturing Inc., catalog number: 1211)
  8. AutoFlow Water Jacket CO2 Incubator (Nuaire, catalog number: NU-4750) 
  9. -20 °C freezer

Software

  1. 2100 Expert (Agilent)
  2. Photoshop CS6 (Adobe)

Procedure

  1. Cell culture
    1. Establish 2D and 3D cultures separately, of CCF-STTG1 cells using RPMI-1640 complete medium. For 3D culture, coat T25 flasks with 100 µl per cm2 GeltrexTM and allow it to solidify in a 37°C incubator for 30 min. Plate CCF-STTG1 cells on top of the solidified GeltrexTM at a density of 5 x 104 cells per cm2. For monolayer or 2D culture, plate equal number of cells directly in T25 flasks (do not coat these flasks with matrix prior to the addition of cells). Incubate the flasks at 37 °C and 5% CO2.
    2. Aspirate the old media and add fresh media to culture flasks every other day. Monitor the cell growth periodically.

  2. RNA isolation
    Note: Make sure that all the supplies used for the RNA isolation such as centrifuge tubes, pipette tips, plastic pipettes and microcentrifuge tubes are sterile, and DNase- and RNase-free.
    1. Begin RNA isolation when the cells in 2D and 3D cultures are ~80-90% confluent (Figure 1). This usually takes about a week, although it may vary with the cell and culture type. Process both culture flasks on the same day in parallel.


      Figure 1. Phase contrast images showing 80-90% confluent CCF-STTG1 cells in monolayer and hydrogel cultures. CCF-STTG1 cells were allowed to grow in A) Monolayer and B) Hydrogel cultures until they reached 80-90% confluency before beginning RNA extraction procedure.

    2. Treat all surfaces with ethanol followed by RNase ZAP prior to UV-including both centrifuge rotors, pipettes, and bench surfaces outside the tissue culture room.
    3. Turn on the centrifuge so it cools to 4 °C.
    4. Turn on and set the water bath at 37 °C.
    5. UV-sterilize the tissue culture hood and the required materials for 30 min before beginning the RNA extraction procedure. Also, have cold 1x PBS ready (for use with cell recovery solution). 
    6. Without removing the media, detach cells from the bottom of the 2D and 3D culture flasks using cell scrapers and transfer them to 50 ml centrifuge tubes. Centrifuge for 5 min at 200 x g at 4 °C. Trypsin was not used in this procedure since it alters gene expression (Chaudhry, 2008).
      Note: Cells can be lysed with TRIzol® reagent in a culture dish or flask directly if the dishes are made of materials that are compatible with TRIzol® reagent. Please check the product details and their compatibility before doing so. 
    7. Decant the supernatant from the tube containing 3D cells. Please make sure to leave equal volume of media in the other tube containing monolayer cell pellet, since these tubes will be processed in parallel under identical conditions.
    8. Separate the astrocytoma cells from the basement membrane matrix with one of the two ways–using TRIzol or cell recovery solution. The method of choice is determined by analyzing the sensitivity of cell lines of interest to both matrix-dissolving reagents (by trial and error method). We found that the cell recovery solution worked well with some cell lines (e.g., HCC70 cell line, Figure 2B), while others were found to be sensitive and died upon exposure (e.g., CCF-STTG1 cell line, Figure 2A). Therefore, we decided to use TRIzol for these cell lines. Since 2D and 3D cultures must be processed identically, 2D or monolayer cultures were also exposed to equal volumes of TRIzol or cell recovery solution although they do not contain matrix.


      Figure 2. Electropherograms showing the quality of total RNA extracted from monolayer and hydrogel cultures. The total RNA was extracted from monolayer and hydrogel cultures of astrocytoma cell line, CCF-STTG1. One microliter of total RNA per sample was analyzed using Agilent Bioanalyzer. A. RNA isolated from CCF-STTG1 cells using TRIzol (Top panel) and cell recovery solution (Bottom panel). B. RNA extracted from HCC70 (breast cancer cell line) cells used as control to show that cell recovery solution works well for HCC70 cells, but not for CCF-STTG1 cells. a: 18s peak, b: 28s peak.

