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Human tissue explants are a valuable tool to study the interactions between host and infectious agents. They reliably mimic many important aspects of tissue cytoarchitecture and functions and allow us the investigation of the mechanisms of microbial pathogenesis under controlled laboratory conditions. One of the advantages of this system is that, unlike isolated cells, infection of tissue blocks with HIV-1 does not require exogenous stimulation with mitogens or activating factors. Here we describe a protocol to infect with HIV-1 human lymphoid tissue from tonsils and cervico-vaginal tissue and maintain them in culture in a non-polarized setting. These ex vivo infected tissues can be used as fruitful models to study HIV-1 pathogenesis and HIV-1 vaginal transmission, respectively, as well as an efficient platform for testing anti-HIV therapeutic and preventative strategies.

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An ex vivo Model of HIV-1 Infection in Human Lymphoid Tissue and Cervico-vaginal Tissue
HIV-1感染人淋巴组织和子宫颈阴道组织的体外模型

微生物学 > 微生物-宿主相互作用 > 离体模型
作者: Andrea Introini
Andrea IntroiniAffiliation: Section of Intercellular Interactions, Program in Physical Biology, NICHD, NIH, Bethesda, USA
For correspondence: andrea.introini@ki.se
Bio-protocol author page: a1164
Christophe Vanpouille
Christophe VanpouilleAffiliation: Section of Intercellular Interactions, Program in Physical Biology, NICHD, NIH, Bethesda, USA
Bio-protocol author page: a1165
Jean Charles Grivel
Jean Charles GrivelAffiliation: Section of Intercellular Interactions, Program in Physical Biology, NICHD, NIH, Bethesda, USA
Bio-protocol author page: a1166
 and Leonid Margolis
Leonid MargolisAffiliation: Section of Intercellular Interactions, Program in Physical Biology, NICHD, NIH, Bethesda, USA
Bio-protocol author page: a1167
Vol 4, Iss 4, 2/20/2014, 4744 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1047

[Abstract] Human tissue explants are a valuable tool to study the interactions between host and infectious agents. They reliably mimic many important aspects of tissue cytoarchitecture and functions and allow us the investigation of the mechanisms of microbial pathogenesis under controlled laboratory conditions. One of the advantages of this system is that, unlike isolated cells, infection of tissue blocks with HIV-1 does not require exogenous stimulation with mitogens or activating factors. Here we describe a protocol to infect with HIV-1 human lymphoid tissue from tonsils and cervico-vaginal tissue and maintain them in culture in a non-polarized setting. These ex vivo infected tissues can be used as fruitful models to study HIV-1 pathogenesis and HIV-1 vaginal transmission, respectively, as well as an efficient platform for testing anti-HIV therapeutic and preventative strategies.

[Abstract]

Material and Reagents

  1. Tonsillar tissue obtained from routine tonsillectomy (Age range of patients: 2-10)
  2. Cervico-vaginal tissue obtained from routine hysterectomy (Age range of patients: 35-55)
  3. Gelfoam 12-7 mm adsorbable gelatin sponge (Pfizer, NDC: 0009-0315-08 )
  4. RPMI 1640 (Life Technologies, Gibco®, catalog number: 31870-025 )
  5. 100x Modified Eagle's medium (MEM)-nonessential amino acids (10 mM) (Life Technologies, Gibco®, catalog number: 11140-035 )
  6. 100x MEM sodium-pyruvate (100 mM) (Life Technologies, Gibco®, catalog number: 11360-070 )
  7. 1,000x Gentamicin (50 mg/ml) (Corning, cellgro®, catalog number: 30-005-CR )
  8. 100x Fungizone (250 μg/ml, amphotericin B) (Life Technologies, Gibco®, catalog number: 15290-018 )
  9. Fetal bovine serum (FBS) (Gemini Bio-products, catalog number: 100-106 )
    Note: We advise testing several lots of serum for culture optimization and use the same lot of FBS for an entire series of experiments. We always test several serum lots on tissues from several donors and select the lot that gives the highest HIV-1 replication. Also, FBS can affect the ability of tissue to secrete cytokines in culture medium.
  10. Phosphate Buffered Saline (PBS) (pH 7.4) (Life Technologies, Gibco®, catalog number: 10010-023 )
  11. Sterile water, cell culture grade (Quality Biological, catalog number: 118-162-101 )
  12. HIV-1 viral preparation(s)
    Note: For most of our experiments we use the following viral preparations: HIV-1BaL and HIV-1LAI.04 obtained from the Virology Quality Assurance Laboratory at Rush University (Chicago, IL). Viral stocks were obtained from the clarified culture medium of peripheral blood mononuclear cell cultures inoculated with either HIV-1BaL or HIV-1LAI.04, originally received from the NIH AIDS Reagent Program. HIV-1 p24gag concentrations were 49 ± 3 ng/ml and 53 ± 3 ng/ml for HIV-1BaL and HIV-1LAI.04 stock, respectively. For more viral preparations used in our experimental setting see Introini et al., 2013 and Vanpouille et al., 2012.
  13. Timentin (GlaxoSmithKline, NDC: 0029-6571-26 ) (see Recipes)
    Note: Timentin is the commercial name of a mix of the antibiotics ticarcillin and clavulanate that are commercially available as individual reagents. These antibiotics efficiently prevent growth of bacteria that can occasionally contaminate tissue samples after surgery. Penicillin and streptomycin can be used instead of Timentin, although they have different properties. For example, Timentin displays low stability at room temperature or 37 °C (about 24 h) therefore, once added to culture medium, it remains active only for the first day of culture.
  14. Culture medium (CM) (see Recipes)

Equipment

  1. Petri dish (100 mm x 20 mm) (BD, Falcon®, catalog number: 353003 )
  2. Petri dish (150 mm x 25 mm) (BD, Falcon®, catalog number: 353025 )
  3. 6-well plates (Corning, Costar®, catalog number: 3506 )
  4. 12-well plates (Corning, Costar®, catalog number: 3513 )
  5. 5, 10-ml pipettes (BD, Falcon®)
  6. Sealed screw-cap 1.5/2-ml tubes (SARSTEDT AG)
  7. Thermomixer with block for 1.5 ml-tubes (Eppendorf)
  8. Forceps or tweezers
  9. Scalpels and blades nos. 10 and 23
  10. Scissors
  11. Flat weighing metallic spatula
  12. 37 °C, 5% CO2 incubator set at 90% humidity
  13. Water bath
  14. 10, 50-ml syringe plunger

Procedure

  1. Allow CM enough time to reach room temperature or put it in a water bath pre-warmed at 37 °C.
  2. Calculate the number of Gelfoam pieces needed per experiment on the basis of the type of tissue used and the number of wells required to perform the experiment (Table 1).

