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3D Culture Protocol for Testing Gene Knockdown Efficiency and Cell Line Derivation
用于检测基因敲减效率和细胞系建立3D培养方法   

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Mauro Sbroggio' Mauro Sbroggio'
Enrico Patrucco Enrico Patrucco
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Cancer Research
Apr 2017

Abstract

Traditional 2D cell cultures with cells grown as monolayers on solid surface still represent the standard method in cancer research for drug testing. Cells grown in 2D cultures, however, lack relevant cell-matrix and cell-cell interactions and ignore the true three-dimensional anatomy of solid tumors. Cells cultured in 2D can also undergo cytoskeletal rearrangements and acquire artificial polarity associated with aberrant gene expression (Edmondson et al., 2014). 3D culture systems that better mimic the in vivo situation have been developed recently. 3D in vitro cancer models (tumorspheres) for studying cancer stem cells have gained increased popularity in the field (Weiswald et al., 2015). Systems that use matrix-embedded or encapsulated spheroids, spheroids cultured in hanging drops, magnetic levitation systems or 3D printing methods are already being widely used in research and for novel drug screening. In this article, we describe a detailed protocol for testing the effect of shRNA-mediated gene silencing on tumorsphere formation and growth. This approach allows researchers to test the impact of gene knockdown on the growth of tumor initiating cells. As verified by our lab, the protocol can be also used for isolation of 3D cancer cell lines directly from tumor tissues.

Keywords: 3D culture (三维培养), Tumorspheres (肿瘤球), Cancer cell line (癌细胞系), shRNA gene silencing (shRNA基因沉默)

Background

3D in vitro cancer cell models represent a bridge experimental method between cell lines and tumors grown in vivo (Pampaloni et al., 2007; Weiswald et al., 2015). 3D characters of solid tumors with heterogeneous access to nutrients or oxygen can only be effectively mimicked by 3D culture systems. In recent years, protocols for tumorsphere culture gained lot of interest. A tumorsphere can be described as a solid, spherical object created from a single progenitor or stem cell. For tumorspheres formation assays, cells are seeded and grown in serum-free media in ultra-low attachment plates (non-adherent conditions), which allows enrichment of cancer cells with stem/progenitor properties (Johnson et al., 2013). Tumorspheres generated from freshly isolated tumor tissue are of special interest in the field because cells from established cell lines typically differ from the primary tumor due to mutations and abnormalities gained during multiple rounds of in vitro passaging. Hereby, we present an optimized protocol for 3D culture-based primary tumor cell isolation and the use of 3D culture to assess the effect of gene silencing on the growth of tumor-initiation cells.

Materials and Reagents

  1. Eppendorf tube
  2. Pipette tips
  3. Vials
  4. Plastic bottles
  5. 50 ml Falcon tube
  6. Petri dishes with clear lid (Fisher Scientific, Fisherbrand, catalog number: FB0875712 )
  7. 6-well plates, Corning Costar Ultra-Low Attachment (Corning, catalog number: 3471 )
  8. 15 ml Falcon tubes (Corning, Falcon®, catalog number: 352196 )
  9. 70 µm cell filter (cell strainer - Corning, Falcon®, catalog number: 352350 )
  10. Ice
  11. Plastic pipette
  12. 24-well plates, Corning Costar Ultra-Low Attachment (Corning, catalog number: 3473 )
  13. 6-well plates (Corning, catalog number: 3506 )
  14. Serological pipettes
  15. Surgical razor blades (FisherBrand High Precision # 22 Style Scalpel Blade, Fisher Scientific, FisherbrandTM, catalog number: 12-000-161 )
  16. Sterile pipets (10 ml) (FisherBrand Sterile Disposable Standard Serological Pipets, Fisher Scientific, FisherbrandTM, catalog number: 13-678-14A )
  17. Cryovials (General Long-Term Storage Cryogenic Tubes 1 ml, Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 5000-1012 )
  18. (optional) Mission pLKO.1-puro-CMV-TurboGFP Positive control Transduction Particles (Sigma-Aldrich, catalog number: SHC003V )
  19. EDTA (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15575-020 )
  20. PBS pH 7.4 (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
  21. Trypsin EDTA (0.05%) (Thermo Fisher Scientific, GibcoTM, catalog number: 25300054 )
  22. SureEntry transduction reagent (QIAGEN, catalog number: 336921 )
  23. Clorox Bleach (Veritiv, catalog number: 30966 )
  24. Heparin Sodium Salt (Sigma-Aldrich, catalog number: H3149-10KU )
  25. DMEM/F12, Glutamax (Thermo Fisher Scientific, GibcoTM, catalog number: 10565018 )
  26. Penicillin/Streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15070063 )
  27. Fetal bovine serum (Thermo Fisher Scientific, GibcoTM, catalog number: 26140079 )
  28. B27 supplement 50x (serum free) (Thermo Fisher Scientific, GibcoTM, catalog number: 17504044 )
  29. bFGF – human Fibroblasts Growth Factor 147 basic (animal free) (Gemini Bio-Products, catalog number: 300-805P )
  30. EGF– Epidermal Growth Factor (human) (Gemini Bio-Products, catalog number: 300-110P )
  31. ROCK inhibitor (Y-27632) (STEMCELL Technologies, catalog number: 72304 )
  32. Heparin solution (STEMCELL Technologies, catalog number: 07980 )
  33. BD Matrigel Matrix Growth Factor Reduced (BD Biosciences, catalog number: 356230 ) or Cultrex® 3-D Culture MatrixTM Reduced Growth Factor Basement Membrane Extract (Trevigen, catalog number: 3445-001-01 )
    Notes:
    1. Aliquot Matrigel or Cultrex Matrix into single-use aliquots in a sterile hood into sterile Eppendorf tubes. We recommend 0.5 ml of Matrigel per Eppendorf tube. When combined with 1.5 ml of ice-cold media, this amount is sufficient for 3D culture using one well in a six-well plate.
    2. Use chilled pipettes and have Eppendorf tubes on ice during procedure.
    3. Store at -80 °C. Thaw on ice or in the refrigerator overnight before use.
  34. Hibernation medium (HibernateTM-A, Thermo Fisher Scientific, GibcoTM, catalog number: A1247501 )
  35. Collagenase IV (Sigma-Aldrich, catalog number: C8051-100MG or similar)
  36. Trypan Blue (TC-10, Bio-Rad Laboratories, catalog number: 1450013 )
  37. Antibiotic-Antimycotic (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 15240096 )
  38. Dispase (1 U/ml, STEMCELL Technologies, catalog number: 07923 )
  39. EDTA (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15575020 )
  40. Accutase (STEMCELL Technologies, catalog number: 07920 )
  41. DMSO (Sigma-Aldrich, catalog number: D2438-50ML )
  42. Ethanol (70% solution, Fisher Scientific, Fisher BioReagentsTM, catalog number: BP8201500 )
  43. Culture medium (see Recipes)
  44. CSC medium (see Recipes)