      1. Using TRIzol reagent:
        1. Add TRIzol® reagent to both tubes so that the sample volume is 10% of the total volume and incubate the samples for 5 min. For example, if the sample volume is 1 ml, then 9 ml of TRIzol should be added. 
        2. Lyse cells by passing them through 21 G x 1¼" needle and 5cc syringe at least 5-6 times.
        3. After cell lysis, add 0.2 ml of chloroform per ml of TRIzol® reagent, invert the tubes to mix the contents for 15 s followed by the incubation for 2-3 min at room temperature. 
        4. Centrifuge at 15,000 x g for 15 min at 4 °C.
        5. Transfer colorless upper phase to the RNase-free tube. The volume of colorless upper phase varies depending upon the volumes of sample and TRIzol/chloroform added in previous steps. For example, in our case, the volume of matrix containing cells was 2.8 ml, so we added 2.8 x 9 ml = 25.2 ml of TRIzol plus 0.2 x 25.2 ml = 5.04 ml of chloroform. So the total volume of the solution was ~33 ml. Out of that, the volume of colorless upper phase came out to be ~50%, i.e., 18 ml (Figure 3).
        6. Add 70% ethanol (equal volume) to a final concentration of 35%. Use absolute or molecular biology grade alcohol to make 70% ethanol to reduce the chances of contamination.
        7. Mix well by inverting the tube to disperse precipitate.


          Figure 3. Colorless upper phase separated after centrifugation step. Followed by incubation with TRIzol and chloroform, the monolayer and hydrogel samples of CCF-STTG1 were centrifuged for 15 min at 15,000 x g at 4 °C to obtain colorless upper phase which contains RNA.

      2. Using cell recovery solution:
        1. Wash cells thrice with cold 1x PBS and add equal amounts of cell recovery solution (227µl per cm2) directly to T25 flasks. 
        2. Scrape the cells using cell scrapers and transfer to a cold 50 ml centrifuge tube. 
        3. Incubate on ice till GeltrexTM is dissolved (up to 1 h, varies for each cell line). Mix the suspension by inverting the tubes every 15 min.
          Note: Please pay attention to exposure times for cell recovery solution used to dissolve GeltrexTM. Some cell lines may be more sensitive to this reagent than others. Optimize treatment times for your cell line before conducting a major experiment.
        4. When the cells settle down at the bottom of the tube, centrifuge the mixture at 200 x g at 4 °C for 5 min.
        5. Wash cells in cold 1x PBS twice. 
    9. Proceed with RNA extraction using the manufacturer’s (Ambion) protocol for isolating total RNA from the animal cells.
    10. Use DNA-freeTM kit to remove DNA contamination and store pure RNA at -80 °C until use. 
    11. Check the quality of extracted RNA integrity with Agilent bioanalyzer (Figure 2).
      Note: The samples with RNA integrity number (RIN) > 8 were considered pure and high quality, and used for experiments. If RNA concentration in the sample is less than 25 ng/μl, Agilent software may not show RIN values (Mueller et al., 2004). In that case, it is essential to determine RNA concentration using another method like Nanodrop. It is also important to confirm RNA integrity by visually inspecting electropherograms provided by Agilent Software even though RIN values are not displayed.

Data analysis

  1. Determine the RNA quality using Agilent bioanalyzer 2100 and RNA 6000 Nano Kit. 
  2. Load RNA samples on the chip using the manufacturer’s (Agilent) instructions and read the chip using 2100 Bioanalyzer Expert Software (Figure 2).

Recipes

  1. RPMI-1640 complete medium
    RPMI 1640 basal medium
    10% FBS
    1. Mix both ingredients and filter sterilize the medium through a 0.8 μm filter unit
      Note: We do not use antibiotics for cell culture in our laboratory but you can add them to the medium at the appropriate concentration (usually 1% of the total volume) depending upon your requirements.
    2. Store at 4 °C and use within a month
  2. Sterile 1x PBS
    1. Dissolve the powdered contents from one pouch of PBS to 1,000 ml of distilled water to get 1x PBS, pH 7.4
    2. Autoclave the solution and store at room temperature

Acknowledgments

We are grateful for the funding support provided by the New Mexico State University Manasse Chair Endowment to E. E. Serrano, and the National Institutes of Health (P50GM68762) grant. We thank V. B. Knight for providing technical assistance for this work. This protocol was modified from previously published RNA extraction methods (Lee et al., 2007; Mroue and Bissell, 2013).

