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May 2018

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Transcytosis Assay for Transport of Glycosphingolipids across MDCK-II Cells
MDCK-II细胞间鞘糖脂运输的跨细胞转运检测   

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Abstract

Absorption and secretion of peptide and protein cargoes across single-cell thick mucosal and endothelial barriers occurs by active endocytic and vesicular trafficking that connects one side of the epithelial or endothelial cell (the lumen) with the other (the serosa or blood). Assays that assess this pathway must robustly control for non-specific and passive solute flux through weak or damaged intercellular junctions that seal the epithelial or endothelial cells together. Here we describe an in vitro cell culture Transwell assay for transcytosis of therapeutic peptides linked covalently to various species of the glycosphingolipid GM1. We recently used this assay to develop technology that harnesses endogenous mechanism of lipid sorting across epithelial cell barriers to enable oral delivery of peptide and protein therapeutics.

Keywords: Transcytosis (跨细胞转运), Endocytic sorting (内吞分选), Glycosphingolipids (鞘脂糖), Epithelial barriers (上皮屏障), Drug delivery (药物运输)

Background

Transport of large molecules across the single-cell thick epithelial barriers lining mucosal surfaces and tight endothelial barriers lining vessels serving the heart muscle and brain occurs by an endocytic process that connects one side of these polarized cells with the other. This process is termed transcytosis (Garcia-Castillo et al., 2017). The absorption and secretion of immunoglobulins by receptor-mediated endocytosis and vesicular transport across the mucosa of the alimentary and respiratory tracts most famously typifies this process. Interest in transcytosis is also stimulated by the potential to harness this pathway for the delivery of therapeutic peptides and proteins across tight epithelial and endothelial barriers (Thuenauer et al., 2017).

Transcytosis is an active (ATP-driven) process. In some cases, transport of large molecules across tight epithelial and endothelial barriers can occur by passive diffusion around the cell through intercellular tight junctions (Fung et al., 2018). But aside from this small level of paracellular leakiness, physiologic meaningful paracellular transport of large molecules around cells occurs only when intercellular tight junctions are dismantled–usually under pathologic conditions. Non-specific and low levels of transcellular transport for large solutes may also occur by fluid-phase endocytosis and miss-sorting of cargo into transcytotic vesicles rather than into the late endosome/lysosome pathway, which receives the bulk of cargo internalized by non-receptor mediated mechanisms. Thus, any assay for transcytosis across healthy epithelial or endothelial barriers in vitro or in vivo needs to control for these confounding non-specific pathways.

We have recently discovered that the endogenous sorting of glycosphingolipids across epithelial barriers can be harnessed for oral delivery of therapeutic peptides in vivo (Garcia-Castillo et al., 2018). Here we describe the in vitro assay using cultures polarized epithelial cells grown on transwell filters that we used to develop the technology that co-opts these endogenous mechanisms of lipid sorting for this purpose. The test compounds used to harness glycolipid sorting are modified to contain a biotin residue to allow for biochemical capture and a fluorophore for detection. Transwell inserts are composed of an inner chamber made with a permeable polycarbonate membrane support where the cells are seeded (Figure 1). After 3-7 days the epithelial cells assemble into a single cell thick monolayer with sealed tight junctions among cells. As such, the inner chamber of the transwell is exposed to the apical membrane of the epithelial monolayer and models the lumenal surface and can hold 200 µl of solution. The outer chamber–the chamber below the permeable support–is exposed to the basolateral surface of the monolayer and models the serosal surface of the epithelial barrier. This chamber holds 1 ml of basolateral solution. The assembly of the monolayer into a “tight” epithelial barrier with sealed functional tight junctions is routinely measured by passive resistance to small ion transport (TER–Transepithelial Resistance) measured by standard direct current electrophysiology or using the alternate current based machine EVOM. Test compounds are added to the apical chamber, and the assay is allowed to proceed to allow for transport across the barrier, where the basolateral chamber is then sampled for the transcytosed analyte using streptavidin-coated beads (Figure 2).