Equipment

  1. Centrifuge (temperature controlled, VWR® benchtop general purpose centrifuge) (VWR, catalog number: 10830-764 )
  2. Pipettes
  3. Forceps
  4. CO2 Cell incubator (BINDER, model: CB 160 )
  5. Biological Safety Level (BSL-2) laminar flow hood (Esco, EscoTechnologies, USA)
  6. TC20 Automated cell counter (Bio-Rad)
  7. Mr. Frosty Freezing Container (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 5100-0001 )
  8. FormaTM 7000 series Ultra Low Temperature Freezers (Thermo Fisher Scientific, Thermo ScientificTM, model: TFM#902 Series )
  9. Liquid nitrogen storage tank
  10. Nikon TE-2000E D-Eclipse Csi confocal microscope running Nikon Elements software (Nikon, model: Eclipse TE2000-E )

Software

  1. Nikon microscope operating software
  2. ImageJ or similar image processing software (https://imagej.nih.gov/ij/)
  3. GraphPad Prism 6.0 software from GraphPad Software, USA
    (https://www.graphpad.com/scientific-software/prism/)
  4. R software (R Core Team, 2015 R: A language and environment for statistical computing)

Procedure

To better mimic in vivo growth of tumor cells, we used a 3D culture system with extracellular matrix protein mixture for tumorspheres development from a single cell. For testing the effect of shRNA-mediated knockdown of a gene of interest on the growth of tumor cells in 3D culture, prepare cells transduced with virus encoding shControl RNA as well as an shRNA specific for a gene of interest.

Note: In the text, the words ‘spheroids’ and ‘tumorspheres’ are used as synonyms.

  1. Transduction of cells with virus encoding shRNA
    1. At the time of transduction, the confluence of the cells should be about 60-70%. Aspirate the medium, wash the plate with sterile PBS and detach cells with Trypsin. Stop the reaction with media containing FBS, centrifuge (300 x g, room temperature; 2 min) and wash the cell pellet with PBS containing SureEntry reagent. For one transduction, use no more than 50,000 cells. Transduce cancer cells in a sterile Eppendorf tube by direct mixing of virus stock solution with cell pellet, washed previously with sterile PBS (pH 7.4) with SureEntry reagent (the final concentration of SureEntry reagent in PBS should be 8 µg/ml). For transduction, use 10 µl of high titer virus (109 TU/ml) per 50,000 cells.
      Notes:
      1. Before mixing the pellet with virus solution, aspirate the supernatant leaving small amount (~10 µl) of PBS with SureEntry just above the cell pellet. Discard pipettes and vials containing residual amount of virus into a plastic bottle containing 10% Clorox bleach solution.
      2. To monitor the efficiency of transduction, control virus can be used (e.g., Mission pLKO.1-puro-CMV-TurboGFP Positive control Transduction Particle, High Titer).
      3. Transduction of cells is always performed in a Biological Safety Level (BSL-2) laminar flow hood. Use precautions for bloodborne human pathogens and decontaminate the area with 10% bleach.
    2. Using forceps carefully transfer the open Eppendorf tube containing the transduction mixture (cells and virus) into a 50 ml Falcon tube. Loosen the cap of Falcon tube to allow gas exchange during incubation. Transfer the Falcon tube containing the Eppendorf tube into an incubator (37 °C, 5% CO2) and incubate for 30 min.
    3. Remove cells from the incubator and transfer them to a sterile hood. Add sufficient amount of culture media (2 or 3 ml) and plate the cells into a 6-well plate or 10 cm Petri dishes.
    4. Change the medium next day (discard the aspirated medium into a bottle containing 10% bleach). Allow transduced cells to recover for 2-4 days (the time of recovery depends on confluency of cells).
    5. Add selection antibiotic at a recommended or pre-tested concentration to get rid of non-transduced cells.
    6. Keep selection antibiotic in culture medium for 12-14 days.
    7. Test the efficiency of gene knockown in transduced cells with appropriate methods – qPCR (mRNA level) or Western blot (protein level).

  2. 3D spheroid culture with cells embedded in ECM protein matrix (Matrigel)
    Note: Although more difficult and time consuming, 3D culture with cells/spheroids embedded in matrix is (in our lab) a preferred method for testing the effect of gene silencing on tumorsphere growth. Growing spheroids embedded in solid or semi-solid protein gel matrix do not have the tendency (or the chance) to clump (Figure 1), which makes quantitative analyses (e.g., counting the number of spheroids or measurement of spheroid diameter) difficult.


    Figure 1. Tumorspheres cultured in CSC media only (left panel, A) or embedded in Matrigel matrix (right panel, B). Note the tendency of tumorspheres to clump when grown in CSC media only. Scale bars represent 50 µm.

    1. Detach cells from 2D plates using Trypsin or other detachment solution for cells. Stop the reaction with media containing FBS and transfer cells from the plate into a 15 ml Falcon tube. Pellet the cells by centrifugation (2 min, 400 x g) at room temperature.
    2. Aspirate the supernatant and re-suspend the cell pellet in sterile PBS.
    3. Filter cell suspension with a 70 µm cell filter and count the number of cells manually or using automated cell counter.
      Note: The number of cells plated per well must be empirically determined for every type of cancer cells. We recommend starting with 5,000 cells/well (24-well plate) or 10,000 cells/well (6-well plate) and adjusting the cell number as needed (toward lower number of cells per well).
    4. Spin down the cells by centrifugation (2 min, 400 x g) at room temperature.
    5. Aspirate the supernatant and re-suspend the cell pellet in sterile culture media (abbreviated here as CSC medium) and place the Falcon tube with cells on ice to chill down.
    6. Pipet ice-cold CSC culture media up-and-down several times to chill the plastic pipette.
    7. Pipette ice-cold Growth Factor Reduced Matrigel (BD Biosciences, USA) or Cultrex Reduced Growth Factor Basement Membrane Extract, PathClear (Trevigen, USA) on ice. With chilled pipette, transfer Matrigel into the vial containing ice-cold culture media and cells. Use at least 1:3 ratio (1 part of Matrigel with 3 parts of ice-cold media with cells).
    8. Mix gently, avoid making bubbles.
    9. Plate the mixture on an ultra-low attachment surface culture plate (Corning, USA) and transfer to the incubator.
    10. Next day, gently add CSC culture media to cover solidified matrix:media with cells. Add fresh media every 3-5 days.
      Note: Aspirate the old media with pipette, not with the vacuum pump (there is a risk of aspiration of Matrigel with spheroids).

  3. 3D spheroid culture (basic, without ECM protein matrix)
    1. Pellet cells by centrifugation at 300 x g for 2 min (at room temperature) and aspirate the supernatant.
    2. Mix cell pellet gently with medium (abbreviated here as CSC medium) and plate cells into an ultra-low attachment plate.
      Note: The number of cells plated per well must be empirically determined for every type of cancer cells. We typically prepare several wells (with serially diluted cells) to find the concentration of cells that can be easily imaged without problems associated with higher-than-optimal density of cells (clumping, too dense spheroids, etc.).
    3. We recommend starting with 5,000 cells/well (24-well plate) or 10,000 cells/well (6-well plate) and adjusting the cell number as needed.
    4. Media replenishment can be performed either by adding extra fresh media (this is preferred in first 2-7 days after cell plating during the time spheres are formed) or by changing media completely. To change the media completely, collect the floating cells from the well and centrifuge at 300 x g, for 2 min. Aspirate the supernatant carefully (cell pellet is sometimes not visible, so leaving small amount of media above the pellet is recommended) and add fresh CSC medium. Mix gently and transfer the cells back to the plate.
    5. Monitor tumorsphere formation daily. Sometimes, cells or spheroids have the tendency to clump in the center of the plate (this is caused by micro vibration of cell culture incubator). In this case, re-suspend the tumorspheres gently by tapping the plate, or mechanically using serological pipettes (in the sterile hood).