Competing interests

The authors declare no competing interests.

References

  1. Baker, B. M. and Chen, C. S. (2012). Deconstructing the third dimension: how 3D culture microenvironments alter cellular cues. J Cell Sci 125(Pt 13): 3015-3024.
  2. Chaudhry, A. M. (2008). Induction of gene expression alterations by culture medium from trypsinized cells. J Biol Sci 8(1): 81-87.
  3. Giatromanolaki, A., Sivridis, E., Mitrakas, A., Kalamida, D., Zois, C. E., Haider, S., Piperidou, C., Pappa, A., Gatter, K. C., Harris, A. L. and Koukourakis, M. I. (2014). Autophagy and lysosomal related protein expression patterns in human glioblastoma. Cancer Biol Ther 15(11): 1468-1478.
  4. Jogalekar, M. P., Cooper, L. G. and Serrano, E. E. (2017). Hydrogel environment supports cell culture expansion of a grade IV astrocytoma. Neurochem Res 42(9): 2610-2624.
  5. Jogalekar, M. P. and Serrano, E. E. (2018). Morphometric analysis of a triple negative breast cancer cell line in hydrogel and monolayer culture environments. PeerJ 6: e4340.
  6. Krex, D., Klink, B., Hartmann, C., von Deimling, A., Pietsch, T., Simon, M., Sabel, M., Steinbach, J. P., Heese, O., Reifenberger, G., Weller, M., Schackert, G. and German Glioma, N. (2007). Long-term survival with glioblastoma multiforme. Brain 130(Pt 10): 2596-2606.
  7. Lee, G. Y., Kenny, P. A., Lee, E. H. and Bissell, M. J. (2007). Three-dimensional culture models of normal and malignant breast epithelial cells. Nat Methods 4(4): 359-365.
  8. Li, H., Fan, X. and Houghton, J. (2007). Tumor microenvironment: the role of the tumor stroma in cancer. J Cell Biochem 101(4): 805-815.
  9. Mroue, R. and Bissell, M. J. (2013). Three-dimensional cultures of mouse mammary epithelial cells. Methods Mol Biol 945: 221-250.
  10. Mueller, O., Lightfoot, S. and Schroeder, A. (2004). RNA integrity number (RIN)–standardization of RNA quality control. Agilent application note: 5989-1165EN.
  11. Pirtoli, L., Cevenini, G., Tini, P., Vannini, M., Oliveri, G., Marsili, S., Mourmouras, V., Rubino, G. and Miracco, C. (2009). The prognostic role of Beclin 1 protein expression in high-grade gliomas. Autophagy 5(7): 930-936.
  12. Reardon, D. A., Rich, J. N., Friedman, H. S. and Bigner, D. D. (2006). Recent advances in the treatment of malignant astrocytoma. J Clin Oncol 24(8): 1253-1265.
  13. Tibbitt, M. W. and Anseth, K. S. (2009). Hydrogels as extracellular matrix mimics for 3D cell culture. Biotechnol Bioeng 103(4): 655-663.
  14. Whiteside, T. L. (2008). The tumor microenvironment and its role in promoting tumor growth. Oncogene 27(45): 5904-5912.
  15. Xu, X., Sabanayagam, C. R., Harrington, D. A., Farach-Carson, M. C. and Jia, X. (2014). A hydrogel-based tumor model for the evaluation of nanoparticle-based cancer therapeutics. Biomaterials 35(10): 3319-3330.

简介

星形细胞瘤是一种在神经系统中发生的浸润性癌,目前缺乏有效的治疗选择。为了开发新的治疗策略,需要更深入地了解肿瘤发生和肿瘤进展的机制。培养系统已经证明,与单层或二维(2D)模型相比,三维(3D)培养模型的使用可能与此目的更相关,因为它们与体内癌症相似RNA测序,微阵列分析和克隆等高通量技术可以为这些系统的相关性提供有用的见解。以前的研究已经报道了这些应用所需的RNA提取方案。我们使用从基底膜基质Geltrex TM 建立的星形细胞瘤的单层和水凝胶培养物中的总RNA中分离。该协议可以被研究人员采用.qqqq。正常和癌细胞系的水凝胶培养物中基因表达的分子基础。
【背景】诊断后5年内患者的生存率较低且几乎不可避免的复发是这种威胁生命的疾病的标志(Krex et al。,2007)。 体外培养系统能够为研究星形细胞瘤中的细胞 - 细胞相互作用和细胞内信号传导事件提供有用的,并且为理解疾病发病机制的认识提供平台是有限的,导致疾病发病机制有限。化合物(Reardon et al。,2006; Baker and Chen,2012)。近年来,三维(3D)体外培养平台因其种类特别受欢迎与体内肿瘤的生物学相关性(Li et al。,2007; Whiteside,2008; Tibbitt和Anseth,2009; Xu et al。, 2014; Jogalekar和Serrano,2018)。使用各种组织工程支架(例如,水凝胶)建立的3D培养物s)与二维(2D)培养物不同,允许以类似于天然组织的方式进行细胞的多层生长,使其成为对候选药物反应的更好预测因子。

