搜索

Bioelectrospray Methodology for Dissection of the Host-pathogen Interaction in Human Tuberculosis
生物电喷雾法剖析人类结核病中宿主与病原体的相互作用   

评审
匿名评审
下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Standard cell culture models have been used to investigate disease pathology and to test new therapies for over fifty years. However, these model systems have often failed to mimic the changes occurring within three-dimensional (3-D) space where pathology occurs in vivo. To truthfully represent this, an emerging paradigm in biology is the importance of modelling disease in a physiologically relevant 3-D environment. One of the approaches for 3-D cell culture is bioelectrospray technology. This technique uses an alginate-based 3-D environment as an inert backbone within which mammalian cells and extracellular matrix can be incorporated. These alginate-based matrices produce highly reproducible results and can be mixed with different extracellular matrix components. This protocol describes a 3-D system incorporating mycobacteria, primary human blood mononuclear cells and collagen-alginate matrix to dissect the host-pathogen interaction in tuberculosis.

Keywords: Bioelectrospray(生物电喷雾), Alginate-based matrices(藻酸盐基质), Multicellular 3-D cell culture(多细胞三维细胞培养), Tuberculosis(结核病), Collagen(胶原), Extracellular matrix(细胞外基质)

Background

Mycobacterium tuberculosis (Mtb) is a pathogen of global public health importance that causes a mortality of 1.8 million people per year and morbidity of 10 million worldwide (WHO, 2016). Despite substantial investment in research, much greater understanding of the host-pathogen interaction is required to improve prevention and treatment. Currently, the pathogen is becoming increasingly resistant to commonly used drugs, with the emergence of extensively drug-resistant Mtb. One of challenges to the tuberculosis (TB) field is the availability of model systems to interrogate the host-pathogen interaction, as widely used animal models do not fully reflect pathology in humans. Hence, there is an urgent need to complement these animal models by developing a physiologically relevant in vitro environment (Bielecka et al., 2017; Tezera et al., 2017). Mtb is an obligate pathogen of man and so we hypothesized that a model system requires human cells, virulent mycobacteria, 3-dimensional organization, extracellular matrix, longitudinal readouts and the ability to modulate the environment over time.

This protocol describes a physiologically relevant in vitro environment by utilizing human cells, extracellular matrix components and live Mtb using a bioelectrospray model to mimic human Mtb infection. This 3-D model is different from other models by using an extracellular matrix that can be released by de-capsulation so that downstream analysis of cells within the matrix can be performed. Furthermore, this methodology has wide potential applicability to investigate infectious, inflammatory and neoplastic diseases and develop novel drug regimens and vaccination approaches.

Materials and Reagents

  1. Female Luer Thread Style Connectors (West Group, catalog number: FTLL210-J1A )
  2. Tissue paper
    Note: Sterilize lab tissue paper by autoclaving and keep it sterile until use.
  3. Nozzle for bioelectrosprayer (0.7 φ) (2 pieces) (Nisco Engineering, catalog number: PE-00577 )
  4. Silicon tubes with connector attached at the end (3 pieces) (VWR, catalog number: 228-0705 )
  5. FalconTM 50 ml conical centrifuge tubes (Corning, Falcon®, catalog number: 352070 )
  6. 75 cm2 flask (CELLSTAR® Cell Culture Flasks, Greiner Bio One International)
  7. SterilinTM 7 ml polystyrene Bijou containers (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 129A )
  8. 2 ml Eppendorf tubes
  9. 20 ml syringe
  10. 5 ml syringe
  11. 0.22 µm syringe filter EMD Millipore MillexTM-GP 33mm (Fisher Scientific, catalog number: 10268401)
    Manufacturer: EMD Millipore, catalog number: SLGP033RS .
  12. Pipette (10 ml, 25 ml) (CELLSTAR® Serological Pipettes , Greiner Bio One International)
  13. Mycobacterium tuberculosis
    Note: Any strain or different species can be used.
  14. Methylated spirit (Fisher Scientific, catalog number: 11492874 )
  15. RPMI 1640 medium (Thermo Fisher Scientific, GibcoTM, catalog number: A1049101 )
  16. EDTA powder (Fisher Scientific, catalog number: 10522965 )
  17. Versene solution (Thermo Fisher Scientific, GibcoTM, catalog number: 15040033 )
  18. Hank’s balanced salt solution (HBSS), no calcium, no magnesium (Thermo Fisher Scientific, GibcoTM, catalog number: 14170112 )
  19. HBSS, calcium, magnesium (Thermo Fisher Scientific, GibcoTM, catalog number: 24020117 )
  20. Human AB serum (Sigma-Aldrich, catalog number: H4522-100ML )
  21. Water (Millipore double distilled water, sterile)
  22. Ultrapure alginate PRONOVATM UP MVG 10 mg (FMC, NovaMatrix, catalog number: 4200106 )
  23. 1 N NaOH cell culture grade solution (Sigma-Aldrich, catalog number: S2770-100ML )
  24. 1 M HEPES (Thermo Fisher Scientific, GibcoTM, catalog number: 15630106 )
  25. 7.5% NaHCO3 solution (Thermo Fisher Scientific, catalog number: 25080060 )
  26. Human collagen type I (3 mg/ml) (Advanced Biomatrix, catalog number: 5007-A )
  27. 0.01 N HCl (pH = 2.0)
  28. Calcium chloride, 96%, extra pure, powder, anhydrous, ACROS OrganicsTM (Thermo Fisher Scientific, catalog number: 10021681)
    Manufacturer: Acros Organics, catalog number: 349615000 .
  29. Sodium citrate dihydrate (Fisher Scientific, catalog number: 10396430 )
  30. 3% alginate (w/v) (see Recipes)
  31. Collagen-alginate mix (1:1 ratio) (see Recipes)
  32. CaCl2 precipitation bath (see Recipes)
  33. De-capsulating solution (see Recipes)