  4. Establishing a new primary 3D spheroid culture line from human tumor tissue
    Note: Before performing an experiment, make sure to obtain an informed consent letter from each patient which acknowledges the use of residual tumor tissue for research. All procedures that involve research with human tumor tissues must be approved by Institutional Review Board (IRB). All collected tissues must be de-identified.
    1. Transfer the native tumor sample from the hospital in sterile PBS or culture media or (the best option) in Hibernation medium (Hibernate®-A, Gibco, Invitrogen), supplemented with Antibiotic-Antimycotic (Gibco, Invitrogen), on ice.
      Note: Tumor samples should be as fresh as possible – the ideal situation is to receive/transfer freshly isolated tumor tissue directly from the surgery room – however, in most cases, the tissue used in experiment is residual (leftover) tissue provided by pathologist.
    2. Wash 2 x or 3 x with sterile PBS supplemented with Antibiotic-Antimycotic (100x) (Gibco Invitrogen). Perform final wash with PBS without antibiotics.
    3. Cut the tumor sample with a surgical blade (Figure 2) into small pieces (1-3 mm) and transfer them into a Falcon tube with ~1 ml of 0.5-1% solution of Collagenase IV (Sigma-Aldrich). Sometimes, collagenase digestion is ‘too strong’ – it is important to monitor sample digestion (take a small portion of the tissue sample in collagenase every 15 min of digestion at 37 °C and check the viability of cells with Trypan blue and TC20 Automated cell counter (Bio-Rad) or equivalent method).


      Figure 2. Scheme of tumor tissue processing

    4. Add sterile PBS to the Falcon tube with the tissue sample to wash out the collagenase by centrifugation (200-500 x g, 3 min, room temperature).
    5. Re-suspend the pellet with sterile PBS and filter cells with 70 µm cell filter. Count cells.
    6. Re-suspend filtered primary cells with i) CSC media or ii) ice cold CSC media mixed with Matrigel (1:3) and plate into Ultra low attachment plates. Use up to 0.5 x 106 cells per well (for 6-well plate).
    7. Monitor the growth of spheroids every day. Add small amount of fresh media every 3 days without disturbing the solid phase with embedded cells. Once spheroids are formed, passage them as described below.
      Notes:
      1. Sometimes it is difficult to establish 3D culture in serum-free media. We recommend preparing one or two wells with cells cultured in CSC media, supplemented with 1% FBS.
      2. Freshly digested tumor tissue can be also used for establishing cancer cell lines for 2D growth. In that case, treat the tumor tissue with collagenase as described above. Once digested, wash collagenase with sterile PBS and without filtration the cells, re-suspend tissue fragments/cells in media containing DMEM/F12, 1% Penicillin/Streptomycin , and 10% FBS). Plate into a Petri dish or 6-well plate.
    8. Change the medium next day to get rid of unattached (dead) cells and debris.
      Note: For 2D cell line generation, we do not pass cells through a cell filter – it is better to seed them in clumps and with the ‘trash’ and wash the unattached cells and debris next day when changing the media.

  5. Passaging the spheroids
    1. Aspirate the media from the plate carefully with the pipette, not vacuum aspirator. Try not to disturb the spheroids in Matrigel.
    2. Collect the Matrigel with embedded spheroids with a 10 ml pipette and transfer it into a fresh 15 ml or 50 ml Falcon Tube (Figure 3).


      Figure 3. Scheme of procedure for tumorspheres passaging

    3. Add 10 ml of Dispase (1 U/ml, StemCell Technologies, Canada) and place the tube into an incubator (37 °C) for 30-60 min. Tap the tube gently several times during digestion.
    4. Once spheroids settle at the bottom of the tube, stop the reaction with 100 µl of 0.5 M EDTA (Gibco, Life Technologies, USA) for final EDTA concentration = 0.005 M and centrifuge (1 min, 300 x g, room temperature).
    5. Discard the supernatant carefully and add 10 ml of sterile PBS to wash. Centrifuge for 1 min (300 x g) and repeat 2 x.
      Note: At this point, pelleted tumorspheres can be used for further expansion in Matrigel, isolation of RNA (for qPCR), or preparation of protein lysates for Western blot.
    6. Discard the PBS leaving just a little bit above the pellet containing tumorspheres and add 5 ml of Accutase (StemCell Technologies, Canada). Do not close the Falcon tube tightly to allow gas exchange inside the incubator.
    7. Place the tube in the incubator (37 °C, 5% CO2) and monitor the enzyme reaction.
      Note: To monitor the enzyme reaction, pipette spheroids several times up and down with 10 ml serological pipette. Properly digested spheroids can be easily re-suspended into single cells.
    8. To prepare single cell suspension, re-suspend pellet from digested spheroid with 5 ml serological pipette. Add sterile PBS to wash and centrifuge (300 x g, 2 min, room temperature). Repeat 2 x.
      Note: At this point, cells can be used for further expansion in Matrigel, FACS analysis, isolation of RNA (for qPCR), or proteins for Western blot.

  6. Cryopreservation of tumorspheres
    Tumorspheres could be cryopreserved in the form of single cell suspension (prepared with Dispase-Accutase treatment) or tumorspheres (Dispase treatment only).
    1. Mix either single cells (0.5 x 106 to 2 x 106) or tumorspheres (tumorspheres from one well from 6-well plate per one cryovial) with fresh CSC culture media (final volume = 1 ml) containing 10% DMSO (Sigma-Aldrich, USA), and place the cryovials immediately into Mr. Frosty Freezing Container (Thermo Scientific).
    2. Transfer Mr. Frosty into a -80 °C freezer. Next day, transfer vials with frozen tumorspheres/cells into liquid nitrogen storage tank. Use the vapor phase for long storage of tumorspheres/cells.

Data analysis

  1. Data collection and image analysis
    In the original article (Strnadel et al., 2017), the images of growing spheres were obtained with Nikon TE-2000E D-Eclipse Csi confocal microscope running Nikon Elements software. Images of multiple fields were taken from each well (from 5 to 20) from which the diameter and the number of tumorspheres were quantified. The diameter (Figure 4) of every tumorsphere was measured (for calibration of the measurement, the scale bar from Nikon microscope operating software was used as described below.). The diameter of cell clumps and single or dead cells (Figure 5) were not measured and the value for these events was set to be 0 (see Statistical analysis).
    Note: Optimally, sufficient number of images to cover the whole well of a culture plate should be taken. If it is not possible, take several representative images from every well. Take the same number of images for shControl and for the shRNA sample.