水凝胶培养系统可以与单层培养物同时进行探索,以进一步了解细胞 - 细胞和细胞 - 基质相互作用,分析细胞基因表达的高通量应用,如RNA测序,微阵列几种方案对具有独特品质的需要的性质有独特的理解,并且需要从单独的培养物和单层和水凝胶培养物中平行分离高质量的RNA。 Muroe和Bissell(Lee et al。,2007; Mroue and Bissell,2013)。然而,关于从3D培养物中提取RNA的信息很少维持在GeltrexTM中,这是一种相对较新的基底膜基质,来自小鼠Engelbreth-Holm-Swarm肿瘤,由Invi制造特罗根。

(CCF-STTG1)对来自细胞系(CCF-STTG1)的干扰因子高度敏感时,现有的方案从多种细胞系中进行细胞分离,包括星形细胞瘤,多发现细胞RNA分离方案的修改可用于从单层和水凝胶中提取总RNA,以及在该方法中使用的其他哺乳动物和两栖动物细胞系。该RNA利用定量聚合酶对关键自噬途径基因Beclin-1( BECN1 )和微管相关蛋白1轻链3β( MAP1LC3B )进行基因表达分析连锁反应(qPCR)(Jogalekar et al。,2017)。我们的研究结果表明,这些基因在星形细胞瘤的水凝胶培养物中适度上调,并且与以前的报道暗示一致。继发性上调与胶质瘤患者预后不良相关(Pirtoli et al。,2009; Giatromanolaki et al。,2014)。

关键字:核酸, 单层, 3D, 水凝胶, 胶质母细胞瘤, 癌症, TRIzol, 细胞回收液, 星形细胞瘤

材料和试剂

  1. 0.8μm150ml过滤装置(Thermo Scientific Nalgene,目录号:125-0080)
  2. Nunc TM 细胞培养处理过滤盖的烧瓶(Nunc TM ,目录号:136196)
  3. Falcon <®>®细胞刮刀,无菌(Corning®,目录号:353085)
  4. Falcon ® 50 ml高净度PP离心管(Corning ®,目录号:352098)&nbsp;
  5. RNase-free Microfuge Tubes(1.5 ml)(Invitrogen TM ,目录号:AM12400)
  6. 5cc BD Luer-LokTM一次性注射器(BD,目录号:309603)
  7. 21 G 11/4针(BD,目录号:305166)
  8. 10 ml血清移液管,无菌(USA Scientific,目录号:1071-0810)
  9. Fisherbrand TM SureOne <<>气溶胶屏障移液器吸头(Fisherbrand TM SureOne TM ,目录号:02-707-404 [1,000μl],02-707-430 [200μl],02-707-432 [20μl])
  10. CCF-STTG1细胞系(ATCC,目录号:CRL-1718)
  11. RPMI-1640 Medium(ATCC,目录号:30-2001)
  12. 胎牛血清(FBS)(ATCC,目录号:30-2020)
  13. LDEV-Free Reduced Growth Factor Basement Membrane Matrix,GeltrexTM(Invitrogen,目录号:A1413202)
  14. 70%乙醇
  15. RNaseZapTMTM RNase去污解决方案(AmbionTM,目录号:AM9780)
  16. TRIzol TM Plus RNA纯化试剂盒(Ambion TM ,目录号:12183555)
  17. 细胞恢复解决方案(康宁,目录号:354253)
  18. 磷酸盐缓冲盐水(PBS)(Sigma-Aldrich,目录号:P3813)&nbsp;
  19. 氯仿(Sigma-Aldrich,目录号:C2432)
  20. 无核酸酶水(未经DEPC处理)(Ambion TM ,目录号:AM9937)
  21. 无DNA DNA去除试剂盒(Ambion TM ,目录号:AM1906)
  22. RNA 6000 Nano Kit(安捷伦,产品目录号:5067-1511)
  23. 乙醇,Pure(Sigma-Aldrich,目录号:E7023)
  24. RPMI-1640完全培养基(见食谱)
  25. 无菌1x PBS(见食谱)