    Table 1. Culture setup
    Tissue type
    No. of Gelfoam pieces (from 1 sponge)
    Culture plate
    Volume of culture medium (per well) (ml)
    No. of blocks (per Gelfoam piece)
    No. of wells (replicates) per experimental condition
    Tonsillar
     4
    6-well
    3
    9
    3 or more
    Cervico-vaginal
    5-6
    12-well
    1
    8
    2 or more

  3. Depending on the number of Gelfoam pieces needed, either fill a 100 mm x 20 mm Petri dish with about 100 ml of CM supplemented with Timentin or a 150 mm x 25 mm Petri dish with about 200 ml (if more than 3 Gelfoam sponges are required).
  4. Put the Gelfoam sponge(s) into the Petri dish using ethanol-sterilized forceps and press the sponge(s) against the bottom of the Petri dish for about 2 min using a bent flat spatula sterilized with ethanol.
    Note: The Gelfoam is extremely brittle when dehydrated. The hydration process should be done carefully especially when pushing down on the foam to chase the air out. The Gelfoam should be as free of air as possible: the presence of air will block the capillaries through which nutrients reach the tissue.
  5. Use ethanol-sterilized scissors to cut the rehydrated Gelfoam sponge(s) into pieces of the appropriate size (Table 1).
  6. Place one piece of Gelfoam into each well using the forceps and add the appropriate amount of CM containing Timentin with a pipette (Table 1).
  7. Place the plates into the incubator.
    Note: The dissection and infection protocols vary depending on the tissue in use. If using tonsils follow option A; if using cervico-vaginal tissue, follow option B.

  1. Tonsillar tissue
    1. Put one tonsil into a 100-mm Petri dish containing CM supplemented with Timentin and cut the tonsil into several large pieces (4–5 pieces).
    2. Transfer one piece into the lid of a Petri dish containing few ml of CM. Cover the Petri dish containing the remaining tonsil pieces to avoid desiccation of tissue.
    3. With a 10- or 23-gauge blade scalpel and forceps, remove cauterized parts and fibroid tissue. Remove bloody/inflamed tissue and any parts containing tonsillolithes and/or with green-brownish color (Figure 1, see Grivel and Margolis, 2009 for additional pictures).


      Figure 1. Preparation of tonsillar tissue for culture
      (Up) In the received surgical specimens the parts that should be removed are residual fibroid tissue from the capsule surrounding palatine tonsils (white), cauterized tissue (brown-black), bloody/inflamed tissue (red) and any other parts with brown-greenish color.
      (Middle) Remove unwanted large parts of tissue with a neat cut holding the specimen with forceps. The dotted line delimitates the cutting surface to remove a layer of cauterized tissue. For small parts it may be easier to directly pinch them with forceps to cut them out.
      (Down) Strips of tissue are dissected into blocks of approximately 8 mm3. 9 blocks are transferred on each Gelfoam piece in a 6-well plate.

    4. Cut the tissue piece into slices of about 2 mm in thickness. Remove any undesirable part as mentioned above, and cut the slices into strips 2 mm thick. Then cut the strips into blocks 2 mm thick. This should result in blocks of roughly 8 mm3
    5. Cut as many blocks as needed, at least 27 blocks are required per experimental condition (Table 1). With the forceps, transfer the blocks into a clean Petri dish containing CM supplemented with Timentin to avoid desiccation of tissue blocks while proceeding with dissection.  
      Note: Since dissection may take long time, it is important to keep adding medium to the Petri dish lid to maintain tissue wet while cutting.
    6. Swirl the plate to randomize the distribution of the blocks.
    7. Take out the six-well plate(s) containing Gelfoam from the incubator and place 9 blocks of tissue on top of each Gelfoam piece using the forceps.
    8. Return the plate(s) to the incubator for overnight culture or proceed with infection (go to step A11).
      Note: We prefer performing infection after overnight incubation to make sure that no bacterial or fungal contamination develops. In addition, a large population of cells egresses tonsillar tissue blocks after dissection. This process is largely completed within the first 24 h of culture.
    9. If tissue was dissected the previous night, aspirate the CM in the six-well plate(s) with a pipette and discard it. Tilt the plate and gently push the Gelfoam back to the upper part of the well to allow the medium to gather at the bottom, aspirate and discard it.
    10. With a pipette, add to each well 3 ml of Timentin-containing CM pre-warmed at 37 °C.
    11. Thaw HIV-1 viral preparation(s) in the water bath pre-warmed at 37 °C. If necessary, dilute viral stock to an appropriate concentration. It is preferable to have a dilution such that the desired inoculum is contained in 5 to 8 μl of medium.
      Note: For most of our experiments we use 6.0 to 7.5 μl of undiluted HIV-1BaL or HIV-1LAI.04 viral stock (Material and Reagents 12) to infect each tonsillar tissue block. It is critical for an efficient infection to allow the minimum time required between thawing HIV-1 viral preparation and depositing the inoculum on tissue blocks.
    12. Pipette the inoculum directly on top of each of the 9 blocks on a Gelfoam piece.
      Note: For an accurate delivery of the inoculum volume we suggest using a reverse pipetting technique and changing tip for every well.
    13. Return the plate to the incubator as soon as infection is completed.

  2. Cervico-vaginal tissue
    1. Put the tissue piece(s) in a 100-mm Petri dish containing CM supplemented with Timentin.
    2. Transfer one piece of tissue into the lid of a Petri dish containing few ml of CM. Cover the Petri dish containing the remaining tissue to avoid desiccation.
    3. With a 23-gauge blade scalpel and forceps separate the mucosa of ectocervix and endocervix from the stromal tissue beneath to obtain strips of mucosa (Figure 2).