Equipment

  1. Oven (Genlab Drying Oven, UK)
  2. Plastic beakers (2.5 L, Thermo Fisher Scientific)
  3. Class I/III biosafety cabinet
  4. Borosilicate crystalizing glass beakers with spout (VWR, catalog number: 216-0068 ) (5 beakers)
  5. Magnetic stirrers (1 cm, 5 pieces)
  6. Sterile scissors
  7. Thermo ScientificTM NalgeneTM Polypropylene Scissor-Type forceps (Thermo Fisher Scientific, Thermo ScientificTM)
  8. 37 °C, 5% CO2 incubator
  9. Centrifuge (Eppendorf, model: 5427 R )
  10. Electrostatic Bead Generator VARv1 (Nisco Engineering, model: VAR v1 )
  11. Test-Tube-rotator (Bibby Scientific, model: STR4 )
  12. PHD ULTRATM CP syringe pump (Harvard Apparatus, model: PHD ULTRATM CP Syringe Pump )
  13. Jack for height adjustment (Nisco Engineering, catalog number: PE-01162 )

Procedure

  1. SOP for bioelectrospray pre-infection
    Note: This is the optimized protocol for encapsulation of peripheral blood mononuclear cells (PBMCs) after overnight infection with Mycobacterium tuberculosis. If the organism of interest is Mycobacterium tuberculosis, the reader should assume that the experiment is done under Class I/III biosafety cabinet in a standard biosafety level 3 lab under approved institutional standards of practice. If the work requires being undertaken in a standard biosafety level 2 lab, one can fumigate the whole machine according to the respective laboratory SOP before taking out from the BSL3 laboratory. The bioelectrospray technique is always performed in a Class I/III biosafety cabinet, with the doors of the bioelectrosprayer kept closed during microsphere generation as an extra level of containment. A Class II biosafety cabinet is inherently not safe to do the procedure. Once the microspheres are formed, they can be transferred for the subsequent steps either to Class II biosafety cabinet or continue in Class I/III biosafety cabinet. The protocol spans three days. Modification for other infections or other biological modelling is possible.
    Day-1
    1. Sterilize the following items in plastic beakers and dry them in an oven.
      1. Five 150 ml Borosilicate glass beakers with spout
      2. Magnetic stirrers (1 cm length) (5 pieces)
      3. Female Luer Thread Style Connectors
      4. Scissors
      5. Forceps (different sizes)
      6. Tissue paper
      7. Sterile silicon tubes with connector attached at the end (3 pieces, 45 cm in length)
    2. Prepare alginate suspension. Alginate is a natural product and there is variability on the product depending on the species of alginate and environmental conditions. In all studies our group have conducted so far, all the procedures were done with medium viscosity G dominant alginate with viscosity above 250 mPas.
    3. Prepare 3% alginate in HBSS without Ca/Mg, with phenol red under sterile conditions and mix it with the buffers to form alginate mix.

    Day-2
    1. Prepare the alginate-collagen matrix as in Recipe 2 (Figure 1).


      Figure 1. Preparation of cells for encapsulation. Cells are recovered from a 75 cm2 flask and pelleted in a Falcon by centrifugation, and then mixed with alginate-collagen matrix in a 7 ml bijou container (usually 25 million cells/5ml of matrix mix). Also see Video 1.

      Video 1. Mixing alginate with PBMCs prior to bioelectrospraying

    2. Extract PBMCs according to standard protocol for separating PBMCs from blood by density centrifugation. One can use either the buffy coat cells which are commercially available or whole blood isolated PBMCs.
    3. Before the final wash, re-suspend the cells in 50 ml of HBSS without Ca/Mg and take 15 µl of cell suspension for cell counting (dilute 10x further if working with leukocyte cones isolated from 500 ml blood). Place the re-suspended cells in the fridge until use.
    4. Count the cells, and calculate the total cells required for final concentration of 5 x 106 cells/ml once re-suspended in cell-alginate-mix.
    5. Pipette off appropriate number of isolated PBMCs and then pellet by spinning at 320 x g, 8 min, 4 °C, in a 50 ml Falcon tube. Discard supernatant and add 30 ml of complete RPMI medium (ampicillin, glutamine) to the PBMC pellet and re-suspend.
    6. Infect PBMCs with appropriate multiplicity of infection (MOI) of Mtb (our experimental standard is MOI 0.1).
    7. Transfer the infected PBMCs into a 75 cm2 flask.
    8. Leave overnight in a 37 °C, 5% CO2 incubator.

    Day-3
    1. Preparation of cells for encapsulation
      1. Next day, take out the flask from the incubator; carefully transfer the contents of the flask (30 ml) to a 50 ml Falcon tube.
      2. Add 5 ml of 5 mM EDTA (or Versene, Thermo Fisher Scientific) to the flask and incubate for 8-10 min at 37 °C, 5% CO2 incubator.
      3. After that time, add 5 ml of HBSS without Ca, Mg (or complete RPMI medium) to the flask to dilute the effect of the detachment solution.
      4. Scrape the bottom surface of the flask with a scraper carefully and lightly to re-suspend all remaining cells.
      5. Transfer the 10 ml contents to the same 50 ml Falcon tube, already containing the medium (total of 40 ml). Finally rinse flask with 10 ml of HBSS without Ca, Mg (or complete RPMI medium) and add to the Falcon.
      6. Pellet cells in the Falcon by centrifugation at 320 x g, 8 min.
      7. Carefully, take the Falcon tube out of centrifuge and decant supernatant. Re-suspend the pelleted cells: e.g., 50 µl per 5 x 106 cells.
      8. Prepare 5 ml alginate-collagen mix in a 7 ml bijou container. You can prepare alginate-collagen mix up to a week prior to the experiment (Video 1).
      9. Mix well your cells with the alginate-collagen mix accordingly in a 7 ml bijou container (Usually 25 million cells/5 ml of alginate).
      10. Keep at 4 °C on ice/in the fridge until bioelectrospraying the cell-alginate suspension.
    2. Bioelectrospray (Figures 2, 3 and 4)


      Figure 2. Nisco electrostatic encapsulator with washed and alcohol sterilized arm, sterile nozzle, sterile silicon tubes and crystalizing glass beakers. 1. Nozzle (0.7 φ) attached to the nozzle holder; 2. Electrostatic accelerator arm for the electrostatic bead generator VARv1; 3. Electrode cable; 4. Silicon tubes with connector attached at the end; 5. Borosilicate crystalizing glass beakers with spout with magnetic stirrers (1 cm); 6. Stirrer; 7. Ring on the electrostatic accelerator arm; 8. Ruler for setting correct needle height.