设备

  1. GilsonTM PETETMAN ClassicTM移液器(Gilson,目录号:F123600 [P20],F123601 [P200],F123602 [P1000],F144802 [P10],F144801 [P2] )
  2. 2100生物分析仪(安捷伦,目录号:G2939BA)
  3. Drummond Pipet-Aid,平原,110V(Drummond,目录号:DP-110)
  4. 生物安全柜(Nuaire,目录号:NU-425-600)
  5. 4°C离心机(Beckman Coulter,目录号:AllegraTM 21 R)&nbsp;
  6. 离心机转子
  7. 37°C水浴(Sheldon Manufacturing Inc.,目录号:1211)
  8. AutoFlow Water Jacket CO 2 孵化器(Nuaire,目录号:NU-4750)&nbsp;
  9. -20°C冰柜

软件

  1. 2100专家(安捷伦)
  2. Photoshop CS6(Adobe)

程序

  1. 细胞培养
    1. 对于使用RPMI-1640完全培养基的CCF-STTG1细胞分别进行2D和3D培养。对于3D培养,用100μl/ cm 2 Geltrex 涂覆T25烧瓶并允许将CCF-STTG1细胞置于固化的Geltrex TM 顶部,密度为5×10 4个细胞/ cm,在37℃培养箱中固化30分钟。对于单层或2D培养,直接在T25烧瓶中培养相同数量的细胞(在加入细胞之前用基质标记烧瓶)。在37℃和5%孵育烧瓶CO 2 。
    2. 定期监测细胞生长。
      每隔一天吸出旧培养基并在培养瓶中加入新鲜培养基。

  2. RNA分离
    注意:确保所有用于RNA分离的供应品,如离心管,移液器吸头,塑料移液管和微量离心管都是无菌的,不含DNase和RNase。
    1. 当2D和3D培养物中的细胞达到约80-90%汇合时开始RNA分离(图1)。这通常需要大约一周,尽管它可能随细胞类型和过程类型而变化。在同一天处理两个培养瓶平行。


      图1.相差图像显示单层和水凝胶培养物中80-90%汇合的CCF-STTG1细胞。允许CCF-STTG1细胞在A)单层和B)水凝胶培养物中生长直至达到80在开始RNA提取程序之前-90%融合。

    2. 在紫外线之前用乙醇处理所有表面,然后用RNase ZAP处理 - 包括离心机转子,移液管和组织培养室外的工作台表面。
    3. 打开离心机使其冷却至4°C。
    4. 打开并将水浴设置在37°C。
    5. 此外,准备冷1x PBS(用于细胞回收溶液)。在开始RNA提取程序之前,对组织培养罩和所需材料进行紫外线灭菌30分钟。
    6. 离心管。在4℃下在200 xg 下离心5分钟。使用转移刮刀并转移至50ml,使用细胞刮刀将胰蛋白酶包括在2D和3D培养瓶中。由于它改变了基因表达,因此未在此程序中使用(Chaudhry,2008)。
      注意:如果培养皿由材料制成,可以在培养皿或培养皿中直接用TRIzol ® 试剂裂解细胞。与TRIzol ® 试剂兼容。请在此之前检查产品详细信息及其兼容性。&nbsp;
    7. 请确保在含有单层细胞沉淀的另一个管中留下等体积的培养基,因为这些管将在相同条件下平行处理。
    8. 使用TRIzol或细胞回收溶液,用两种方法中的一种将星形细胞瘤细胞与基底膜基质分开。选择的方法通过分析两种基质与表面的目标细胞系来确定。我们发现细胞回收溶液对某些细胞系(例如,HCC70细胞系,图2B)效果很好,而其他细胞系被发现敏感并在暴露后死亡(例如<其中,CCF-STTG1细胞系,图2A。我们决定将TRIzol用于这些细胞系。由于2D和3D培养必须独特处理,2D或单层培养也暴露于等体积的TRIzol细胞。恢复解决方案,虽然它们不含矩阵。