      Figure 2. Preparation of cervico-vaginal tissue for culture
      (Up) The endocervix is the part of the uterus lining the lumen of the canal that provides communication between the uterine cavity and that of the vagina. The portion of the cervix that projects into the vagina through the upper anterior vaginal wall is called ectocervix. In the received cervico-vaginal tissue specimens the endocervical mucosa is readily recognized as the part with darker-red color and coated by thick mucus, whereas the ectocervical mucosa has a clearer color.
      (Middle) The surgical specimen can be cut into smaller pieces to facilitate the removal of the mucosa. Holding the stromal tissue with forceps, separate it from the mucosa cutting the tissue specimens in the longitudinal direction (indicated by the arrows) with respect to the mucosal layer. The dotted line delimitates the cutting surface and should be located 2-3 mm below the epithelial edge of the tissue specimen.
      (Down) Strips of mucosa are dissected into blocks of approximately 8 mm3. After infection 8 blocks are transferred on each Gelfoam piece in a 12-well plate.

    4. Cut the mucosa strips into strips of 2 mm in thickness, remove and discard as much connective-muscular tissue as possible, leaving only a layer 2 mm thick comprising the mucosal epithelium, sub-mucosa and part of the underlying stroma. Cut the strips into blocks 2 mm thick. This should result in blocks of roughly 8 mm3.
    5. Cut as many blocks as needed, at least 16 blocks are required per experimental condition (Table 1). With the forceps, transfer the blocks into a clean Petri dish containing CM supplemented with Timentin to avoid desiccation of tissue blocks while proceeding with dissection.  
      Note: For most of our experiments we pool blocks from the endocervix and ectocervix. One may consider keeping them separated because of their different structural and immunologic features (Pudney et al., 2005). Also, the endocervix usually continues to produce mucus in culture and this might interfere with subsequent assays. Note that in the received cervico-vaginal tissue specimens the endocervix is much smaller than the ectocervix.
    6. Swirl the plate to randomize the distribution of the blocks.
    7. With the forceps, transfer the blocks into sterile 1.5-ml conical tube(s) (maximum 16 blocks per tube).
    8. Thaw HIV-1 viral preparation(s) in the water bath pre-warmed at 37 °C. If necessary, dilute the viral stock to an appropriate concentration and transfer it into the tube(s) containing the blocks.
      Notes:
      a. Cervico-vaginal tissue is best infected at the time of dissection. We recommend including an experimental condition in which both the virus and a powerful antiviral drug are combined. This is a useful control to ascertain whether the virus actually replicates rather than being adsorbed and released by the tissue.
      b. For most of our experiments we use 500 μl of undiluted HIV-1BaL viral stock (Material and Reagents 12) to infect 12-16 cervico-vaginal tissue blocks. We add lamivudine 10 μM to HIV-1 preparation and to CM during the entire culture length as control for HIV-1 release.
    9. Transfer the tubes into a Thermomixer pre-warmed at 37 °C and set mixing at 300 revolutions per minute. Incubate for 2 h, gently inverting the tubes few times every half an hour.
      Note: It is critical for an efficient infection to allow the minimum time required between thawing HIV-1 viral preparation and transferring the tubes to 37 °C.
    10. Fill the wells of a 6-well plate (or more plates if more than six tubes were used for infection) with 3-4 ml of PBS.
    11. Discard the viral preparation from the tube(s). Using the forceps, gently transfer the blocks to the 6-well plate lid to remove the excess of viral preparation remained on the blocks, and then transfer the blocks into a well containing PBS.
    12. Swirl gently with a pipette and wait for 1 min. With a pipette, aspirate the PBS taking care not to aspirate the blocks.
    13. Add 3-4 ml of PBS to the well, swirl gently with a pipette tip and wait for 1 min.
    14. Repeat steps B12-13 three times.
      Note: These washing steps are necessary because HIV-1 replication in cervico-vaginal tissue is not as high as in tonsillar tissue and can be masked by the subsequent release of viruses absorbed on the tissue.
    15. Take a 12-well plate out of the incubator and transfer 8 blocks on top of each Gelfoam piece using the forceps.
    16. Return the plate to the incubator.

Common to A and B

  1. Three days after infection with HIV-1, harvest an aliquot of medium from each of the replicate wells and transfer it into a screw cap tube.
    Note: We usually pool aliquots of replicate wells.
  2. Using ethanol-sterilized forceps put back any block of tissue that may have dropped from the Gelfoam.
  3. With a pipette, discard the remaining medium from the plate. Tilt the plate, gently bring the Gelfoam to the upper part of the well and let the medium flow down to the bottom, aspirate and discard it.
  4. With a pipette, add the adequate volume of fresh CM pre-warmed at 37 °C (Table 1).
    Note: It is not required to supplement CM with Timentin at this stage.
  5. Return the plate(s) to the incubator.
  6. Store medium aliquots at -80 °C.
  7. Repeat steps 1 to 5 every 3 days until day 12-15.
  8. At day 12-15 post-infection collect tissue blocks using forceps and process them as required by the experiment.
  9. For RNA extraction we usually keep tissue blocks in RNA later overnight at 4 °C before storing them at -80 °C. For flow cytometry we immediately process tissue blocks by digesting them with an enzymatic cocktail to obtain a single-cell suspension.
    Note: During culture, tissue outgrows into the Gelfoam pieces. Also individual cells may migrate out of the tissue into the gel. One can analyze the part of tissue that is above the gel separately by pinching it out with the forceps. The cells within the gel can be squeezed out using the forceps or a syringe plunger. However, one should keep in mind that what extracted from the gel is not just a fraction of migrated cells but rather a mixture of such cells and tissue structures.
  10. Dispose plates with any residual CM, tissue blocks and Gelfoam as per the institution’s biosafety practices required for handling human infectious waste.
  11. A number of techniques can be used to assess HIV-1 production and replication in tissue blocks. Our standard read-out is to measure HIV-1 p24gag in culture supernatants harvested throughout culture time using a fluorescent bead-based immunoassay (Biancotto et al., 2009).
    Note: Tissues from different donors infected with the same inoculum of the same HIV-1 stock may give different results as measured by p24gag accumulation in culture supernatant (Table 2). This may be due to several factors, such as the number and the activation status of HIV-1 target cells and the presence of co-infecting pathogens, which affect HIV-1 replication and production in tissue blocks. Therefore it is pivotal in the set up of different conditions of an experiment to use tissue from only one donor (donor-matched conditions).