      Figure 3. Biolelectrospraying microspheres. Matrix in syringe is injected to the bioelectrosprying machine and microspheres are formed. A syringe filled with matrix was set up on syringe pump (A) so that it will inject the matrix into silicon tube (B) connected to the electrostatic bead generator. The syringe driver is sitting on jack (C) for height adjustment. E. Unused crystalizing glass beakers on the roof of the bioelectrospray machine; F. Housing with doors to enclose the electrostatic bead generator; G. High-voltage switch on/off (white, on/green, off) which is left of potentiometers for optional peristaltic pump, agitator speed and voltage on electrode. Voltage indicator displaying 7.0 kV. Biobin (H) and old media bottle (I) containing surfanios (10%) for discarding biohazardous waste.


      Figure 4. Microspheres are formed in HBSS solution with 100 mM CaCl2. A mix of collagen-alginate with cells in 2 mm diameter silicon tube at a specific rate and microspheres are formed in the gelling bath. Also see Video 3.

      1. Items required:
        1. Bioelectrospray sterile items (Listed above Procedure A Day-1)
        2. Nisco Electrostatic Encapsulator with washed and alcohol sterilized arm
        3. 50 ml Falcon tube
        4. 1 M CaCl2 solution
        5. HBSS with Ca, Mg
        6. HBSS without Ca/Mg
      2. Procedure:
        1. Set the machine up at the rear of the MSC class I/III with the syringe driver on the jack adjacent to it, so that the syringe driver is equal height to the top of the bioelectrosprayer (see Videos 2 and 3).

          Video 2. Setting up the bioelectrospray system

          Video 3. Bioelectrospray system in operation with microspheres being formed

        2. Adjust the syringe driver speed according to the diameter of the syringe (e.g., appropriate rate for 5 ml syringe to give 10 ml/h). The adjustment of the speed varies dependent on the syringe brand.
        3. Prepare the Borosilicate glass beaker by placing magnetic stirrer inside.
        4. Open the sterile silicon tubes and the nozzles and connect them to the bioelectrospray needle held in the arm of encapsulator.
        5. Run 50 ml HBSS without Ca/Mg through the tubing using a 20 ml syringe slowly into empty Borosilicate glass beaker. This will wash the system and check the connection of the needle in the encapsulator arm.
        6. Dry the arm with sterile tissue.
        7. Aspirate the cell-matrix mixture into a 5 ml syringe slowly. Alginate is very viscous and so this must be performed with patience. Avoid creating bubbles. The air enclosed will be ultimately end inside the microspheres, causing them to float.
        8. Connect the 5 ml syringe to the tube, and inject it slowly until it nears the bioelectrospray needle at the end.
        9. Pour 100 mM CaCl2 in HBSS (without Ca/Mg) into one of the beakers until it is half-full. Put beaker under the arm of encapsulator.
        10. Place the syringe in the syringe driver and ensure driving screw abuts end of syringe. Close the doors of the bioelectrosprayer fully.
        11. Start bioelectrospraying by turning on the voltage and stirrer of the encapsulator, and initiating the syringe pump at 10 ml/h. Alginate-collagen mix will be ejected through the needle and the spheroids will be collected in the gelling bath. We use 7 kV voltage and 70% stirring speed. The voltage, stirring speed, affects the diameter of the microspheres and alginate type and nozzle size and further information can be found in the work of Workman and colleagues (Workman et al., 2014).
          Safety Note: The electrostatic bead generator has an electric charge. Therefore, do not touch any parts when the generator is on to prevent exposure to high voltage (low current) electricity.
        12. Once the syringe contents have been fully expelled by syringe driver, it is necessary to drive alginate mix from tubing dead space through the bioelectrosprayer needle or continue with second batch of the cell-matrix suspension.
        13. Stop the bioelectrosprayer, replace the syringe with one containing 5 ml HBSS (without Ca/Mg), and recommence bioelectrosprayer and driver at 10 ml/h until all collagen-alginate mix is expelled and HBSS reaches the needle. This will be clear from colour and microspheres no longer form in gelling bath.
        14. Once all alginate is bioelectrosprayed, decant microspheres to 50 ml tubes by pouring. Allow them to settle and then remove as much supernatant CaCl2 solution from the Falcon as possible to the waste bottle with a 5 ml pipette. Microspheres take about 2 min to settle and so centrifugation is not required, and may damage the spheres (Figure 5).
        15. Add HBSS with Ca, Mg to the microspheres to total volume 50 ml. Stand Falcons in racks.
        16. Wash the microspheres 2 x by removing HBSS with Ca, Mg with a pipette and then adding again (Video 4).


          Figure 5. Microspheres in HBSS with Ca/Mg after transferred from the gelling bath. Also see Video 4.

          Video 4. Decanting microspheres after generation

        17. Aliquot microspheres to the appropriate tissue culture plate or sterile Eppendorf tubes. Aliquoting is performed using a 1 ml pipette with the end cut off with sterile scissors, to give an orifice sufficiently large to pipette up microspheres. Keep the Falcon with microspheres agitated during pipetting to keep a constant concentration within the media and avoid settling during aliquoting.
        18. Pipette off HBSS from wells/Eppendorfs to leave microspheres only.
        19. Add RPMI 1640 medium supplemented with 10% AB serum, and incubate at 37 °C for the duration of the experiments. Each microsphere has ~600 µm diameter and the viability of the cells after a complete procedure is 95%.
        20. If setting up a second experimental condition (e.g., uninfected cells, different cell augmentation), then replace silicone tube and repeat steps as above. A maximum of 8 experimental conditions can be readily undertaken in one day. One should plan 1 h per 5 ml matrix, giving a total of up to 10 h for the final generation of microspheres.

  2. De-capsulation of cells
    1. Aspirate the microspheres into a 50 ml centrifuge tube and allow them to settle at the bottom of the tube. Then discard the supernatant carefully.
    2. Wash the microspheres with HBSS without Ca/Mg twice. Let the microspheres settle at the bottom of the tube, then remove the supernatant.
    3. Add 10 ml de-capsulating solution to the capsules.
    4. Mix the suspension thoroughly prior to incubation at room temperature up to 15 min. Shake the microspheres intermittently.
    5. When the microspheres are completely de-capsulated, their absence is visible with the naked eye. Fill the remaining tube with HBSS without Ca/Mg (or complete RPMI) at room temperature.
    6. Centrifuge the de-capsulated cells to pellet them at 320 x g for 5 min and discard the supernatant.
    7. Wash the cell pellet by re-suspending in HBSS followed by centrifugation.
    8. Further use of collagenase is usually not necessary.
    9. The de-capsulated cells can be further cultured as a monolayer or used for downstream analysis.