Figure 1. Schematic representation of MDCK-II transwell inserts and transcytosis assay


Figure 2. Capture and read of biotinylated peptide–fusions from basolateral media (Adapted from Figure 1A in Garcia-Castillo et al., 2018. Creative Commons Attribution License)

Mid-picomolar sensitivity can be achieved with this assay. Our basic approach can be used to measure transport by other molecules that enter the transcytotic pathway–such as for IgG that binds to the Fcγ-receptor FcRn that traffics in both directions across polarized epithelial monolayers (Nelms et al., 2017). The strength of our assay is the ability to directly, quantitatively, and sensitively measure the amount of compound transcytosed. Non-specific paracellular leak between cells versus real transport through cells is controlled in several ways, such as by using an analyte that cannot engage the transcytotic pathway (in our case the reporter peptide lacking fusion to the glycolipid carrier, or by fusion to a glycolipid with ceramide structure that cannot enter the transcytotic pathway), or by inhibiting endocytosis or transcytosis via a 4 °C temperature block, by chemical inhibition of endocytosis using Dyngo, and by siRNA knockdown of genes responsible for transcytosis as described in Garcia-Castillo et al. (2018).

Materials and Reagents

Notes:

  1. Material and reagents are stored as per the manufacturer’s recommendation. 
  2. Fluorescent peptides and glycosphingolipid-peptide fusions are used as described in Garcia-Castillo et al., 2018. The assay requires the presence of both a fluorophore and a biotin covalently attached to compound you want to measure (see Figure 3). Peptide fusions containing biotin and fluorophore attached to ganglioside GM1 were synthesized in-house using specialized methods described in (Garcia-Castillo et al., 2018). Precursor peptides were synthesized by New England Peptide (Gardner, MA, USA), and gangliosides were purchased from Dr. Sandro Sonnino (U. Milan, Italy). However, in theory, any macromolecule (peptide, protein, nucleic acid or chemical) containing both a fluorophore and biotin will work in this assay. An example compound is shown below. Here, lower-cased amino acids are depicting D-isomers. Since glycine (shown as upper-cased G) does not have chirality, the peptide essentially contains no L-amino acids that can be susceptible to degradation in vivo.


    Figure 3. Structure of peptide-lipid fusion constructs used in transcytosis assay 

  1. Pipette tips (USA Scientific, catalog numbers: 1126-7810 , 1120-8810 , 1121-3810 )
  2. 0.4 μm pore size Transwell® inserts (Corning, catalog number: 3413 )
    Note: Transwell® plates are kept at room temperature.
  3. 96-well Assay Plate (No Lid, Black Flat Bottom, Non-treated, polystyrene) (Corning, Costar®, catalog number: 3916 )
  4. Aluminium-foil (Fisher Scientific, FisherbrandTM, catalog number: S05356A )
  5. MDCK-II cells (Parental line from American Type Culture Collection) (ATCC, catalog number: CCL-34 ) (Kind gift from Dr. Steven Claypool)
  6. Fatty acid-free bovine serum albumin (Sigma-Aldrich, catalog number: A6003-10G ) (lyophilized powder, essentially fatty acid free)
  7. PierceTM Streptavidin magnetic beads (Thermo Fisher Scientific, catalog number: 88817 )
    Note: Beads are stored at 4 °C per the manufacturer's recommendation.
  8. Formamide (Sigma-Aldrich, catalog number: F9037-100ML )
  9. Dimethylformamide (DMF) (Sigma-Aldrich, catalog number: 227056 )
  10. Biotin (Sigma-Aldrich, catalog number: B4501-1G )
  11. DMEM 4.5 g/L D-glucose (Corning, catalog number: 10-013-CVR )
  12. Pen/Strep, 100x (Thermo Fisher Scientific, GibcoTM, catalog number: 15140148 )
  13. Fetal Bovine Serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: A3160502 )
  14. Tris base (Sigma-Aldrich, catalog number: T1503-1KG )
  15. NaCl (Sigma-Aldrich, catalog number: S9625-10KG )
  16. Tween 20 (Sigma-Aldrich, catalog number: P2287-500ML )
  17. 0.25% Trypsin-EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25200072 )
  18. PBS (Thermo Fisher Scientific, GibcoTM)
  19. EDTA (Sigma-Aldrich, catalog number: E4884-500G )
  20. MDCK-II complete growth media (see Recipes)
  21. Basolateral solution (see Recipes)
  22. Apical solution (see Recipes)
  23. TBS 10x (see Recipes)
  24. 1x TBS-T (see Recipes)
  25. 0.5 M EDTA pH 8.0 (see Recipes)
  26. Elution buffer (see Recipes)