    Table 2. Reference values of HIV-1 p24gag inoculum and relative production in tissue explants. HIV-1 p24gag production is indicated as cumulative value of the p24gag values measured in culture supernatant sampled every 3 days over 12 days of culture (day6+day9+day12). The value of p24gag measured at day 3 was excluded because it does also contain p24gag released from the HIV-1 inoculum absorbed on tissue blocks together with newly produced p24gag as a result of viral replication. The value of HIV-1 production in cervico-vaginal tissue infected with HIV-1BaL is normalized by the p24gag value measured in culture supernatant of donor-matched blocks infected and cultured in the presence of lamivudine 10 μM. Infection of cervico-vaginal tissue with HIV-1LAI.04 does not lead to detectable HIV-1 replication in our experimental setting. HIV-1 p24gag was measured using a fluorescent bead-based immunoassay (Biancotto et al., 2009).
    Tissue type
    Virus
    Inoculum per block (pg of HIV-1 p24gag)
    No. of blocks
    HIV-1 p24gag
     production (ng/ml)
    Tonsillar
    HIV-1BaL
    367.5
    27
    1-10

    HIV-1LAI.04
    397.5
    27
    10-100
    Cervico-vaginal
    HIV-1BaL
    1531.2
    16
    1-5
    HIV-1LAI.04
    1656.2
    16
    Undetectable

Notes

  1. For our experiments we use tissues collected from indicated surgeries that are not harvested for the purpose of the study and therefore can be considered as 'pathological waste'. In any case, human tissues must be harvested according to a protocol approved by an ethical review board. Collecting personal and medical data, such as use of drugs, menstrual cycle, sexual behavior, use of contraceptives, etc. from tissue donors may provide important information to interpret experimental results. For an example see Saba et al., 2013. Collection of these data poses ethical issues concerning informed consent and privacy that must be addressed in the protocol for tissue harvest.
  2. The tissue delivered to the laboratory should be in good condition, having a “healthy” pinkish color and lacking large blood clots and greenish necrotic centers. The state of the tissue is also dependent on how quickly it is delivered for processing. It is important for long-term culture to start the experiment with a healthy tissue. Tonsillar tissue is soft and prone to deterioration. According to our experience, this tissue should be delivered within 3-5 h post-surgery. We have noticed that cooling on ice during delivery makes it less suitable for long-term culture. Therefore we maintain tonsillar tissues in PBS at room temperature during delivery. Cervico-vaginal tissue is much sturdier and we successfully infected with HIV-1 and cultured tissues delivered 24 h post-surgery. We use ice-cold RPMI 1640 as delivery medium.
  3. The operator should be informed on the risks of working with blood-borne pathogens and receive proper training to work in a Biosafety Level 2 laboratory in order to handle tissue specimens, even if the experiments do not involve the use of HIV-1.
  4. Take note, and pictures for the more meticulous experimenters, of any feature of tissue specimens that you suspect may affect experimental results, such as bloody, cauterized and necrotic parts. We suggest keeping the medium in which the dissection is performed as well as any unused piece(s) of tissue because they might be useful to assess cell viability, for archiving DNA/RNA or histological analysis.
  5. The values of HIV-1 inoculum reported in Table 2 were optimized to perform infection with our viral stocks of tissue specimens delivered to the laboratory and processed following standard procedures to minimize any variability not due to the intrinsic nature of the tissue. Nevertheless, the production of HIV-1 varies remarkably between tissues from different donors. Therefore we invite the readers to take the values reported in Table 2 as a reference, bearing in mind that multiple inter-donor factors affect the outcome of an infection. For this reason we recommend to use exclusively donor-matched experimental conditions and perform an appropriate statistical analysis on data averaged from multiple donors.
  6. The experimental system described above is non-polarized meaning that penetration of HIV-1 may occur from all sides of a tissue block. The aim of this system is not to simulate HIV-1 transmission through the epithelium but rather to investigate the mechanisms that regulate HIV-1 spreading in tissues. For cervico-vaginal tissue, this culture system may still model HIV-1 transmission as there is evidence in vivo that HIV-1 can reach the subepithelial layer of the mucosa through pre-existing microabrasions of the epithelium or caused by sexual intercourse. Experimental settings of polarized infection with HIV-1 and culture of tissue explants have been described by others in Dezzutti and Hladik, 2013. Advantages and disadvantages of both systems as a model of HIV-1 transmission are described in Merbah et al., 2011.

Recipes

  1. Timentin
    Prepare a working 100x stock solution by adding 100 ml of sterile water to a 3.1 g vial of Timentin. Aliquot in sterile screw-cap tubes, freeze and keep aliquots at -30 °C. Unused thawed aliquots should be discarded.
  2. Culture medium (CM)
    Remove 90 ml (set aside for other use) from a 500 ml bottle of RPMI1640
    add
    75 ml of FBS
    5 ml of 100x MEM-nonessential amino acids
    5 ml of 100x fungizone
    5 ml of 100x MEM-sodium pyruvate
    500 μl of 1,000x gentamicin
    Stored at 4 °C
    Supplement CM with Timentin only before use. Dilute 1 ml of freshly thawed 100x Timentin in 99 ml of CM. Prepare the volume needed for the experiment on the basis of the indications in Table 1 considering that extra CM is required to prepare Gelfoam and cut tissues.
    Note: We usually prepare large volume of CM (4 bottles) that we store at 4 °C and use to culture tissues within 2 weeks from preparation. Older CM can be used to keep tissue blocks during dissection, after being supplemented with Timentin.

Acknowledgments

This protocol was adapted from the following publications: Introini et al. (2013) and Grivel and Margolis (2009). This work was supported by the Intramural program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development.