Data analysis

Once the microspheres are formed, one can consider them as an individual’s cells/collection of cells as the microspheres are permeable, like sponges. Microspheres can be set up in 96-well plates for assays testing viability, necrosis and apoptosis. Mycobacterial growth can be measured by luminescence if the bacterium has a luminescence reporter plasmid, or directly by counting colony forming units on Middlebrook 7H11 agar after de-capsulation. Cellular composition can be analyzed by flow cytometry after de-capsulation and paraformaldehyde fixation, and RNA analysis by de-capsulation and cellular lysis with Trizol. Data is analyzed according to standard workflows, our routine is a minimul of 2 separate donors with each experimental variable in triplicate.

Recipes

  1. 3% alginate (w/v)
    1. Measure the alginate in aseptic conditions in BSC-II
    2. Place approximately 1.5 g of purified sodium alginate (MVG from NovaMatrix with high glucuronic acid content ≥ 60%, viscosity > 200 mPas, and endotoxin ≤ 100 EU/g) in a sterile 50 ml tube
      1. Weigh sterile empty Falcon tube
      2. Weigh the Falcon tube with alginate
      3. Determine the weight of alginate by subtracting the weight of empty Falcon tube
      4. Add the appropriate volume of HBSS without Ca/Mg, for final percentage of 3%
    3. Vortex the tube for approximately 3 min to partially dissolve the alginate powder then place the tube on an orbital mixer at 10 x g overnight or for two nights in a cold room
    4. Store the alginate solution at 4 °C for short-term storage (1-2 weeks) or at -20 °C for long-term storage (one year)
  2. Collagen-alginate mix (1:1 ratio)
    1. Prepare the following buffers
      1. 0.05 N NaOH in 0.2 M HEPES by mixing 2.5 ml NaOH with 10 ml HEPES (stock 1 M) and adding 37.5 ml of endotoxin free water for final 50 ml solution of NaOH/HEPES solution
      2. 100 ml 7.5% NaHCO3
    2. Human collagen (3 mg/ml dissolved aqueous solution in 0.01 N HCl [pH = 2.0])
    3. Mix the HEPES/NaOH buffer, NaHCO3, and alginate as follows:
      1. 3% alginate: 50% of the total mix
      2. HEPES/NaOH: 4.5% of the total mix
      3. 7.5% NaHCO3: 9% of the total mix
      4. Filter sterilize by 0.22 µm filter (Fisher Scientific)
      5. Add the human collagen (3 mg/ml): 36.5% of the total mix
    4. Store the collagen-alginate solution at 4 °C for short-term storage (1-2 weeks) or at -20 °C for long-term storage (up to a year)
  3. CaCl2 precipitation bath
    1. Dissolve 147 g of CaCl2·2H2O and 23.8 g of HEPES in 1 L of Milli-Q H2O
    2. Adjust the pH level to 5-6
    3. Sterilize the solution using a 0.22 μm filter
    4. Precipitation fluid can be stored at room temperature (6-12 months)
    5. On the day of the experiment, mix 5 ml concentrate with 45 ml of HBSS without Ca/Mg for working solution
  4. De-capsulating solution
    1. Prepare and sterilize the following solutions
      1. 1 M of NaCitrate (add 294.1 g in 1 L of Milli-Q H2O)
      2. 1 M of EDTA (add 292.2 g in 1 L of Milli-Q H2O)
      3. 1 M of citric acid (add 192.1 g in 1 L of Milli-Q H2O)
      4. Store solutions in cell culture grade plastic containers at room temperature (6-12 months)
    2. Make 55 mM NaCitrate and 10 mM EDTA in HBSS with Ca, Mg and adjust the pH to 7.2
    3. To prepare 100 ml of de-capsulating solution
      Mix 5.5 ml of NaCitrate and 1 ml of EDTA in HBSS with Ca, Mg and adjust the pH to 7.2-7.4 and store at 4 °C for up to 2 weeks

Acknowledgments

We would like to thank S. N. Jayasinghe from University College London, United Kingdom for all the technical support and advice on the bioelectrospray technology. This work is funded by the grant from the US National Institute for Health R33AI102239, the UK National Centre for the 3Rs NC/L001039/1 and the Antimicrobial Resistance Cross Council Initiative supported by the seven research councils MR/N006631/1.

References

  1. Al Shammari, B., Shiomi, T., Tezera, L., Bielecka, M. K., Workman, V., Sathyamoorthy, T., Mauri, F., Jayasinghe, S. N., Robertson, B. D., D'Armiento, J., Friedland, J. S. and Elkington, P. T. (2015). The extracellular matrix regulates granuloma necrosis in tuberculosis. J Infect Dis 212(3): 463-473.
  2. Bielecka, M. K., Tezera, L. B., Zmijan, R., Drobniewski, F., Zhang, X., Jayasinghe, S. and Elkington, P. (2017). A bioengineered three-dimensional cell culture platform integrated with microfluidics to address antimicrobial resistance in tuberculosis. mBio 8:e02073-16.
  3. Tezera, L. B., Bielecka, M. K., Chancellor, A., Reichmann, M. T., Shammari, B. A., Brace, P., Batty, A., Tocheva, A., Jogai, S., Marshall, B. G., Tebruegge, M., Jayasinghe, S. N., Mansour, S. and Elkington, P. T. (2017). Dissection of the host-pathogen interaction in human tuberculosis using a bioengineered 3-dimensional model. eLife 6:e21283.
  4. WHO. (2016). Global tuberculosis report 2016.
  5. Workman, V. L., Tezera, L. B., Elkington, P. T. and Jayasinghe, S. N. (2014). Controlled generation of microspheres incorporating extracellular matrix fibrils for three-dimensional cell culture. Adv Funct Mater 24(18): 2648-2657.