Equipment

  1. Portable Pipet-Aid® XP Pipette Controller (Drummond Scientific, model: 4-000-101 )
  2. Incubator Forma Scientific CO2 water-jacketed incubator (Thermo Fisher Scientific, model: FormaTM Series II , catalog number: 3110)
  3. Centrifuge (Eppendorf, model: 5415C )
  4. EVOM2 Epithelial Voltohmmeter (World Precision Instruments, model: EVOM2 )
  5. TECAN Spark multimode microplate reader (Tecan Trading, model: Spark® )
  6. MagneSphere® Technology Magnetic Separation Stand (Promega, catalog number: Z5342 )
  7. Sonicator (Stainless Steel AquaSonic Ultrasonic cleaner, VWR, model: 50T )

Software

  1. GraphPad Prism 7
    GraphPad Software 7825 Fay Avenue, Suite 230 La Jolla, CA 92037 USA (https://www.graphpad.com/scientific-software/prism/)

Procedure

  1. Day 1: Plating and polarizing cells
    1. Trypsinization
      1. Aspirate MDCK-II growth media from the stock cell culture flask used for passaging. 
      2. For a T-75 flask of MDCK-II cells, add 5-10 ml of warmed, sterile PBS. Gently tilt/rotate flask to wash the cells. Remove PBS.
      3. For a T-75 flask, add 3 ml of 0.25% Trypsin-EDTA. Tilt/rotate flask to make sure cell surface is evenly coated.
      4. Return to a 37 °C incubator with 5% CO2 under humidified conditions for 5-10 min.
      5. Observe under a light microscope with phase contrast. If cells are properly detached, they will appear round and floating in suspension.
      6. Collect cell suspension in conical tube and pipet cells 6-8 times up and down. No clumps should be observed. Cell suspension should be a homogeneous mixture. 
    2. Add MDCK-II cells at a density of 200,000 cells in 200 μl in complete growth media (see Recipes) to the apical side of a Transwell® insert (12-well 0.4 μm pore size Transwell®-65 mm). In addition, 1 ml complete growth media is added to the basolateral chamber.
      1. Two Transwell® inserts are prepared for (Untreated) controls, 2 Transwell® inserts for reporter peptide, and 2 Transwell® per glycosphingolipid-peptide fusion tested. 
      2. Each condition is tested as biological duplicates (i.e., 2 Transwell® inserts per condition). Experiments must always include untreated and reporter-peptide controls.
    3. Incubate the plate for 2-3 days to allow for polarization at 37 °C with 5% CO2 under humidified conditions.