    1. Take multiple images from 3D culture. Make sure that the image has an embedded scale bar from microscope operating software.
    2. Open images in ImageJ software. The icon menu bar will pop up.
      Note: ImageJ is freely available image processing software (freeware). Similar image processing programs can also be used.
    3. Click on “Oval” selection icon located on ImageJ menu bar.
    4. Measure the diameter of every sphere (in pixels) by fitting the oval selection gate to each particular spheroid. The width of spheroid represents its diameter (d).
    5. Measure the length of scale bar (click Straight line icon in ImageJ menu). Compare measured diameter values (in pixels) with a known length of scale bar. Calculate the diameter of every spheroid.


      Figure 4. Measurement of tumorspheres diameter. Images were taken and opened in ImageJ program. The width of circle, fitted accurately to a particular tumorsphere represents its diameter (d). Scale bar was used to calibrate the measurement (Scale bar represents 100 µm).


      Figure 5. Representative images of tumorspheres established from pancreatic cancer cells transduced with viruses encoding Control shRNA (A) or shRNA of a gene of interest (in this case PEAK1; pseudopodium-enriched atypical kinase 1) (B). The dramatic effect of PEAK1 gene silencing is visible when tumorsphere size is compared between the two groups. Clumped cells and single cell events were excluded from the analysis. Scale bars represent 100 µm.

  2. Statistical analysis
    The data were plotted and analyzed in GraphPad Prism 6.0 software as described in the `Statistical analysees’ section of the original paper (Strnadel et al., 2017). For users more experienced in bio-statistical methods and familiar with R software, we recommend the following strategy of data processing: [Since] the probability density of the sphere diameter is – due to the presence of clumps – bimodal (Figure 6 for an illustration), the Wilcoxon two-sample test seems more appropriate than the t-test used in the original paper. It is also worth mentioning that the equality of the probability of clumps in shControl and shRNA may be tested by the two-sample test for the equality of proportions.
    For the illustration, we present results of data analysis for the following data set of tumorsphere diameters.
    shControl: 213 µm, 124 µm, 316 µm, 173 µm, 284 µm, 373 µm, 311 µm, 404 µm, 440 µm, 351 µm, 204 µm, 231 µm, 147 µm,151 µm,160 µm, 227 µm, 151 µm, 151 µm, 89 µm, 333 µm, 338 µm, 80 µm, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0 µm).
    shRNA: 84 µm, 102 µm, 107 µm, 102 µm, 124 µm, 84 µm, 93 µm, 102 µm, 129 µm, 107 µm, 102 µm, 102 µm, 107 µm, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0 µm).


    Figure 6. Probability density of sphere diameter, estimated by the kernel density estimator

    A kernel density estimate of the probability density of the sphere diameter for shControl and shRNA is shown in Figure 6. A Kernel density estimate is a smoothed histogram. Bi-modality of the distribution, caused by the presence of clumps (i.e., the 2D cells) is clearly visible. Boxplot with swarmplot of the data is presented in Figure 7.
    The Wilcoxon test of the equality of the population median of the diameter in shControl and shRNA leads to the P-value of 0.00006 and the difference between the medians of shControl and shRNA is 124.4 µm.


    Figure 7. Boxplot with swarmplot of the data

    The question whether the population proportion of clumps is the same in shControl as in shRNA, when addressed by the two sample test of proportions, leads to the P-value of 0.1036 and the 95 percent confidence interval for the difference of proportion between shControl and shRNA (-0.5716, -0.0884). The proportion of clumps in shControl is 0.3529, whereas 0.6829 in shRNA population.
    The data analysis was performed in R software, using the functions wilcox.test, prop.test, density, boxplot and beeswarm.

Notes

The fresh tumor tissues provided by pathologists are typically non-sterile or contaminated. Based on our experience, we highly recommend sterilizing the tumor tissue before processing. You can sterilize the tissue with 70% Ethanol (submerge the tissue into 70% Ethanol for 3-5 sec or spray the tumor tissue with Ethanol). Wash the tissue with excess amount of sterile PBS immediately after sterilization with Ethanol, and then continue with collagenase treatment.

Recipes

  1. Culture medium used for transduction of cells with shRNA encoding virus
    Note: Mix all of the components in sterile hood and store at 4 °C.
    500 ml DMEM/F12, Glutamax (Gibco, Thermo Fisher Scientific, USA)
    1% Penicillin/Streptomycin (Gibco, Thermo Fisher Scientific, USA)
    10% Fetal bovine serum (Gibco, Thermo Fisher Scientific, USA)
  2. CSC medium
    Notes:
    1. Mix all of the components in a sterile hood and store at 4 °C for up to 2 weeks. Add fresh aliquots of bFGF and EGF every time media is used.
    2. *Store aliquots at -20 °C.
    3. ** Protect aliquots from light and store at -20 °C.
    4. Heparin is an anticoagulant (prevents cell clumping) and increases the stability and functionality of FGF. Store at 2-8 °C.
    485 ml of DMEM/F12 GlutaMAX (Gibco, Life Technologies, USA)
    5 ml Penicillin/Streptomycin 100x (Gibco, Life Technologies, USA)
    10 ml of B27 supplement 50x (serum free) (Gibco, Life Technologies, USA)
    *bFGF – human Fibroblasts Growth Factor 147 basic (animal free) (Gemini Bio-Products, USA), final concentration 20 ng/ml
    *EGF– Epidermal Growth Factor (human) (Gemini Bio-Products), final concentration 20 ng/ml
    **ROCK inhibitor (Y-27632) (StemCell Technologies, Canada), final concentration 10 µM
    Heparin solution (Stemcell Technologies, Canada), final concentration 2 µg/ml

Acknowledgments

These experimental protocols have been previously published and are presented here in original and modified forms from Strnadel et al., 2017. Authors want to thank Prof. Richard Klemke from Department of Pathology (University of California, San Diego) for his support. Special thanks goes to Dr. Vratislav Horak from IAPG, Libechov, Czech Republic, Dr.Michal Kalman, Dr.Juraj Marcinek and Prof.Lukas Plank from Clinic of Surgery and Transplant Center, Jessenius Faculty of Medicine in Martin, and Prof. Ludovit Laca and Dr. Jan Janik from Clinic of Surgery and Transplant Center, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava.This work was supported by grants, listed in the original article (Strnadel et al., 2017), grants from Research and Development Support Agency (Grant no. APVV‑15‑0217) and the project ‘Biomedical Center Martin’, (ITMS code 26220220187), co‑financed from EU sources. This publication is also the result of the project implementation: "CENTER OF TRANSLATIONAL MEDICINE", ITMS: 26220220021 supported by the Operational Programme Research and Innovation funded by the ERDF. All authors declare no conflict of interest.

References

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  4. R Core Team (2015). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. (https://www.R-project.org/)
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  6. Weiswald, L. B., Bellet, D. and Dangles-Marie, V. (2015). Spherical cancer models in tumor biology. Neoplasia 17(1): 1-15.