简介

标准细胞培养模型已被用于调查疾病病理学和测试新疗法超过五十年。 然而,这些模型系统通常未能模拟在体内发生病理学的三维(3-D)空间内发生的变化。 为了真实地表示这一点,生物学中新兴的范例是在生理相关的三维环境中建模疾病的重要性。 3-D细胞培养的方法之一是生物电喷雾技术。 该技术使用基于藻酸盐的3-D环境作为惰性骨架,其中可以并入哺乳动物细胞和细胞外基质。 这些基于藻酸盐的基质产生高度可重复的结果,并且可以与不同的细胞外基质组分混合。 该方案描述了一种结合分枝杆菌,原代人血液单核细胞和胶原 - 藻酸盐基质的3-D系统,以解剖结核病中宿主病原体的相互作用。
【背景】结核分枝杆菌(Mtb)是全球公共卫生重要性的病原体,每年造成180万人死亡,全球发病率达到1000万(世卫组织,2016年)。尽管在研究方面进行了大量投资,但需要更多地了解宿主 - 病原体的相互作用才能改善预防和治疗。目前,病原菌对常用药物的耐药性越来越高,出现广泛耐药Mtb。结核病(TB)领域的挑战之一是可用于询问宿主 - 病原体相互作用的模型系统,因为广泛使用的动物模型不能完全反映人类的病理学。因此,迫切需要通过开发与生理相关的体外环境(Bielecka等人,2017年; Tezera等人)来补充这些动物模型。,2017)。 Mtb是人的专性病原体,因此我们假设模型系统需要人类细胞,有毒分枝杆菌,3维组织,细胞外基质,纵向读数和随时间调节环境的能力。
 该协议通过使用人类细胞,细胞外基质组分和活Mtb使用生物电喷雾模型来模拟人类Mtb感染来描述生理相关的体外环境。该3-D模型与其他模型不同,其通过使用可通过解封装释放的细胞外基质,以便可以进行基质内的细胞的下游分析。此外,该方法具有广泛的潜在适用性来调查感染性,炎症性和肿瘤性疾病,并开发新的药物方案和疫苗接种方法。

关键字:生物电喷雾, 藻酸盐基质, 多细胞三维细胞培养, 结核病, 胶原, 细胞外基质

材料和试剂

  1. 女性Luer线型连接器(西集团,目录号:FTLL210-J1A)
  2. 组织纸
    注意:通过高压灭菌灭菌实验室纸巾,并保持无菌直到使用。
  3. 喷嘴用于生物电喷雾器(0.7φ)(2件)(Nisco Engineering,目录号:PE-00577)
  4. (3件)(VWR,目录号:228-0705)
  5. Falcon TM 将50ml圆锥形离心管(Corning,Falcon ®,目录号:352070)
  6. 75厘米<2>烧瓶(CELLSTAR 细胞培养瓶,Greiner Bio One International)
  7. Sterilin TM 7ml聚苯乙烯Bijou容器(Thermo Fisher Scientific,Thermo Scientific TM,目录号:129A)
  8. 2 ml Eppendorf管
  9. 20毫升注射器
  10. 5 ml注射器
  11. 0.22μm注射器过滤器EMD Millipore Millex TM -GP 33mm(Fisher Scientific,目录号:10268401)
    制造商:EMD Millipore,目录号:SLGP033RS。
  12. 移液管(10ml,25 ml)(CELLSTAR 血清学移液器,Greiner Bio One International)
  13. 结核分枝杆菌
    注意:可以使用任何菌株或不同种类。
  14. 甲基化精神(Fisher Scientific,目录号:11492874)
  15. RPMI 1640培养基(Thermo Fisher Scientific,Gibco TM,目录号:A1049101)
  16. EDTA粉末(Fisher Scientific,目录号:10522965)
  17. Versene溶液(Thermo Fisher Scientific,Gibco TM,目录号:15040033)
  18. 汉克平衡盐溶液(HBSS),无钙,无镁(Thermo Fisher Scientific,Gibco TM,目录号:14170112)
  19. HBSS,钙,镁(Thermo Fisher Scientific,Gibco TM,目录号:24020117)
  20. 人AB血清(Sigma-Aldrich,目录号:H4522-100ML)
  21. 水(Millipore双蒸水,无菌)
  22. 超纯海藻酸盐PRONOVA TM UP MVG 10 mg(FMC,NovaMatrix,目录号:4200106)
  23. 1N NaOH细胞培养级溶液(Sigma-Aldrich,目录号:S2770-100ML)
  24. 1 M HEPES(Thermo Fisher Scientific,Gibco TM ,目录号:15630106)
  25. 7.5%NaHCO 3溶液(Thermo Fisher Scientific,目录号:25080060)
  26. 人类胶原I型(3mg / ml)(Advanced Biomatrix,目录号:5007-A)
  27. 0.01N HCl(pH = 2.0)
  28. 氯化钙,96%,超纯,粉末,无水,ACROS Organics TM(Thermo Fisher Scientific,目录号:10021681)
    制造商:Acros Organics,目录号:349615000。
  29. 柠檬酸钠二水合物(Fisher Scientific,目录号:10396430)
  30. 3%藻酸盐(w / v)(见食谱)
  31. 胶原 - 藻酸盐混合物(1:1比例)(参见食谱)
  32. CaCl 2沉淀浴(参见食谱)
  33. 脱壳解决方案(见配方)

设备

  1. 烤箱(Genlab干燥炉,英国)
  2. 塑料烧杯(2.5升,赛默飞世尔科技)
  3. I / III级生物安全柜
  4. 硼硅酸盐晶体玻璃烧杯(VWR,目录号:216-0068)(5个烧杯)
  5. 磁力搅拌器(1厘米,5片)
  6. 无菌剪刀
  7. Thermo Scientific TM Nalgene TM聚丙烯剪刀式镊子(Thermo Fisher Scientific,Thermo Scientific TM )
  8. 37℃,5%CO 2培养箱
  9. 离心机(Eppendorf,型号:5427 R)
  10. 静电珠发生器VARv1(Nisco Engineering,型号:VAR v1)
  11. 测试管旋转器(Bibby Scientific,型号:STR4)
  12. PHD ULTRA TM CP注射泵(Harvard Apparatus,型号:PHD ULTRA TM注射泵)
  13. 杰克高度调整(Nisco Engineering,目录号:PE-01162)