  2. Day 3: Preparation of testing glycosphingolipid compounds
    1. Centrifuge the tube containing lyophilized compound at 13,000 rpm (~13,800 x g) for 3 min to pellet the material to the bottom of tube.
    2. Add 1 part volume of DMF (actual volume depends on stock concentration–for our compounds, this is ~60 μl), sonicate for 30-60 sec, followed by ~5 sec vortex.
      Notes: 
      1. Sonication is done in a bath sonicator (VWR, AquaSonic, model 50T ).
      2. You want to reach a high final concentration in the ~50-200 μM range (in a tube containing approximately 100 μg of lyophilized peptide [MW ~2,103], add 50 μl of DMF and 100 μl of water to obtain a stock solution in the ~50-200 μM range). The concentration of all test compounds is determined using NanoDrop (For the Alexa Flour 488 used in our studies, we use absorbance at 495 nm).
      3. The solution may appear cloudy and orange-red at this point.
    3. Add 2 parts H2O (for example 120 μl water to give a final volume of 180 μl) followed by a quick 5 sec vortex to reach a final 33% DMF in H2O.
      Notes: 
      1. The solution should be bright green and clear of particulates. If it is orange, then add either more DMF or H2O until it turns green. (The color change we observed is for an Alexa Flour 488 attached to the reporter peptide) 
      2. At this point, this stock solution will be used to make the final dilution into “Apical” media for adding onto cells.
    4. To prepare this Apical test solution, dilute the appropriate volume of stock into Apical Solution (see Recipes) to reach a final concentration of 0.1 μM.
      Note: The goal is to have a final 1:1 ratio of lipid to dfBSA.

  3. Transcytosis in MDCKII cells
    1. Check electrical resistance of MDCK-II Transwell® inserts after 2-3 days using EVOM Epithelial Voltohmmeter to measure integrity of tight junctions.
      Note: Acceptable electrical resistances of MDCK-II monolayers used for transcytosis experiments is in the 250Ω-300Ω range.
    2. Prepare apical and basolateral solutions (see Recipes).
    3. Wash transwells 2 times using serum-free DMEM. Replace media with apical (200 μl) and basolateral (1 ml) solutions in the respective chamber. 
    4. Allow cells to equilibrate for 15 min in a 37 °C/5% CO2 cell culture incubator.
    5. Replace apical chamber with 200 μl apical solution containing 0.1 μM reporter peptide or 0.1 μM glycosphingolipid fusion.
      Note: Reporter peptide and glycosphingolipid fusion stocks range from 30 μM to 150 μM in 33% DMF/66% H2O. 
    6. Incubate for 3 h in a 37 °C/5% CO2 cell culture incubator.
    7. To quantify transcytosed peptide and lipid-peptide fusions, collect basolateral media in pre-labeled Eppendorf tubes and proceed to streptavidin pull-down assay. 
    8. To calculate the apparent permeability coefficient (PAPP, cm/sec), apical chamber solution is also collected in pre-labeled Eppendorf tubes. A standard curve ranging from 0 nM to 200 nM is used to interpolate concentration of peptide or lipid-peptide fusion in apical chamber after 3 h.

  4. Streptavidin Pull-down Assay of basolateral media samples
    1. Wash Streptavidin magnetic beads 3 times with TBS-T.
      Notes:
      1. Beads are never vortexed. They are brought into solution in stock tube by gentle inverting.
      2. Ten microliter beads are needed per sample.
      Example: For 12 samples, place 120 μl Streptavidin beads in an Eppendorf tube and wash 3 times each with 1 ml TBS-T using magnetic rack.
    2. Resuspend 10 μl beads in 50 μl TBS then add to 1 ml basolateral sample
      Example: For 12 samples, resuspend 120 μl Streptavidin beads by adding 600 μl TBS. Then, add 50 μl Streptavidin beads to each basolateral sample.
    3. Incubate basolateral samples with streptavidin beads overnight at 4 °C with head-over rotation covered in foil.

  5. Day 4: Elution and read-out of basolateral samples
    1. Collect the beads using a magnetic rack and wash 3 times with TBS-T.
      Note: Each wash is done with 1 ml TBS-T and beads mixed by inverting.
    2. Bound peptide or lipid-peptide fusions are eluted from beads by addition of 220 μl elution buffer (See Recipes) and boiling.
      Notes:
      1. Invert the beads to ensure that they are in solution.
      2. After beads are in solution, boil for 2 min at 65 °C in a heat block.
    3. After boiling, collect the beads using a magnetic rack. 
    4. Pipet 100 μl of each sample x 2 (technical replicates) on a black 96-well plate.
    5. Fluorescence is read on a TECAN Spark microplate reader for Alexa-488 channel and against a standard curve for each compound. 
      1. Settings:

      2. Sample standard curves (Figure 4)


        Figure 4. Sample standard curves for peptide (blue) and lipid-peptide fusion (red) used to calculate the amount of transcytosed compound. Plotted are the mean ± SEM of 3 technical replicates.