简介

细胞在固体表面生长为单层的传统二维细胞培养仍然代表了药物检测癌症研究的标准方法。然而,在2D培养物中生长的细胞缺乏相关的细胞基质和细胞 - 细胞相互作用,并且忽略实体肿瘤的真实三维解剖结构。在2D中培养的细胞也可经历细胞骨架重排并获得与异常基因表达相关的人造极性(Edmondson等人,2014)。最近开发出更好地模拟体内情况的3D文化系统。用于研究癌症干细胞的3D体外肿瘤模型(肿瘤球体)在该领域已经获得了越来越多的普及(Weiswald等人,2015)。使用基质嵌入或封装的球体,悬滴培养的球体,磁悬浮系统或3D打印方法的系统已经广泛用于研究和新药筛选。在本文中,我们描述了测试shRNA介导的基因沉默对肿瘤球体形成和生长的影响的详细方案。这种方法允许研究人员测试基因敲低对肿瘤起始细胞生长的影响。正如我们实验室所证实的那样,该方案也可用于直接从肿瘤组织中分离3D癌细胞系。

【背景】3D体外肿瘤细胞模型代表了细胞系与体内生长的肿瘤之间的桥接实验方法(Pampaloni等人,2007; Weiswald等人,等)。,2015)。只有通过3D培养系统才能有效地模拟具有不同营养物质或氧气的实体瘤的3D特征。近年来,肿瘤球文化协议引起了很大的兴趣。肿瘤球可以被描述为由单个祖细胞或干细胞创建的固体球形物体。对于肿瘤球体形成测定,将细胞接种并在无血清培养基中在超低附着平板(非粘附条件)下生长,这允许富集具有干/祖性质的癌细胞(Johnson等人,2013)。由新鲜分离的肿瘤组织产生的肿瘤球在该领域中具有特殊意义,因为来自已建立的细胞系的细胞通常与原发肿瘤不同,这是由于在多轮体外传代期间获得的突变和异常。因此,我们提出了基于3D培养的原发性肿瘤细胞分离的优化方案以及使用3D培养来评估基因沉默对肿瘤起始细胞生长的影响。

关键字:三维培养, 肿瘤球, 癌细胞系, shRNA基因沉默

材料和试剂

  1. Eppendorf管
  2. 移液器吸头
  3. 样品瓶
  4. 塑料瓶
  5. 50毫升Falcon管
  6. 带有清洁盖子的培养皿(Fisher Scientific,Fisherbrand,目录号:FB0875712)
  7. 6孔板,Corning Costar超低附着物(Corning,目录号:3471)
  8. 15ml Falcon管(Corning,Falcon ,目录号:352196)
  9. 70微米细胞过滤器(细胞过滤器 - Corning,Falcon ,目录号:352350)

  10. 塑料移液器
  11. 24孔板,康宁Costar超低附着物(Corning,目录号:3473)
  12. 6孔板(康宁,目录号:3506)
  13. 血清移液器
  14. 手术刀片(FisherBrand高精度#22型刀片刀片,Fisher Scientific,Fisherbrand TM TM,目录号:12-000-161)
  15. 无菌移液管(10ml)(FisherBrand Sterile Disposable Standard Serological Pipets,Fisher Scientific,Fisherbrand TM,目录号:13-678-14A)
  16. 冷冻管(一般长期储存低温管1 ml,Thermo Fisher Scientific,Thermo Scientific TM,目录号:5000-1012)
  17. (可选)Mission pLKO.1-puro-CMV-TurboGFP阳性对照转导颗粒(Sigma-Aldrich,目录号:SHC003V)
  18. EDTA(Thermo Fisher Scientific,Invitrogen TM,目录号:15575-020)
  19. PBS pH 7.4(Thermo Fisher Scientific,Gibco TM,目录号:10010023)
  20. 胰蛋白酶EDTA(0.05%)(Thermo Fisher Scientific,Gibco TM,目录号:25300054)
  21. SureEntry转导试剂(QIAGEN,目录号:336921)
  22. Clorox Bleach(Veritiv,目录号:30966)
  23. 肝素钠盐(Sigma-Aldrich,目录号:H3149-10KU)
  24. DMEM / F12,Glutamax(Thermo Fisher Scientific,Gibco TM,目录号:10565018)
  25. 青霉素/链霉素(Thermo Fisher Scientific,Gibco TM,目录号:15070063)
  26. 胎牛血清(Thermo Fisher Scientific,Gibco TM,目录号:26140079)
  27. B27补充剂50x(无血清)(Thermo Fisher Scientific,Gibco TM,产品目录号:17504044)
  28. bFGF-人成纤维细胞生长因子147碱性(无动物)(Gemini生物产品,目录号:300-805P)
  29. EGF-表皮生长因子(人)(Gemini Bio-Products,目录号:300-110P)
  30. ROCK抑制剂(Y-27632)(STEMCELL Technologies,目录号:72304)
  31. 肝素溶液(STEMCELL Technologies,目录号:07980)
  32. BD基质胶基质生长因子减少(BD Biosciences,目录号:356230)或Cultrex 3-D培养基TM减少生长因子基底膜提取物(Trevigen,目录号: 3445-001-01)
    备注:
    1. 将Matrigel或Cultrex基质等分成无菌罩中的一次性等分试样到无菌Eppendorf管中。我们建议每个Eppendorf管0.5 ml Matrigel。当与1.5毫升冰冷培养基结合使用时,这个量足以在一个六孔板中使用一个孔进行3D培养。
    2. 使用冷冻移液器并在操作过程中将Eppendorf管置于冰上。
    3. 在-80°C下保存。 在冰上或冰箱里解冻后使用
  33. 休眠介质(Hibernate TM -A,Thermo Fisher Scientific,Gibco TM,目录号:A1247501)
  34. 胶原酶IV(Sigma-Aldrich,目录号:C8051-100MG或类似物)
  35. 台盼蓝(TC-10,Bio-Rad Laboratories,目录号:1450013)
  36. 抗生素 - 抗真菌药(100x)(Thermo Fisher Scientific,Gibco TM,目录号:15240096)
  37. Dispase(1U / ml,STEMCELL Technologies,目录号:07923)
  38. EDTA(Thermo Fisher Scientific,Invitrogen TM,目录号:15575020)
  39. Accutase(STEMCELL Technologies,目录号:07920)
  40. DMSO(Sigma-Aldrich,目录号:D2438-50ML)
  41. 乙醇(70%溶液,Fisher Scientific,Fisher BioReagents TM,目录号:BP8201500)
  42. 培养基(见食谱)
  43. CSC中等(见食谱)

设备

  1. 离心机(温控型,VWR台式通用离心机)(VWR,目录号:10830-764)
  2. 移液器
  3. 镊子
  4. CO 2细胞培养箱(BINDER,型号:CB 160)
  5. 生物安全级别(BSL-2)层流罩(Esco,EscoTechnologies,USA)
  6. TC20自动细胞计数器(Bio-Rad)
  7. Frosty冷冻容器先生(赛默飞世尔科技Thermo Scientific TM,产品目录号:5100-0001)
  8. Forma TM 7000系列超低温冷冻机(Thermo Fisher Scientific,Thermo Scientific TM,型号:TFM#902系列)
  9. 液氮储罐
  10. Nikon TE-2000E运行Nikon Elements软件的D-Eclipse Csi共聚焦显微镜(尼康,型号:Eclipse TE2000-E)