程序

  1. 生物电喷雾预感染的SOP
    注意:这是在用结核分枝杆菌过夜感染后外周血单核细胞(PBMC)的封装的优化方案。如果感兴趣的生物体是结核分枝杆菌,读者应该假定实验是在经过认可的实践机构标准的标准生物安全3级实验室中根据I / III级生物安全柜进行的。如果工作需要在标准的生物安全二级实验室进行,则可以在从BSL3实验室取出之前,根据相应的实验室SOP对整个机器进行熏蒸。生物电喷雾技术总是在I / III级生物安全柜中执行,生物电喷雾器的门在微球产生期间保持关闭,作为额外的遏制水平。第二类生物安全柜本身就不能做到这一点。一旦形成微球体,可以将其转移到II级生物安全柜中,或继续进入I / III级生物安全柜。协议三天。修改其他感染或其他生物建模是可能的。
    第1天
    1. 在塑料烧杯中灭菌以下物品,并在烤箱中烘干。
      1. 五个150毫升硼硅酸盐玻璃烧杯与喷口
      2. 磁力搅拌器(1厘米长)(5件)
      3. 女性Luer螺纹样式连接器
      4. 剪刀
      5. 镊子(不同大小)
      6. 组织纸
      7. 带有连接器的无菌硅管末端(3个,长45厘米)
    2. 准备藻酸盐悬浮液。海藻酸盐是一种天然产物,产品的变异性取决于藻酸盐的种类和环境条件。在我们所有的研究中,我们的团队已经进行了所有的程序,所有的程序是用粘度高于250 mPas的中等粘度的G显性藻酸盐进行的。
    3. 在没有Ca / Mg的HBSS中,在无菌条件下用酚红制备3%藻酸盐并将其与缓冲液混合以形成藻酸盐混合物。

    第2天
    1. 准备藻酸盐 - 胶原蛋白基质,如方案2(图1)。


      图1.用于包封的细胞的制备从75cm 2烧瓶中回收细胞并通过离心在Falcon中沉淀,然后与藻酸盐 - 胶原基质混合7毫升bijou容器(通常2500万个细胞/ 5毫升的基质混合物)。另见视频1.

      Video 1. Mixing alginate with PBMCs prior to bioelectrospraying

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player


    2. 根据标准方案提取PBMC,通过密度离心将PBMC与血液分离。可以使用市售的全血分离的PBMC的血沉棕黄层细胞。
    3. 在最后洗涤之前,将细胞重新悬浮在50ml CaCl 2中的HBSS中,并取15μl细胞悬浮液进行细胞计数(如果使用从500ml血液中分离的白细胞锥体进一步稀释10倍)。将再悬浮的细胞放在冰箱中直到使用。
    4. 计数细胞,并计算重新悬浮在细胞 - 藻酸盐混合物中的5×10 6细胞/ ml终浓度所需的总细胞数。
    5. 取出合适数量的分离的PBMC,然后通过在50mL Falcon管中以320×g,8分钟,4℃旋转沉淀。弃去上清液,加入30ml完全RPMI培养基(氨苄青霉素,谷氨酰胺)至PBMC颗粒并重新悬浮。
    6. 感染具有适合的Mtb感染(MOI)的PBMC(我们的实验标准是MOI 0.1)。
    7. 将感染的PBMC转移到75厘米烧瓶中。
    8. 在37℃,5%CO 2培养箱中放置过夜。

    第3天
    1. 制备用于封装的细胞
      1. 第二天,从培养箱中取出烧瓶;小心地将烧瓶内容物(30ml)转移到50ml Falcon管中。
      2. 向烧瓶中加入5ml 5mM EDTA(或Versene,Thermo Fisher Scientific),并在37℃,5%CO 2培养箱中孵育8-10分钟。
      3. 之后,加入5ml不含Ca,Mg(或完全RPMI培养基)的HBSS至烧瓶中以稀释分离溶液的作用。
      4. 用刮刀仔细刮擦烧瓶底面,轻轻重新悬挂所有残留的细胞
      5. 将10ml内容物转移到已经含有培养基(总共40ml)的相同的50ml的Falcon管中。最后用不含Ca,Mg(或完全RPMI培养基)的10ml HBSS冲洗烧瓶,并加入Falcon。
      6. 通过以320×g离心,8分钟
        在Falcon中的颗粒细胞
      7. 小心地将Falcon管从离心机中取出并倒出上清液。再次悬浮沉淀的细胞:例如,每5×10 6个细胞50μl。
      8. 准备5毫升海藻酸钠胶原混合物在一个7毫升bijou容器。您可以在实验前一周准备藻酸盐胶原蛋白胶囊(视频1)。
      9. 将其细胞与藻酸盐 - 胶原蛋白混合物相应地混合在7毫升的容器中(通常为2500万个细胞/ 5毫升藻酸盐)。
      10. 在冰上/冰箱中保持4°C,直到生物电喷雾细胞藻酸盐悬浮液。
    2. 生物电喷雾(图2,3和4)


      图2.具有洗涤和酒精消毒臂,无菌喷嘴,无菌硅管和结晶玻璃烧杯的Nisco静电封装剂。 1。喷嘴(0.7φ)附着在喷嘴座上; 2.静电加速器臂用于静电珠发生器VARv1;电极电缆; 4.带末端连接器的硅管;硼硅酸盐晶体玻璃烧杯,带有搅拌器(1厘米);搅拌器7.静电加速器臂环; 8.用于设定针头高度的标尺。


      图3.生物电喷雾微球。 将注射器中的基质注入生物电动机中,形成微球。在注射泵(A)上设置填充有基质的注射器,以便将基质注入连接到静电珠发生器的硅管(B)中。注射器驱动器坐在插孔(C)上进行高度调节。 E.生物电喷雾器屋顶上未使用的结晶玻璃烧杯; F.带门的外壳,用于封闭静电珠发生器; G.高压开关开/关(白色,开/绿色,关闭),电位计留在可选的蠕动泵,搅拌器速度和电极上的电压。电压指示器显示7.0 kV。生物垃圾(H)和含有表面活性剂(10%))的旧培养基瓶(I),用于丢弃生物危害废物。


      图4.微球在具有100mM CaCl 2的HBSS溶液中形成。 胶原藻酸盐与2mm直径硅管中的细胞以特定比例混合,并在胶凝浴中形成微球体。另见视频3.