      3. A basolateral standard curve for each compound being tested is made in elution buffer ranging from 0 pM to 1,000 pM.
        1. Make 1 μM solutions of peptide and lipid-peptide stocks in elution buffer.
        2. Make 1 ml of 10 nM solutions (10 μl 1 μM + 990 μl elution buffer).
        3. Make serial dilutions with elution buffer beginning with 1,000 pM:
          1,000 pM = 100 μl 10 nM + 900 μl elution buffer
          500 pM = 500 μl 1,000 pM + 500 μl elution buffer
          250 pM = 500 μl 500 pM + 500 μl elution buffer
          125 pM = 500 μl 250 pM + 500 μl elution buffer
          62.5 pM = 500 μl 125 pM + 500 μl elution buffer
          31.5 pM = 500 μl 62.5 pM + 500 μl elution buffer
          15.6 pM = 500 μl 31.5 pM + 500 μl elution buffer
          0 pM = elution buffer

Data analysis

GraphPad Prism software is used to calculate the concentration of lipid-peptide fusion. As detailed above, a standard curve for each test compound is used to relate fluorescence intensity to a known concentration of peptide or lipid-peptide fusion. Absorbance at 495 nm is initially used to calculate stock concentrations. For detailed instructions on graphing and interpolating standard curves, the reader is referred to the GraphPad software website.
To calculate the apparent permeability coefficient (PAPP, cm/sec) a standard curve ranging from 0 nM to 200 nM is used to interpolate concentration of peptide or lipid-peptide solution remaining in the apical chamber after a 3 h continuous incubation.

The apparent permeability coefficient (PAPP, cm/sec) is calculated across cell monolayers grown in transwells based on the appearance rate of lipid-peptide fusions in the basolateral compartment over time:
Papp (cm/sec) = [VD/(A x MD)] x (DMR/Dt)
Papp (cm/sec) = [cm3/(cm2 x mol)] x (mol/sec)

VD = apical (donor) volume (cm3) (e.g., 0.2 ml = 0.2 cm3)
MD = apical (donor) amount (mol)
A = membrane surface area (cm2) of apical (donor) chamber (i.e., transwell surface area = 0.33 cm2 in this protocol)
DMR/Dt = the amount of compound (mol) transferred to the basolateral (receiver) compartment over time (sec).

Notes

  1. This protocol is also used routinely in our lab with human intestinal T84 cells and human colonic Caco-2 cells.
  2. Tight junction integrity must be confirmed using EVOM measurements.
  3. To dissolve reporter peptide and glycosphingolipid-reporter peptide fusions first add DMF and sonicate for 30-60 sec, then add water.
  4. Assay is done in serum-free DMEM. 
  5. The molar ratio 1:1 of compound to defatted-BSA is used.
  6. When preparing compound solutions, keep solutions at 37 °C and never place diluted lipids on ice as they may form micelles. 
  7. A typical control for paracellular leak is a 4 °C temperature block to stop endocytosis/transcytosis machinery.
  8. We use this assay in our lab to test the effect of gene silencing (using esiRNAs) and small molecule drugs (as is described for Dyngo-4a in Garcia-Castillo et al., 2018). 
  9. Using this assay, we performed structure-function studies of glycosphingolipid-peptide fusions containing modifications to the ceramide moiety. Also, using this assay, we tested for transcytosis of the incretin hormone GLP-1 (glucagon-like peptide 1).