软件

  1. 尼康显微镜操作软件
  2. ImageJ或类似的图像处理软件( https://imagej.nih.gov/ij/ ) />
  3. 美国GraphPad Software公司的GraphPad Prism 6.0软件。
    https://www.graphpad.com/scientific-software/prism/
  4. R软件(R核心团队,2015 R: A语言和统计计算环境

程序

为了更好地模拟体内肿瘤细胞的生长,我们使用具有细胞外基质蛋白混合物的3D培养系统用于从单细胞发育的肿瘤球体。为了测试在三维培养中shRNA介导的感兴趣基因敲低对肿瘤细胞生长的影响,制备用编码shControl RNA的病毒转导的细胞以及对目的基因特异的shRNA。

注:在文中,“球体”和“肿瘤球体”这两个词用作同义词。

  1. 用编码shRNA的病毒转导细胞
    1. 在转导时,细胞的汇合应该是约60-70%。吸出培养基,用无菌PBS清洗平板并用胰蛋白酶分离细胞。用含FBS的培养基停止反应,离心(300xg室温,2分钟),用含有SureEntry试剂的PBS洗涤细胞沉淀。对于一次转导,使用不超过50,000个细胞。通过直接混合病毒原液和细胞沉淀,在无菌Eppendorf管中转染癌细胞,先用SureEntry试剂(PBS中SureEntry试剂的终浓度应为8μg/ ml)用无菌PBS(pH7.4)洗涤。对于转导,每50,000个细胞使用10μl高滴度病毒(10 9 TU / ml)。
      备注:
      1. 在将沉淀与病毒溶液混合之前,将上清液吸出,留下少量(〜10μl)含SureEntry的PBS于细胞沉淀上方。将含有残留量的病毒的移液管和小瓶弃于含有10%Clorox漂白剂溶液的塑料瓶中。
      2. 为监测转导效率,可使用对照病毒(例如,Mission pLKO.1-puro-CMV-TurboGFP阳性对照转导颗粒,高滴度)。
      3. 细胞的转导总是在生物安全水平(BSL-2)层流罩中进行。对血液传播的人类病原体采取预防措施,并使用10%漂白剂对该区域进行消毒。
    2. 使用镊子小心地将含有转导混合物(细胞和病毒)的开放Eppendorf管转移到50ml Falcon管中。在孵化期间松开Falcon管的盖子以允许气体交换。将含有Eppendorf管的Falcon管转移到培养箱(37℃,5%CO 2)中孵育30分钟。
    3. 从培养箱中取出细胞并将它们转移到无菌罩中。加入足够量的培养基(2或3毫升),并将细胞放入6孔板或10厘米培养皿中。
    4. 第二天更换培养基(将吸入培养基丢弃到含有10%漂白剂的瓶子中)。允许转导的细胞恢复2-4天(恢复的时间取决于细胞汇合)。

    5. 在推荐或预先测试的浓度下添加选择性抗生素以除去未转导的细胞。

    6. 保持在培养基中选择抗生素12-14天。
    7. 用适当的方法检测转导细胞中基因敲入的效率 - qPCR(mRNA水平)或蛋白质印迹(蛋白质水平)

  2. 细胞嵌入ECM蛋白质基质(Matrigel)中的3D球体培养
    注意:虽然更困难和更耗时,但在基质中嵌入细胞/球体的3D培养(在我们的实验室中)是检测基因沉默对肿瘤球体生长的影响的首选方法。嵌入固体或半固体蛋白质凝胶基质中的生长球体不具有凝集的趋势(或机会)(图1),这使定量分析(例如,计数球体的数量或测量球体直径)困难。 /


    图1.只在CSC培养基中培养的肿瘤球(左图,A)或嵌入基质胶基质(右图,B)。注意只有在CSC培养基中生长时,肿瘤球的聚集趋势。比例尺代表50μm。

    1. 使用胰蛋白酶或其他细胞分离溶液从2D平板上分离细胞。停止与含有FBS的培养基的反应,并将细胞从平板转移到15ml Falcon管中。
      在室温下通过离心(2分钟,400μg×g)使细胞沉淀
    2. 吸出上清液并重悬细胞沉淀在无菌PBS中。
    3. 用70微米细胞过滤器过滤细胞悬液,并手动计数细胞数量或使用自动细胞计数器。
      注意:必须根据经验确定每种类型的癌细胞每孔接种的细胞数量。我们建议从5,000个细胞/孔(24孔板)或10,000个细胞/孔(6孔板)开始并根据需要调整细胞数量(向每孔细胞数量较少)。
    4. 通过在室温下离心(2分钟,400μg×g)使细胞旋转下来。
    5. 吸出上清液并将细胞沉淀物重新悬浮在无菌培养基(此处缩写为CSC培养基)中,并将具有细胞的Falcon管置于冰上冷却。

    6. 移动冰冷的CSC培养基上下数次以冷却塑料吸管。
    7. 在冰上移取冰冷生长因子还原基质胶(BD Biosciences,USA)或Cultrex Reduced Growth Factor Basement Membrane Extract,PathClear(Trevigen,USA)。用冷冻移液管将Matrigel转移到含有冰冷培养基和细胞的小瓶中。使用至少1:3的比例(1份Matrigel和3份含细胞的冰冷培养基)。
    8. 轻轻混合,避免产生气泡。
    9. 将混合物放在超低附着表面培养板(美国康宁)上并转移到培养箱中。
    10. 第二天,轻轻地添加CSC培养基以覆盖固化的基质:含细胞的培养基。每3-5天添加新鲜的媒体。
      注意:使用移液器吸取旧介质,而不是使用真空泵(可能会吸入带球体的基质胶)。

  3. 3D球体培养(基本的,没有ECM蛋白质基质)
    1. 通过在300gxg离心2分钟(在室温下)离心沉淀细胞并吸出上清液。
    2. 用培养基(此处缩写为CSC培养基)轻轻混合细胞沉淀,并将细胞铺在超低附着板中。
      注意:必须根据经验确定每种类型的癌细胞每孔接种的细胞数量。我们通常会准备好几个孔(连续稀释的细胞),以便找到可以轻松成像的细胞浓度,而不会产生与细胞密度高于最佳密度相关的问题(结块,太密集的球体等)。 br />
    3. 我们建议从5,000个细胞/孔(24孔板)或10,000个细胞/孔(6孔板)开始并根据需要调整细胞数量。
    4. 培养基补充可以通过添加额外的新鲜培养基(在形成细胞的时间期间在细胞培养后的前2-7天内优选)或完全改变培养基来进行。为了完全改变培养基,从孔中收集浮动细胞并以300×g 离心2分钟。仔细吸取上清液(细胞团有时不可见,因此建议在培养基上留下少量培养基)并添加新鲜的CSC培养基。
      轻轻混匀并将细胞移回平板。
    5. 每天监测肿瘤球形成。有时,细胞或球状体在板的中心有聚集的趋势(这是由细胞培养箱的微振动引起的)。在这种情况下,通过轻敲平板轻轻重新悬浮肿瘤球体,或使用血清移液管(无菌罩)机械地重新悬浮肿瘤球。