      1. 所需物品:
        1. 生物电喷雾无菌物品(上述步骤A Day-1)
        2. Nisco静电封装机,带有洗涤和酒精消毒的手臂
        3. 50ml Falcon管
        4. 1M CaCl 2溶液
        5. HBSS与Ca,Mg
        6. HBSS无Ca / Mg
      2. 程序:
        1. 将机器放置在MSC I / III类型的后面,并使其与其相邻的插孔上的注射器驱动器相连,以使注射器驱动器与生物电极器的顶部相等(见视频2和3)。 >
          Video 2. Setting up the bioelectrospray system

          To play the video, you need to install a newer version of Adobe Flash Player.

          Get Adobe Flash Player


          Video 3. Bioelectrospray system in operation with microspheres being formed

          To play the video, you need to install a newer version of Adobe Flash Player.

          Get Adobe Flash Player


        2. 根据注射器的直径(例如)调节注射器驱动器速度,适当的速率为5ml注射器,以提供10ml / h)。速度的调整取决于注射器品牌。
        3. 通过放置磁力搅拌器来制备硼硅酸盐玻璃烧杯。
        4. 打开无菌硅管和喷嘴,并将其连接到保持在密封剂臂上的生物电喷雾针。
        5. 使用20毫升注射器,将50毫升不含Ca / Mg的HBSS通过管道缓慢进入空的硼硅酸盐玻璃烧杯。这将清洗系统并检查封装臂中针的连接。
        6. 用无菌纸巾干燥手臂。
        7. 将细胞基质混合物慢慢吸入5ml注射器。海藻酸盐非常粘稠,所以必须耐心地进行。避免造成气泡。封闭的空气将最终终止于微球内部,使其浮起来
        8. 将5ml注射器连接到管中,并缓慢注射,直到其末端接近生物电喷雾针。
        9. 将HBSS(不含Ca / Mg)中的100mM CaCl 2水溶液倒入烧杯中,直到半满。将烧杯放在封装器的臂下。
        10. 将注射器置于注射器驱动器中,并确保驱动螺钉紧靠注射器的末端。完全关闭生物电喷雾器的门。
        11. 通过打开密封剂的电压和搅拌器开始生物电喷雾,并以10ml / h启动注射泵。藻酸盐 - 胶原蛋白混合物将通过针头喷射,球体将被收集在胶凝浴中。我们使用7 kV电压和70%搅拌速度。电压,搅拌速度影响微球的直径和藻酸盐类型和喷嘴尺寸,并且可以在Workman及其同事(Workman等人,2014年)的工作中找到进一步的信息。 > 安全注意事项:静电珠发生器具有电荷。因此,当发电机接通时,不要触摸任何部件,以防止暴露于高电压(低电流)电力。
        12. 一旦注射器内容物被注射器驱动器完全排出,就需要通过生物电喷雾针将管道死腔的藻酸盐混合物驱动,或者继续使用第二批细胞基质悬浮液。
        13. 停止生物电喷雾器,用含有5毫升HBSS(不含Ca / Mg)的注射器更换注射器,并以10ml / h重新开始生物电喷雾器和驱动器,直到所有胶原 - 藻酸盐混合物排出,HBSS到达针头。这将从颜色和微球清除,不再在凝胶浴中形成。
        14. 一旦所有的藻酸盐被生物电喷雾,倾倒的微球通过倾倒到50ml管中。允许它们沉淀,然后用5毫升移液管将尽可能多的上清CaCl 2溶液从Falcon中取出到废物瓶中。微球需要约2分钟来沉淀,因此不需要离心,可能会损坏球体(图5)。
        15. 将HBSS与Ca,Mg一起加入到微球中,总体积为50ml。在机架上架着猎鹰。
        16. 用移液管用Ca,Mg去除HBSS然后再次加入,以清洗微球2 x(视频4)。


          图5.从凝胶浴转移后,Ca / Mg在HBSS中的微球。 另请参阅视频4.

          Video 4. Decanting microspheres after generation

          To play the video, you need to install a newer version of Adobe Flash Player.

          Get Adobe Flash Player


        17. 将等分试样微球放入合适的组织培养板或无菌Eppendorf管中。使用1毫升移液管进行等分试样,用无菌剪刀切断,给出足够大的孔口,以移液微球。保持猎鹰在移液时搅动微球,保持在培养基内不变的浓度,并避免等分中的沉降。
        18. 从井/ Eppendorfs取出HBSS,只留下微球。
        19. 加入补充有10%AB血清的RPMI 1640培养基,并在37℃下孵育实验。每个微球的直径约为600μm,完整程序后细胞的活力为95%。
        20. 如果设置第二个实验条件(例如,未感染的细胞,不同的细胞增加),则替换硅胶管并重复如上所述的步骤。一天最多可以容纳8个实验条件。应该计划每5ml基质1小时,总共最多10小时用于最终产生微球。

  2. 细胞脱壳
    1. 将微球吸入50ml离心管中,使其在管的底部沉降。然后仔细丢弃上清
    2. 用不含Ca / Mg的HBSS洗涤微球两次。让微球沉淀在管的底部,然后除去上清液
    3. 向胶囊中加入10 ml脱胶溶液。
    4. 在室温下孵育15分钟前彻底混合悬浮液。间歇地摇动微球
    5. 当微球完全脱胶后,肉眼看不见它们。在室温下,不用Ca / Mg(或完全RPMI)的HBSS填充剩余的管
    6. 离心脱胶细胞以320 x g将其沉淀5分钟,弃掉上清液。
    7. 通过重新悬浮在HBSS中,然后离心分离细胞沉淀。
    8. 通常不需要进一步使用胶原酶。
    9. 脱壳细胞可以作为单层进一步培养或用于下游分析。