References

  1. Biancotto, A., Brichacek, B., Chen, S. S., Fitzgerald, W., Lisco, A., Vanpouille, C., Margolis, L. and Grivel, J. C. (2009). A highly sensitive and dynamic immunofluorescent cytometric bead assay for the detection of HIV-1 p24. J Virol Methods 157(1): 98-101.
  2. Dezzutti, C. S. and Hladik, F. (2013). Use of human mucosal tissue to study HIV-1 pathogenesis and evaluate HIV-1 prevention modalities. Curr HIV/AIDS Rep 10(1): 12-20.
  3. Grivel, J. C. and Margolis, L. (2009). Use of human tissue explants to study human infectious agents. Nat Protoc 4(2): 256-269.
  4. Introini, A., Vanpouille, C., Lisco, A., Grivel, J. C. and Margolis, L. (2013). Interleukin-7 facilitates HIV-1 transmission to cervico-vaginal tissue ex vivo. PLoS Pathog 9(2): e1003148. 
  5. Merbah, M., Introini, A., Fitzgerald, W., Grivel, J. C., Lisco, A., Vanpouille, C. and Margolis, L. (2011). Cervico-vaginal tissue ex vivo as a model to study early events in HIV-1 infection. Am J Reprod Immunol 65(3): 268-278.
  6. Pudney, J., Quayle, A. J. and Anderson, D. J. (2005). Immunological microenvironments in the human vagina and cervix: mediators of cellular immunity are concentrated in the cervical transformation zone. Biol Reprod 73(6): 1253-1263. 
  7. Saba, E., Grivel, J.C., Vanpouille, C., Brichacek, B., Fitzgerald, W., Margolis, L. and Lisco, A. (2010). HIV-1 sexual transmission: early events of HIV-1 infection of human cervico-vaginal tissue in an optimized ex vivo model. Mucosal Immunol 3(3): 280-290.
  8. Saba, E., Origoni, M., Taccagni, G., Ferrari, D., Doglioni, C., Nava, A., Lisco, A., Grivel, J. C., Margolis, L. and Poli, G. (2013). Productive HIV-1 infection of human cervical tissue ex vivo is associated with the secretory phase of the menstrual cycle. Mucosal Immunol 6(6): 1081-1090.
  9. Vanpouille, C., Lisco, A., Introini, A., Grivel, J.C., Munawwar, A., Merbah, M., Schinazi, R. F., Derudas, M., McGuigan, C., Balzarini, J. and Margolis, L. (2012). Exploiting the anti-HIV-1 activity of acyclovir: suppression of primary and drug-resistant HIV isolates and potentiation of the activity by ribavirin. Antimicrob Agents Chemother 56(5): 2604-2611.

材料和试剂

  1. 从常规扁桃体切除术获得的扁桃体组织(患者年龄范围:2-10)
  2. 从常规子宫切除术获得的宫颈阴道组织(患者年龄范围:35-55)
  3. Gelfoam 12-7mm可吸附明胶海绵(Pfizer,NDC:0009-0315-08)
  4. RPMI 1640(Life Technologies,Gibco ,目录号:31870-025)
  5. 100x改良Eagle培养基(MEM) - 非必需氨基酸(10mM)(Life Technologies,Gibco ,目录号:11140-035)
  6. 100×MEM丙酮酸钠(100mM)(Life Technologies,Gibco ,目录号:11360-070)
  7. 1,000x庆大霉素(50mg/ml)(Corning,cellgro ,目录号:30-005-CR)
  8. 100x Fungizone(250μg/ml,两性霉素B)(Life Technologies,Gibco ,目录号:15290-018)
  9. 胎牛血清(FBS)(Gemini Bio-products,目录号:100-106)
    注意:我们建议测试几批血清进行文化优化,并对整个系列实验使用相同批次的FBS。我们总是在几个供体的组织上测试几个血清批次,并选择给出最高HIV-1复制的批次。此外,FBS可影响组织在培养基中分泌细胞因子的能力。
  10. 磷酸盐缓冲盐水(PBS)(pH 7.4)(Life Technologies,Gibco ,目录号:10010-023)
  11. 无菌水,细胞培养级(Quality Biological,目录号:118-162-101)
  12. HIV-1病毒制剂
    注意:对于我们的大多数实验,我们使用从病毒学质量保证实验室获得的以下病毒制备物:HIV-1 subAbL和HIV-1 subAID0.04 Rush大学(芝加哥,IL)。病毒原种获自用HIV-1 SubLaL或HIV-1 SubLa.04.04接种的外周血单核细胞培养物的澄清培养基,最初来自NIH AIDS试剂程序。对于HIV-1 subLaL和HIV-1 subAgL 1,HIV-1 p24agag浓度为49±3ng/ml和53±3ng/ml。/sub> stock。对于我们的实验设置中使用的更多病毒制剂,参见Introini等人,2013和Vanpouille等人,2012年。
  13. Timentin(GlaxoSmithKline,NDC:0029-6571-26)(参见配方)
    注意:Timentin是作为单独试剂可商购的抗生素替卡西林和克拉维酸的混合物的商品名。这些抗生素有效地防止在手术后偶尔可能污染组织样品的细菌的生长。可以使用青霉素和链霉素代替特美汀,尽管它们具有不同的性质。例如,特美汀在室温或37℃(约24小时)显示出低稳定性,因此,一旦加入到培养基中,其仅在培养的第一天保持活性。
  14. 培养基(CM)(见Recipes)

设备

  1. 培养皿(100mm×20mm)(BD,Falcon,目录号:353003)
  2. 培养皿(150mm×25mm)(BD,Falcon ,目录号:353025)
  3. 6孔板(Corning,Costar ,目录号:3506)
  4. 12孔板(Corning,Costar ,目录号:3513)
  5. 5,10-ml移液管(BD,Falcon )
  6. 密封螺旋盖1.5/2ml管(SARSTEDT AG)
  7. 具有用于1.5ml管(Eppendorf)的块的热混合器
  8. 镊子或镊子
  9. 手术刀和刀片 10和23
  10. 剪刀
  11. 平称重金属刮刀
  12. 37℃,设定在90%湿度的5%CO 2培养箱中
  13. 水浴
  14. 10,50 ml注射器活塞

程序

  1. 让CM有足够的时间达到室温或将其放在37°C预热的水浴中
  2. 根据所用组织的类型和进行实验所需的孔数(表1),计算每次实验所需的明胶海绵片数量(表1)。

    表1.文化设置
    组织类型
    海藻块数(1海绵)
    文化板
    培养基体积(每孔)(ml)
    块数(每个明胶块)
    每个实验条件下的孔数(重复)
    Tonsillar
      4
    6井
    3
    9
    3或更多
    宫颈阴道
    5-6
    12孔
    1
    8
    2以上