      图2.显示从单层和水凝胶培养物中提取的总RNA质量的电泳图。从星形细胞瘤细胞系CCF-STTG1的单层和水凝胶培养物中提取总RNA。每个样品一微升总RNA A.使用TRIzol(上图)和细胞回收溶液(下图)从CCF-STTG1细胞分离的RNA。从用作对照的HCC70(乳腺癌细胞系)细胞中提取的RNA以显示回收溶液适用于HCC70细胞,但不适用于CCF-STTG1细胞.a:18s峰,b:28s峰。

      1. 使用TRIzol试剂:
        1. 将TRIzol®试剂加入两个管中,使样品体积为总体积的10%,样品中加入5分钟。例如,如果样品体积为1 ml,则加入9 ml TRIzol应该添加。&nbsp;
        2. 将细胞通过21 G x 11/4“针头和5cc注射器至少5-6次裂解细胞。
        3. 细胞裂解后,每毫升TRIzol®试剂加入0.2毫升氯仿,倒置试管混合内容物15分钟,然后在室温下孵育2-3分钟。
        4. 在4℃以15,000 x g 离心15分钟。
        5. 无色上层相的体积变为无RNase管。无色上相的体积根据样品的体积和前面步骤中添加的TRIzol /氯仿而变化。例如,在我们的情况下,含有2.8 ml细胞的基质的体积,所以我们加入2.8×9毫升= 25.2毫升TRIzol加0.2×25.2毫升= 5.04毫升氯仿。所以溶液的总体积为~33毫升。其中,无色上相的体积出现了〜 50%,即,18 ml(图3)。
        6. 加入70%乙醇(等体积)至终浓度为35%。使用绝对或分子生物级酒精制成70%乙醇,以减少污染的机会。
        7. 通过倒置管子以分散沉淀物来充分混合。


          图3.离心步骤后分离无色上层相。然后用TRIzol和氯仿孵育,CCF-STTG1的单层和水凝胶样品在15,000 xg 离心15分钟在4°C下获得含有RNA的无色上层相。

      2. 使用细胞回收方案:
        1. 用冷的1x PBS洗涤细胞三次,并将等量的细胞回收溶液(227μl/ cm 2)直接加入T25烧瓶中。&nbsp;
        2. 使用细胞刮刀刮擦细胞并转移到冷的50ml离心管中。&nbsp;
        3. 每15分钟翻转一次,混合悬浮液。
          在冰上孵育直至GeltrexTM溶解(最多1小时,每个细胞系的品种)。 注意:请注意用于溶解Geltrex的细胞回收溶液的暴露时间 TM 。某些细胞系可能对在进行主要实验之前,优化细胞系的治疗时间。
        4. 当细胞在管底部沉降时,将混合物在200℃下在4℃下离心5分钟。
        5. 在冷的1x PBS中洗涤细胞两次。&nbsp;
    9. 使用制造商(Ambion)方案进行RNA提取,以从动物细胞中分离总RNA。
    10. 使用DNA-freeTM试剂盒去除DNA污染,并在-80°C下储存纯RNA直至使用。&nbsp;
    11. 使用Agilent bioanalyzer检查提取的RNA完整性的质量(图2)。
      注意:RNA完整性编号(RIN)> 8的样品被认为是纯的和高质量的,并用于实验。如果样品中的RNA浓度小于25 ng /μl,安捷伦软件可能不会显示RIN值(Mueller 等 ,2004)。在这种情况下,使用Nanodrop等其他方法确定RNA浓度至关重要。通过视觉确认RNA完整性也很重要。检查安捷伦软件提供的电泳图,即使没有显示RIN值。

数据分析

  1. 使用Agilent bioanalyzer 2100和RNA 6000 Nano Kit确定RNA质量。&nbsp;
  2. 使用制造商(安捷伦)的说明在芯片上加载RNA样品,并使用2100 Bioanalyzer Expert软件在芯片上读取(图2)。