数据分析

一旦微球形成,人们可以将它们视为个体的细胞/细胞的收集,因为微球是可渗透的,如海绵。微孔可以设置在96孔板中用于测定活力,坏死和凋亡的测定。如果细菌具有发光报道质粒,或者直接通过在脱胶后在Middlebrook 7H11琼脂上计数菌落形成单位,则可以通过发光测量分枝杆菌生长。细胞组成可以通过流式细胞仪在脱胶和多聚甲醛固定后进行分析,通过脱胶和Trizol细胞裂解进行RNA分析。根据标准工作流程分析数据,我们的程序是每个实验变量一式三份的2个独立供体的最小值。

食谱

  1. 3%藻酸盐(w / v)
    1. 在BSC-II
      无菌条件下测量藻酸盐
    2. 将约1.5g纯化的藻酸钠(来自NovaMatrix的MVG,高葡萄糖醛酸含量≥60%,粘度> 200mPas,内毒素≤100EU/ g)放入无菌50ml管中
      1. 称取无菌空猎鹰管
      2. 用海藻酸盐称重猎鹰管
      3. 通过减去空的Falcon管的重量来确定藻酸盐的重量
      4. 加入不含Ca / Mg的适当体积的HBSS,最终百分比为3%
    3. 将管旋转约3分钟,以部分溶解藻酸盐粉末,然后将管置于10 x 10 g的轨道混合器上,或在冷藏室中放置两晚或以上。
    4. 将藻酸盐溶液储存在4°C以短期储存(1-2周)或-20°C长期储存(一年)
  2. 胶原 - 藻酸盐混合物(1:1比例)
    1. 准备以下缓冲区
      1. 通过将2.5ml NaOH与10ml HEPES(储备1M)混合,并向最终的50ml NaOH / HEPES溶液溶液中加入37.5ml无内毒素游离水,在0.2M HEPES中加入0.05N NaOH。
      2. 100ml 7.5%NaHCO 3
    2. 人胶原蛋白(3mg / ml溶于0.01N HCl溶液的水溶液[pH = 2.0])
    3. 将HEPES / NaOH缓冲液,NaHCO 3和藻酸盐如下混合:
      1. 3%藻酸盐:总混合物的50%
      2. HEPES / NaOH:总混合物的4.5%
      3. 7.5%NaHCO 3:总混合物的9%
      4. 用0.22μm过滤器过滤灭菌(Fisher Scientific)
      5. 加入人体胶原蛋白(3毫克/毫升):总混合物的36.5%
    4. 将胶原 - 藻酸盐溶液储存在4°C以短期储存(1-2周)或-20°C进行长期储存(长达一年)
  3. CaCl 2沉淀浴
    1. 溶解147g CaCl 2·2H 2 O和23.8g HEPES在1L Milli-Q H 2 O中的溶液
    2. 将pH值调至5-6
    3. 使用0.22μm的过滤器
      消毒溶液
    4. 沉淀液可以在室温下储存(6-12个月)
    5. 在实验当天,将5ml浓缩物与45ml HBSS混合,不含Ca / Mg用于工作溶液
  4. 解封装解决方案
    1. 准备和消毒以下解决方案
      1. 1M柠檬酸钠(在1升Milli-Q H 2 O中加入294.1g)
      2. 1M EDTA(在1升Milli-Q H 2 O中加入292.2g)
      3. 1M柠檬酸(在1L Milli-Q H 2 O中加入192.1g)
      4. 在室温(6-12个月)的细胞培养级塑料容器中储存溶液
    2. 用Ca,Mg在HBSS中制备55mM硝酸钠和10mM EDTA,并将pH调节至7.2
    3. 准备100ml脱壳溶液
      在Ca,Mg的HBSS中混合5.5ml硝酸钠和1ml EDTA,并将pH调节至7.2-7.4并在4℃下储存长达2周

致谢

我们要感谢英国伦敦大学学院的S.N. Jayasinghe所有关于生物电喷雾技术的技术支持和建议。这项工作由美国国家卫生研究所R33AI102239,英国国家3R / NCA / L001039 / 1国家中心和抗菌药物抗性交叉委员会倡议资助,由七个研究委员会MR / N006631 / 1支持。

参考

  1. Al Shammari,B.,Shiomi,T.,Tezera,L.,Bielecka,MK,Workman,V.,Sathyamoorthy,T.,Mauri,F.,Jayasinghe,SN,Robertson,BD,D'Armiento, Friedland,JS和Elkington,PT(2015)。&nbsp; 细胞外基质调节结核病肉芽肿坏死。 J. Infect Dis 212(3):463-473。
  2. Bielecka,MK,Tezera,LB,Zmijan,R.,Drobniewski,F.,Zhang,X.,Jayasinghe,S.and Elkington,P。(2017)。&lt; a class =“ke-insertfile”href = http://www.ncbi.nlm.nih.gov/pubmed/28174307“target =”_ blank“>与微流体结合的生物工程三维细胞培养平台,以解决结核病中的抗菌素耐药性。 8:e02073-16。
  3. Tezera,LB,Bielecka,MK,Chancellor,A.,Reichmann,MT,Shammari,BA,Brace,P.,Batty,A.,Tocheva,A.,Jogai,S.,Marshall,BG,Tebruegge, Jayasinghe,SN,Mansour,S.和Elkington,PT(2017)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/28063256”target = “_blank”>使用生物工程三维模型解剖人类结核病中的宿主病原体相互作用。 eLife 6:e21283。
  4. 谁。 (2016)。 2016年全球结核病报告
  5. Workman,VL,Tezera,LB,Elkington,PT和Jayasinghe,SN(2014)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/25411575 “target =”_ blank“>控制产生含有细胞外基质原纤维的微球用于三维细胞培养。 24(18):2648-2657。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright Tezera et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Tezera, L. B., Bielecka, M. K. and Elkington, P. T. (2017). Bioelectrospray Methodology for Dissection of the Host-pathogen Interaction in Human Tuberculosis. Bio-protocol 7(14): e2418. DOI: 10.21769/BioProtoc.2418.
  2. Tezera, L. B., Bielecka, M. K., Chancellor, A., Reichmann, M. T., Shammari, B. A., Brace, P., Batty, A., Tocheva, A., Jogai, S., Marshall, B. G., Tebruegge, M., Jayasinghe, S. N., Mansour, S. and Elkington, P. T. (2017). Dissection of the host-pathogen interaction in human tuberculosis using a bioengineered 3-dimensional model. eLife 6:e21283.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。