  3. 根据所需的明胶海绵块的数量,或者用约200ml补充有特美汀的CM填充100mm×20mm培养皿或者用约200ml填充150mm×25mm培养皿(如果需要超过3个海藻糖海绵 )。
  4. 使用乙醇灭菌钳将明胶海绵放入培养皿中,使用用乙醇灭菌的弯曲平刮刀将海绵压在培养皿底部约2分钟。
    注意:当脱水时,明胶海绵极脆。 水合过程应该小心,特别是当向下推泡沫以追逐空气时。 明胶海绵应该尽可能没有空气:存在 空气会阻塞营养物质通过其进入组织的毛细血管。
  5. 使用乙醇灭菌剪刀将再水化的明胶海绵切成适当大小的片(表1)。
  6. 使用镊子将一块明胶海绵放入每个孔,并用移液管加入适量的含有特美汀的CM(表1)。
  7. 将板放入培养箱。
    注意:解剖和感染方案因使用的组织而异。 如果使用扁桃体遵循选项A; 如果使用宫颈阴道组织,按照选项B.
  1. 扁桃体组织
    1. 将一个扁桃体放入一个100毫米的陪替氏培养皿中含有补充了特美汀的CM,并将扁桃体切成几个大片(4-5片)。
    2. 转移一块进入含有几毫升CM的培养皿的盖子。盖住含有剩余扁桃体片的培养皿,以避免组织干燥。
    3. 用10或23号刀片手术刀和镊子,去除烧灼部分和纤维组织。去除血腥/发炎的组织和任何含有扁桃体和/或绿棕色的部分(图1,参见Grivel和Margolis,2009的附加图片)。


      图1.制备用于培养的扁桃体组织
      (上)在接受的外科手术标本中,应该切除的部分是来自胶囊周围腭扁桃体(白色),烧灼组织(棕黑色),血腥/发炎组织(红色)绿色。
      (中)用镊子夹住样品的整齐切口去除不需要的大部分组织。虚线界定切割表面以移除烧灼组织层。对于小部件,可能更容易用钳子直接夹住它们以将其切除。
      (向下)将组织条切割成约8mm 3的块。将9个块转移到6孔板中的每个明胶片上。

    4. 将组织切成约2mm厚的切片。如上所述去除任何不需要的部分,并将切片切成2mm厚的条。然后将条切成2 mm厚的块。这将导致大约8毫米 3 的块。
    5. 根据需要切割多个块,每个实验条件需要至少27个块(表1)。用镊子,将块转移到含有CM补充的干净培养皿中,以避免组织块脱水,同时进行解剖。  
      注意:由于解剖可能需要很长时间,因此重要的是要在培养皿盖上加入培养基,以便在切割时保持组织湿润。
    6. 旋转板以随机化块的分布。
    7. 从培养箱中取出含有Gelfoam的六孔板,并使用镊子在每个Gelfoam片的顶部放置9块组织。
    8. 将板放回培养箱过夜培养或进行感染(转到步骤A11)。
      注意:我们优选在过夜孵育后进行感染,以确保没有细菌或真菌污染发展。此外,大量的细胞在解剖后流出扁桃体组织块。这个过程大部分在前24小时的文化中完成。
    9. 如果前一天晚上解剖组织,用移液管吸取六孔板中的CM,并丢弃。倾斜板,轻轻地推动明胶海绵回到井的上部,让介质聚集在底部,吸出并丢弃它。
    10. 用移液管,向每个孔中加入在37℃预热的3ml含有特美汀的CM。
    11. 解冻HIV-1病毒制剂在37°C预热的水浴中。如有必要,将病毒原液稀释至适当浓度。优选具有稀释物,使得所需的接种物包含在5至8μl的培养基中 注意:对于我们的大多数实验,我们使用6.0至7.5μl未稀释的HIV-1BaL或HIV-1LAI.04病毒原液(材料和试剂12)感染每个扁桃体组织块。对于有效的感染,至关重要的是允许解冻HIV-1病毒制剂和将接种物沉积在组织块上之间所需的最短时间。
    12. 将接种物直接移植到Gelfoam片上的9个块的顶部 注意:为了精确输送接种体积,我们建议使用反向移液技术,并更换每个孔的尖端。
    13. 感染完成后立即将培养板放回孵育器。

  2. 宫颈阴道组织
    1. 将组织片放在含有CM补充的Timentin的100-mm培养皿中。
    2. 转移一块组织到含有几毫升CM的培养皿的盖子。 盖住含有剩余组织的培养皿以避免干燥。
    3. 用23号刀片手术刀和镊子分离外宫颈粘膜和子宫颈粘膜从下面的基质组织获得粘膜条(图2)。


      图2.准备用于培养的宫颈阴道组织
      (Up)宫颈内膜是子宫内衬管腔的一部分,提供子宫腔和阴道间的连通。子宫颈的通过上前阴道壁突入阴道的部分称为外宫颈。在接受的宫颈阴道组织标本中,宫颈内粘膜容易被识别为具有深红色的部分,并且被厚粘液包裹,而外宫颈粘膜具有更清晰的颜色。
      (中)可以将外科手术标本切成更小的片以便于除去粘膜。用钳子保持基质组织,将其与粘膜切割组织标本在纵向(由箭头指示)相对于粘膜层。虚线限定切割表面并且应位于组织标本的上皮边缘下方2-3mm处。
      (向下)将粘膜条切割成约8mm 3的块。感染后,在12孔板中的每个凝胶块上转移8个块
    4. 将粘膜条切成2mm厚的条,除去并丢弃尽可能多的结缔组织肌肉组织,仅留下包含粘膜上皮,粘膜下层和部分基底基质的2mm厚的层。将条切成2 mm厚的块。这将导致大约8mm 3 的块。
    5. 根据需要切割多个块,每个实验条件需要至少16个块(表1)。用镊子,将块转移到含有CM补充的干净培养皿中,以避免组织块脱水,同时进行解剖。  
      注意:对于我们的大多数实验,我们从endocervix和ectocervix集合块。可以考虑保持它们分离,因为它们具有不同的结构和免疫特征(Pudney等人,2005)。此外,宫颈内膜通常在培养物中继续产生粘液,这可能干扰后续测定。注意,在接收的宫颈阴道组织标本中,宫颈内膜远小于外宫颈。
    6. 旋转板以随机化块的分布。
    7. 用镊子,将块转移到无菌的1.5毫升锥形管(s)(每管最多16块)。
    8. 解冻HIV-1病毒制剂在37°C预热的水浴中。如有必要,将病毒原液稀释至适当浓度,并将其转移到含有块的管中 注意:
      a。 Cervico阴道组织在解剖时最好被感染。我们建议包括实验条件,其中将病毒和强大的抗病毒药物组合。这是一个有用的控制,以确定病毒是否实际复制,而不是被组织吸附和释放。
      b。 对于我们的大多数实验,我们使用500μl未稀释的HIV-1BaL病毒原液(材料和试剂12)感染12-16个宫颈阴道组织块。我们在HIV-1制剂中添加拉米夫定10μM,在整个培养长度期间添加CM作为HIV-1释放的对照。
    9. 将管转移到在37℃预热的Thermomixer中,并以300转/分钟设定混合。孵育2小时,每半小时轻轻倒转管数次。
      注意:对于有效的感染,允许在解冻HIV-1病毒制备物和将管转移至37℃之间所需的最短时间是至关重要的。
    10. 用3-4ml PBS填充6孔板的孔(如果超过6个管用于感染,则更多的板)。
    11. 从试管中弃去病毒制剂。 使用镊子,轻轻地将块传递到6孔板盖以去除残留在块上的过量的病毒制备物,然后将块转移到含有PBS的孔中。
    12. 用吸管轻轻旋转,等待1分钟。 用移液管吸入PBS,小心不要吸入块。
    13. 加入3-4毫升PBS的孔,用吸管尖轻轻旋转,等待1分钟。
    14. 重复步骤B12-13三次。
      注意:这些洗涤步骤是必要的,因为在宫颈阴道组织中的HIV-1复制不像在扁桃体组织中那样高,并且可以被随后释放的吸收在组织上的病毒掩盖。
    15. 取12孔板从孵化器,并使用镊子转移8块在每个Gelfoam片的顶部。
    16. 将板放回培养箱。