  4. 从人类肿瘤组织建立一个新的初级3D球体培养系统
    注:在进行实验之前,请确保从每位患者获得知情同意书,承认使用残留肿瘤组织进行研究。所有涉及人类肿瘤组织研究的程序都必须经过Institutional Review Board(IRB)的批准。所有收集的组织必须被去除识别。
    1. 将来自医院的天然肿瘤样品在无菌PBS或培养基中或在冬眠培养基(最佳选择)中(补充有抗生素 - 抗真菌药物(Gibco,Invitrogen)(Hibern's -A,Gibco,Invitrogen) ),在冰上。
      注意:肿瘤样本应尽可能新鲜 - 理想情况是直接从手术室接收/转移新鲜分离的肿瘤组织 - 然而,在大多数情况下,实验中使用的组织是剩余(剩余)组织由病理学家。
    2. 用补充有抗生素 - 抗真菌药(100x)(Gibco Invitrogen)的无菌PBS洗2次或3次。
      用不含抗生素的PBS进行最后清洗
    3. 用手术刀(图2)将肿瘤样品切成小块(1-3mm),并用〜1ml的0.5-1%胶原酶IV(Sigma-Aldrich)溶液将它们转移到Falcon管中。有时,胶原酶消化“太强” - 重要的是监测样品消化(在37℃下每15分钟消化胶原酶中的一小部分组织样品,并用台盼蓝和TC20自动细胞检查细胞的生存力计数器(Bio-Rad)或同等方法)。


      图2.肿瘤组织处理方案

    4. 将无菌PBS加入具有组织样品的Falcon管中以通过离心(200-500×g,3分钟,室温)洗出胶原酶。
    5. 用无菌PBS重悬沉淀并用70μm细胞过滤器过滤细胞。计数细胞。
    6. 用i)CSC培养基或ii)与Matrigel(1:3)混合的冰冷的CSC培养基重新悬浮过滤的原代细胞,并且平板成超低附着板。
      每个孔使用多达0.5×10 6个细胞(6孔板)。
    7. 每天监测球状体的生长情况。每隔3天添加少量新鲜培养基,而不会干扰嵌入细胞的固相。一旦形成球体,按照下面的描述通过它们。
      备注:
      1. 有时很难在无血清培养基中建立3D培养。我们建议在CSC培养基中培养一至两个培养孔,并补充1%FBS。
      2. 新鲜消化的肿瘤组织也可以用于建立用于2D生长的癌细胞系。在那种情况下,如上所述用胶原酶处理肿瘤组织。一旦消化,用无菌PBS洗涤胶原酶并且不用过滤细胞,在含有DMEM / F12,1%青霉素/链霉素和10%FBS的培养基中重悬细胞碎片/细胞。放入培养皿或6孔板中。

    8. 第二天更换媒体以摆脱未连接的(死亡)细胞和碎片。
      注意:对于二维细胞系的生成,我们不会通过细胞过滤器传递细胞 - 最好是在更换介质时将它们种植成团块和“垃圾”,并在第二天清洗未附着的细胞和碎片。 /

  5. 传递球体
    1. 用移液管小心地从培养皿中吸出培养基,而不是真空吸气器。尽量不要打扰Matrigel中的球状体。
    2. 用10毫升移液管收集带有嵌入球体的Matrigel,并将其转移到新鲜的15毫升或50毫升Falcon试管中(图3)。


      图3.肿瘤球体传代的程序图

    3. 加入10ml分散酶(1U / ml,StemCell Technologies,Canada)并将管置于培养箱(37℃)中30-60分钟。
      在消化过程中轻轻敲击管子几次
    4. 一旦球体沉降在管底部,用100μl0.5MEDTA(Gibco,Life Technologies,USA)停止反应,最终EDTA浓度为0.005M,并离心(1分钟,300xg) ,室温)。
    5. 小心弃去上清,加10 ml无菌PBS洗涤。离心1分钟(300×g)并重复2次。
      注:此时,沉淀的肿瘤球可用于基质胶中的进一步扩增,分离RNA(用于qPCR)或用于蛋白质印迹的蛋白质裂解物的制备。
    6. 弃去PBS,稍微留在含有肿瘤球的沉淀上方,并加入5ml Accutase(StemCell Technologies,Canada)。请勿紧密关闭Falcon管以允许在培养箱内进行气体交换。
    7. 将管置于培养箱(37℃,5%CO 2)中并监测酶反应。
      注意:为了监测酶反应,用10毫升血清移液管上下吸移球体数次。正确消化的球体可以轻松地重新悬浮成单个细胞。
    8. 为了制备单细胞悬液,用5毫升血清移液管从消化的球体中重悬沉淀。加入无菌PBS洗涤并离心(300xg,2分钟,室温)。重复2次。
      注意:此时,细胞可用于进一步扩增Matrigel,FACS分析,分离RNA(用于qPCR)或用于蛋白质印迹的蛋白质。

  6. 肿瘤球的低温保存
    肿瘤球可以以单细胞悬液(用Dispase-Accutase处理制备)或肿瘤球(仅用于Dispase处理)的形式冷冻保存。
    1. 将新鲜的CSC培养基与单细胞(0.5×10 6至2×10 6)或肿瘤球体(来自6孔板每个冷冻管的一个孔的肿瘤球体)混合(终体积= 1ml),含有10%DMSO(Sigma-Aldrich,USA),并立即将冷冻管置于Frosty冷冻容器(Thermo Scientific)中。
    2. 将Frosty先生转移到-80°C的冷冻箱中。第二天,将带有冷冻肿瘤球/细胞的小瓶转移到液氮储罐中。使用汽相长时间储存肿瘤球/细胞。

数据分析

  1. 数据收集和图像分析
    在原始文章(Strnadel等人,2017)中,使用运行Nikon Elements软件的尼康TE-2000E D-Eclipse Csi共聚焦显微镜获得成长球体的图像。从每个孔(从5到20个)取得多个场的图像,从中量化肿瘤球的直径和数量。测量每个肿瘤区域的直径(图4)(为了校准测量,如下所述使用来自Nikon显微镜操作软件的比例尺)。未测量细胞团块和单个或死亡细胞的直径(图5),并将这些事件的值设置为0(参见统计分析)。
    注意:最理想的是,应该有足够数量的图像覆盖培养板的整个孔。如果不可能,请从每口井取几张有代表性的图像。采用相同数量的shControl图像和shRNA样本。

    1. 从3D文化中获取多个图像。确保图像具有嵌入式显微镜操作软件的比例尺。
    2. 在ImageJ软件中打开图像。图标菜单栏会弹出。
      注:ImageJ是免费提供的图像处理软件(免费软件)。类似的图像处理程序也可以使用。
    3. 点击位于ImageJ菜单栏上的“Oval”选择图标。
    4. 通过将椭圆选择门适配到每个特定的球体,测量每个球体的直径(以像素为单位)。
      。球体的宽度代表其直径(d)。
    5. 测量比例尺的长度(单击ImageJ菜单中的直线图标)。将测量的直径值(以像素为单位)与已知长度的比例尺进行比较。计算每个球体的直径。

      “”
      图4.肿瘤直径的测量在ImageJ程序中拍摄并打开图像。圆形的宽度,准确地拟合到特定的肿瘤区域代表其直径(d)。使用比例尺校准测量(比例尺代表100μm)。