食谱

  1. RPMI-1640完整介质
    RPMI 1640基础培养基
    10%FBS
    1. 混合两种成分并通过0.8μm过滤装置过滤灭菌培养基
      注意:我们的实验室不使用抗生素进行细胞培养,但您可以根据您的要求将其添加到适当浓度的培养基中(通常为总体积的1%)。
    2. 储存在4°C并在一个月内使用
  2. 无菌1x PBS
    1. 将粉末状内容物从一袋PBS溶解到1,000ml蒸馏水中,得到1x PBS,pH7.4
    2. 高压灭菌溶液并在室温下储存

致谢

感谢您对此项工作的帮助。此协议已从之前的健康与人文研究所进行了修改。公布的RNA提取方法(Lee et al。,2007; Mroue and Bissell,2013)。

竞争利益

作者宣称没有竞争利益。

参考

  1. 贝克,BM和Chen,CS(2012)解构第三维:.文化的微环境如何改变3D细胞提示。 J Cell Sci 125(Pt 13):3015-3024。
  2. 基因表达的改变的乔德里,AM(2008)。诱导培养基胰蛋白酶消化从细胞。 J Biol Sci 8(1):81-87。
  3. Giatromanolaki,A.,Sivridis,E.,Mitrakas,A.,Kalamida,D.,Zois,CE,海德尔,S.,Piperidou,C.,PAPP-A,A.,Gatter,KC,哈里斯,AL和Koukourakis,MI (2014)。自噬和在人脑胶质瘤溶酶体相关蛋白的表达模式。 Cancer Biol Ther 15(11):1468-1478。
  4. Jogalekar,MP,库珀,LG和拉诺,EE(2017)。水凝胶环境支持细胞培养物中扩展IV级星形细胞瘤。 Neurochem Res 42(9):2610-2624。
  5. Jogalekar,MP和拉诺,EE(2018)。一个三阴性乳腺癌细胞系的形态测定分析在水凝胶和单层培养环境中。 PeerJ 6:e4340。
  6. Krex,D.,Klink,B.,Hartmann,C.,von Deimling,A.,Pietsch,T.,Simon,M.,Sabel,M.,Steinbach,JP,Heese,O.,Reifenberger,G。, Weller,M.,Schackert,G。和German Glioma,N。(2007)。 Long-多形性胶质母细胞瘤的术语存活率。 脑 130(Pt 10):2596-2606。
  7. Lee,GY,Kenny,PA,Lee,EH和Bissell,MJ(2007)。三 - 正常和恶性乳腺上皮细胞的维度培养模型。 Nat Methods 4(4):359-365。
  8. Li,H.,Fan,X。和Houghton,J。(2007)。肿瘤微环境:肿瘤基质在癌症中的作用。 J Cell Biochem 101(4):805-815。
  9. Mroue,R。和Bissell,MJ(2013)。小鼠哺乳动物上皮细胞的三维培养方法Mol Biol 945:221-250。
  10. Mueller,O。,Lightfoot,S。和Schroeder,A。(2004)。 ) - RNA质量控制的标准化。安捷伦应用简报:5989-1165 ZH。
  11. Pirtoli,L.,Cevenini,G.,Tini,P.,Vannini,M.,Oliveri,G.,Marsili,S.,Mourmouras,V.,Rubino,G。和Miracco,C。(2009)。 href =“http://www.ncbi.nlm.nih.gov/pubmed/19556884"target =”_ blank“> Beclin 1蛋白在高级别胶质瘤中的预后作用。 自噬 5(7):930-936。
  12. Reardon,DA,Rich,JN,Friedman,HS和Bigner,DD(2006)。最新进展治疗恶性星形细胞瘤。 J Clin Oncol 24(8):1253-1265。
  13. Tibbitt,MW和Anseth,KS(2009)。 Hydrogels作为细胞外基质模拟3D细胞培养。 Biotechnol Bioeng 103(4):655-663。
  14. Whiteside,TL(2008)。肿瘤微环境及其在促进肿瘤生长中的作用。
  15. 基于水凝胶的肿瘤模型,用于评估基于纳米颗粒的癌症治疗方法。 Biomaterials 35(10):3319-3330。
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引用:Jogalekar, M. P. and Serrano, E. E. (2019). Total RNA Isolation from Separately Established Monolayer and Hydrogel Cultures of Human Glioblastoma Cell Line. Bio-protocol 9(14): e3305. DOI: 10.21769/BioProtoc.3305.
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