A和B共有

  1. 感染HIV-1后三天,从每个重复孔中收获一份培养基,并将其转移到螺旋盖管中。
    注意:我们通常会将重复井的等分试样混合。
  2. 使用乙醇灭菌镊子放回可能从明胶海绵掉下的任何块组织。
  3. 用移液管,从板上丢弃剩余的介质。 倾斜板,轻轻地带上明胶海绵到井的上部,让介质向下流到底部,吸出并丢弃它。
  4. 用移液管加入足够体积的预热至37℃的新鲜CM(表1) 注意:在这个阶段不需要补充CM与Timentin。
  5. 将板放回培养箱。
  6. 将培养基等分试样储存在-80℃。
  7. 每3天重复步骤1至5,直到第12-15天。
  8. 在感染后12-15天,使用镊子收集组织块,并根据实验需要处理它们。
  9. 对于RNA提取,我们通常在4℃下将组织块保持在RNA中过夜,然后将其储存在-80℃。 对于流式细胞术,我们立即通过用酶混合物消化组织块来处理组织块 获得单细胞悬液。
    注意:在文化期间,组织长出到明胶海绵片。单个细胞也可以从组织迁移到凝胶中。可以通过用镊子夹紧凝胶,分别分析凝胶上方的组织部分。凝胶内的细胞可以使用镊子或注射器柱塞挤出。然而,应该记住,从凝胶中提取的不仅仅是迁移细胞的一部分,而是这些细胞和组织结构的混合物。
  10. 根据机构处理人类传染性废物所需的生物安全措施,将板块与任何残留的CM,组织块和明胶海绵一起处理。
  11. 许多技术可用于评估HIV-1在组织块中的产生和复制。我们的标准读出是使用基于荧光珠的免疫测定法(Biancotto等人,2009)在整个培养时间收获的培养物上清液中测量HIV-1p24ag gag。
    注意:用相同HIV-1原种的相同接种物感染的来自不同供体的组织可以通过培养物上清液中的p24 gag积累测量得到不同的结果(表2)。这可能是由于几个因素,例如HIV-1靶细胞的数量和活化状态以及共感染病原体的存在,其影响HIV-1在组织块中的复制和产生。因此,在实验的不同条件的设置中使用来自仅一个供体的组织(供体匹配条件)是至关重要的。

    表2. HIV-1p24 gag接种物的参考值和在组织外植体中的相对产量 HIV-1 p24 gag 生产被表示为累积的在培养12天(第6天+第9天+第12天)中每3天取样培养上清液中测量的p24 gag值的值。排除了在第3天测量的p24 gag的值,因为它还包含从HIV-1接种物释放的p24 gag从新吸收的组织块连同新产生的p24 > gag 作为病毒复制的结果。通过在感染的供体匹配的块的培养物上清液中测量的p24qag值来归一化感染有HIV-1 subLaL的子宫颈阴道组织中的HIV-1产生的值,在拉米夫定10μM的存在下培养。感染HIV-1 sub-LAI.04的子宫颈 - 阴道组织不会导致我们的HIV-1复制 实验设置。 使用基于荧光珠的免疫测定法测量HIV-1p24ag gag(Biancotto等人,2009)。
    组织类型
    病毒
    每块的接种物(pg HIV-1 p24 sub gag )
    块数
    HIV-1 p24 gag
      production(ng/ml)
    Tonsillar
    HIV- 1BaL
    367.5
    27
    1-10

    HIV-1 LAI.04
    397.5
    27
    10-100
    宫颈阴道
    HIV- 1BaL
    1531.2
    16
    1-5
    HIV- 1LAI.04
    1656.2
    16
    检测不到