      “”
      图5.由编码对照shRNA(A)或感兴趣基因的shRNA(在这种情况下为PEAK1;富伪蛋白非典型激酶1)的病毒转导的胰腺癌细胞建立的肿瘤球的代表性图像(B) >。当两组之间的肿瘤大小比较时,PEAK1基因沉默的显着效果是可见的。从分析中排除了聚集的细胞和单细胞事件。比例尺代表100μm。

  2. 统计分析
    如原始论文的“统计分析”部分(Strnadel等人,2017)所述,在GraphPad Prism 6.0软件中绘制和分析数据。对于在生物统计方法方面更有经验且熟悉R em软件的用户,我们推荐采用以下数据处理策略:[由于]由于团块的存在,球体直径的概率密度为 - 双峰(图6为例子),Wilcoxon双样本测试似乎比原始文章中使用的测试更适合。另外值得一提的是shControl和shRNA中团块的概率相等可以通过对样本比例相等的双样本检验来检验。
    为了说明,我们提供了以下数据集的肿瘤球直径的数据分析结果。
    shControl:213μm,124μm,316μm,173μm,284μm,373μm,311μm,404μm,440μm,351μm,204μm,231μm,147μm,151μm ,160微米,227微米,151微米,151微米,89微米,333微米,338微米,80微米,0,0,0,0,0,0,0,0,0,0,0,0微米) 。
    的的shRNA: 84微米,102微米,107微米,102微米,124微米,84微米,93微米,102微米,129微米,107微米,102微米,102微米,107微米,0, 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0, 0,0μm)。

    “”
    图6.球体直径的概率密度,由核密度估计器估算

    图6显示了shControl和shRNA的球体直径概率密度的核密度估计。核密度估计是一个平滑的直方图。由团块的存在(即,二维细胞)引起的分布的双模式清晰可见。 Boxplot与数据的swarmplot如图7所示。
    对于shControl和shRNA中的直径的中值的平均值的Wilcoxon测试导致0.00006的em值,并且shControl和shRNA的中值之间的差值为124.4μm。


    图7.带有数据交换空间的Boxplot

    shControl中的群体比例是否与shRNA中的群体比例相同,当通过两个样本比例测试得出时,导致0.1036的95%置信区间和95%置信区间的95%置信区间shControl和shRNA之间的比例差异(-0.5716,-0.0884)。
    在shControl中团块的比例是0.3529,而在shRNA群体中是0.6829。
    数据分析在R软件中进行,使用函数 wilcox.test , prop.test , density , boxplot 和 beeswarm 。

笔记

病理学家提供的新鲜肿瘤组织通常是非消毒或污染的。基于我们的经验,我们强烈建议在加工前对肿瘤组织进行消毒。您可以用70%乙醇对组织进行消毒(将组织浸入70%乙醇中3-5秒或用乙醇喷雾肿瘤组织)。用乙醇灭菌后立即用过量的无菌PBS清洗组织,然后继续胶原酶处理。

食谱

  1. 用于编码病毒的shRNA转导细胞的培养基
    注意:将所有组件放在无菌罩中,并在4°C下保存

    500ml DMEM / F12,Glutamax(Gibco,Thermo Fisher Scientific,USA) 1%青霉素/链霉素(Gibco,Thermo Fisher Scientific,USA)
    10%胎牛血清(Gibco,赛默飞世尔科技,美国)
  2. CSC中等
    注意:
    1. 将所有组分混合在无菌罩中并在4°C下储存长达2周。每次使用培养基时添加新鲜的bFGF和EGF的等分试样。
    2. *将等分试样储存在-20°C。
    3. **保护等分样品不受光照影响,并保存在-20°C。
    4. 肝素是一种抗凝剂(可防止细胞结块)并增加FGF的稳定性和功能。存放在2-8°C。
    485 ml DMEM / F12 GlutaMAX(Gibco,Life Technologies,USA)
    5毫升青霉素/链霉素100x(Gibco,Life Technologies,USA)
    10ml B27补充剂50x(无血清)(Gibco,Life Technologies,USA)
    * bFGF-人成纤维细胞生长因子147碱性(无动物)(Gemini Bio-Products,USA),终浓度20ng / ml
    * EGF-表皮生长因子(人)(Gemini Bio-Products),最终浓度为20ng / ml。
    ** ROCK抑制剂(Y-27632)(StemCell Technologies,Canada),最终浓度为10μM
    肝素溶液(Stemcell Technologies,Canada),终浓度2μg/ ml

致谢

这些实验方案已经在以前发表过,并在此以原始形式和修改后的形式从Strnadel et al。,2017发表。作者要感谢来自加利福尼亚大学圣地亚哥分校病理学系的Richard Klemke教授)为他的支持。特别感谢来自捷克共和国Libachov IAPG的Vratislav Horak博士,来自马丁的Jessenius医学院外科和移植中心诊所的Dr.Michal Kalman,Dr.Juraj Marcinek和Luukas Plank教授以及Ludovit Laca教授以及来自布拉迪斯拉发Comenius大学Martin Martin医学院外科和移植中心诊所的Jan Janik博士。这项工作得到了原始文章(Strnadel 等)中列出的赠款的支持。 2017),研究与发展支持机构(APVV-15-0217)的拨款以及由欧盟资助的共同出资的“生物医学中心Martin”项目(ITMS代码26220220187)。本出版物也是项目实施的结果:“翻译医学中心”,ITMS:26220220021,由ERDF资助的运营计划研究与创新提供支持。所有作者声明不存在利益冲突。

参考

  1. Edmondson,R.,Broglie,J. J.,Adcock,A.F。和Yang,L。(2014)。 三维细胞培养系统及其在药物发现和基于细胞的生物传感器中的应用 Assay Drug Dev Technol 12(4):207-218。
  2. Johnson,S.,Chen,H.和Lo,P.(2013)。 体外肿瘤球体形成分析。 Bio-protocol 3(3):e325。
  3. Pampaloni,F.,Reynaud,E.G。和Stelzer,E.H。(2007)。 第三个方面弥补了细胞培养和活组织之间的差距。 Nat Rev Mol Cell Biol 8(10):839-845。
  4. R核心团队(2015)。 R:统计计算的语言和环境。< / a> R统计计算基金会,奥地利维也纳。 ( https://www.R-project.org/
  5. Strnadel,J.,Choi,S.,Fujimura,K.,Wang,H.,Zhang,W.,Wyse,M.,Wright,T.,Gross,E.,Peinado,C.,Park,HW,Bui ,J.,Kelber,J.,Bouvet,M.,Guan,KL和Klemke,RL(2017)。 eIF5A-PEAK1信号调控YAP1 / TAZ蛋白表达和胰腺癌细胞生长 < Cancer Res 77(8):1997-2007。
  6. Weiswald,L.B.,Bellet,D。和Dangles-Marie,V。(2015)。 肿瘤生物学中的球形癌模型 <瘤形态 17( 1):1-15。
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引用:Strnadel, J., Woo, S. M., Choi, S., Wang, H., Grendar, M. and Fujimura, K. (2018). 3D Culture Protocol for Testing Gene Knockdown Efficiency and Cell Line Derivation. Bio-protocol 8(11): e2874. DOI: 10.21769/BioProtoc.2874.
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