笔记

  1. 对于我们的实验,我们使用从指示的手术收集的组织,其不是为了研究的目的而收获的,因此可以被认为是"病理性废物"。在任何情况下,人体组织必须根据伦理审查委员会批准的方案收获。从组织供体收集个人和医疗数据,例如使用药物,月经周期,性行为,使用避孕药等可以提供重要的信息来解释实验结果。例如,参见Saba等人,2013年。收集这些数据提出了关于知情同意和隐私的伦理问题,必须在组织收获方案中解决。
  2. 输送到实验室的组织应处于良好状态,具有"健康"的粉红色,并且缺乏大血块和绿色坏死中心。组织的状态还取决于其被递送用于处理的速度。对于长期培养来说,用健康的组织开始实验是重要的。扁桃体组织是软的并且易于变质。根据我们的经验,这种组织应该在手术后3-5小时内交付。我们注意到,在交付期间在冰上冷却使其不太适合长期培养。因此,我们在传送过程中在室温下在PBS中维持扁桃体组织。宫颈阴道组织更坚固,我们成功感染艾滋病毒1和培养组织24 h后手术。我们使用冰冷的RPMI 1640作为输送介质
  3. 应该告知操作者关于使用血源性病原体的风险,并接受适当的培训以在生物安全2级实验室中工作,以便处理组织标本,即使实验不涉及使用HIV-1。
  4. 注意和图片为更细致的实验者,任何特点的组织标本,你怀疑可能会影响实验结果,如血腥,烧灼和坏死部分。我们建议保留进行解剖的介质以及任何未使用的组织,因为它们可能有助于评估细胞活力,归档DNA/RNA或组织学分析。
  5. 优化表2中报告的HIV-1接种物的值以进行感染,其中将病毒储存的组织标本递送至实验室,并按照标准程序进行处理以最小化不是由于组织的固有性质引起的任何变化。然而,HIV-1的产生在来自不同供体的组织之间显着变化。因此,我们邀请读者将表2中报告的值作为参考,同时铭记多个捐赠者间因素会影响感染的结果。因此,我们建议只使用捐献者匹配的实验条件,并对来自多个捐献者的平均数据进行适当的统计分析
  6. 上述实验系统是非极化的,意味着HIV-1的穿透可以发生在组织块的所有侧面。该系统的目的不是模拟通过上皮的HIV-1传输,而是调查调节组织中HIV-1传播的机制。对于子宫颈阴道组织,这种培养系统仍然可以模拟HIV-1传输,因为在体内有证据证明HIV-1可以通过预先存在的上皮微阵列到达粘膜的上皮上层造成性交。具有HIV-1的极化感染和组织外植体培养物的实验设置已由Dezzutti和Hladik,2013中的其他人描述。这两种系统作为HIV-1传播模型的优点和缺点在Merbah等人, ,

食谱

  1. 特美汀
    通过加入100 ml无菌水到3.1 g小瓶的特美汀制备一个工作的100x储备溶液。在无菌螺帽管中等分,冷冻并保持等分在-30℃。未使用的解冻等分试样应丢弃。
  2. 培养基(CM)
    从500 ml的RPMI1640瓶中取出90 ml(留作其他用途)
    添加
    75 ml FBS
    5ml的100×MEM非必需氨基酸
    5ml 100x的菌剂
    5ml的100×MEM-丙酮酸钠 500μl1,000x庆大霉素
    储存在4°C
    仅在使用前补充CM与特门汀。稀释1毫升新鲜解冻的100x特美汀在99毫升CM。考虑到需要额外的CM来制备明胶海绵和切割的组织,根据表1中的指示准备实验所需的体积。
    注意:我们通常准备大量的CM(4瓶),我们存储在4°C,并使用从培养2周内培养组织。较旧的CM可以用于在解剖期间保持组织块,在补充之后 Timentin。

致谢

该协议改编自以下出版物: Introini等人(2013)和Grivel和Margolis(2009)。 这项工作得到了Eunice Kennedy Shriver国家儿童健康和人类发展研究所的校内方案的支持。

参考文献

  1. Biancotto,A.,Brichacek,B.,Chen,S.S.,Fitzgerald,W.,Lisco,A.,Vanpouille,C.,Margolis,L.and Grivel,J.C。(2009)。 用于检测HIV-1 p24的高灵敏度和动态免疫荧光细胞计数珠测定法。 J Virol Methods 157(1):98-101。
  2. Dezzutti,C. S.和Hladik,F。(2013)。 使用人体粘膜组织研究HIV-1发病机制并评估HIV-1预防模式。 HIV/AIDS Rep 10(1):12-20
  3. Grivel,J.C。和Margolis,L。(2009)。 使用人体组织外植体研究人类感染因子。 Nat Protoc 4(2):256-269。
  4. Introini,A.,Vanpouille,C.,Lisco,A.,Grivel,J.C.and Margolis,L。(2013)。 白细胞介素7促进艾滋病毒1传播到子宫颈阴道组织离体 > 。 PLoS Pathog 9(2):e1003148。
  5. Merbah,M.,Introini,A.,Fitzgerald,W.,Grivel,JC,Lisco,A.,Vanpouille, ,L.(2011)。 离体宫颈阴道组织作为研究早期事件的模型HIV-1 infection。 Am J Reprod Immunol 65(3):268-278。
  6. Pudney,J.,Quayle,A.J.and Anderson,D.J。(2005)。 人类阴道和宫颈中的免疫微环境:细胞免疫的介质集中在子宫颈转化区。 73(6):1253-1263。 
  7. Saba,E.,Grivel,J.C.,Vanpouille,C.,Brichacek,B.,Fitzgerald,W.,Margolis,L。和Lisco,A。(2010)。 HIV-1性传播:人类子宫颈阴道组织HIV-1感染的早期事件优化的离体模型。粘膜免疫 3(3):280-290。
  8. Saba,E.,Origoni,M.,Taccagni,G.,Ferrari,D.,Doglioni,C.,Nava,A.,Lisco,A.,Grivel,JC,Margolis,L.and Poli, )。 离体人类子宫颈组织的生产性艾滋病毒1感染与离体相关联月经周期的分泌期。粘膜免疫 6(6):1081-1090。
  9. Vanbouille,C.,Lisco,A.,Introini,A.,Grivel,JC,Munawwar,A.,Merbah,M.,Schinazi,RF,Derudas,M.,McGuigan,C.,Balzarini, L.(2012)。 利用阿昔洛韦的抗艾滋病毒1活性:抑制原发性和耐药性艾滋病毒分离株和利巴韦林的活性增强。抗微生物剂化学 56(5):2604-2611。
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How to cite this protocol: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Introini, A., Vanpouille, C., Grivel, J. C. and Margolis, L. (2014). An ex vivo Model of HIV-1 Infection in Human Lymphoid Tissue and Cervico-vaginal Tissue. Bio-protocol 4(4): e1047. DOI: 10.21769/BioProtoc.1047; Full Text
  2. Introini, A., Vanpouille, C., Lisco, A., Grivel, J. C. and Margolis, L. (2013). Interleukin-7 facilitates HIV-1 transmission to cervico-vaginal tissue ex vivo. PLoS Pathog 9(2): e1003148.




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