Recipes

  1. MDCK-II complete growth media
    DMEM
    10% FBS
    1x Pen/Strep
    Store at 4°C
  2. Basolateral solution (40 ml)
    40 ml DMEM
    400 mg defatted-BSA (df-BSA) (final concentration: 1% [w/v])
    Prepared fresh on the day of assay
  3. Apical solution (40 ml)
    40 ml DMEM
    24.6 μl basolateral solution (Recipe 2, df-BSA final concentration: 0.1 μM)
    Prepared fresh on the day of assay
  4. TBS 10x (1 L)
    24 g Tris base
    88 g NaCl
    Dissolve in 900 ml distilled water and pH 7.4
    Add distilled water to a final volume of 1 L
    Store at room temperature
  5. 1x TBS-T (1 L)
    100 ml 10x TBS
    1 ml Tween 20
    900 ml distilled water
    Store at room temperature
  6. 0.5 M EDTA pH 8.0 (1 L)
    186.1 g Na2EDTA to 800 ml MilliQ H2O
    Add NaOH to reach pH 8.0
    Add MilliQ H2O up to 1 L
  7. Elution buffer (95% Formamide, 10 mM EDTA, 0.4 mg/ml biotin) (100 ml)
    95 ml 100% formamide
    2 ml 0.5 M EDTA, pH 8.0
    40 mg biotin
    3 ml MilliQ H2O

Acknowledgments

This project was supported by an NIH F-32 DK111072-01 to M.D.G-C; DK084424, DK048106, DK104868, and an unrestricted innovator grant from Novo Nordisk to W.I.L; DK104868 to D.C. We acknowledge the Harvard Digestive Disease Center DK034854. We are grateful to all members of the Lencer and Chinnapen laboratories for their helpful discussions.

Competing interests

The authors declare that there are no conflicts of interest or competing interests.

References

  1. Fung, K. Y. Y., Fairn, G. D. and Lee, W. L. (2018). Transcellular vesicular transport in epithelial and endothelial cells: Challenges and opportunities. Traffic 19(1): 5-18. 
  2. Garcia-Castillo, M. D., Chinnapen, D. J. and Lencer, W. I. (2017). Membrane transport across polarized epithelia. Cold Spring Harb Perspect Biol 9(9): a027912. 
  3. Garcia-Castillo, M. D., Chinnapen, D. J., Te Welscher, Y. M., Gonzalez, R. J., Softic, S., Pacheco, M., Mrsny, R. J., Kahn, C. R., von Andrian, U. H., Lau, J., Pentelute, B. L. and Lencer, W. I. (2018). Mucosal absorption of therapeutic peptides by harnessing the endogenous sorting of glycosphingolipids. Elife 7: e34469.
  4. Nelms, B., Dalomba, N. F. and Lencer, W. (2017). A targeted RNAi screen identifies factors affecting diverse stages of receptor-mediated transcytosis. J Cell Biol 216(2): 511-525. 
  5. Thuenauer, R., Muller, S. K. and Romer, W. (2017). Pathways of protein and lipid receptor-mediated transcytosis in drug delivery. Expert Opin Drug Deliv 14(3): 341-351.

简介

单细胞厚粘膜和内皮屏障上的肽和蛋白质货物的吸收和分泌通过活性内吞和囊泡运输发生,其连接上皮细胞或内皮细胞(管腔)的一侧与另一侧(浆膜或血液)。 评估该途径的测定必须通过弱的或受损的细胞间连接强有力地控制非特异性和被动的溶质通量,所述细胞间连接将上皮细胞或内皮细胞密封在一起。 在这里,我们描述了一种体外>细胞培养Transwell测定法,用于与各种鞘糖脂GM1共价连接的治疗性肽的转胞吞作用。 我们最近使用该测定开发了技术,该技术利用跨上皮细胞屏障的脂质分选的内源机制,以实现肽和蛋白质治疗剂的口服递送。

【背景】
大分子穿过覆盖粘膜表面的单细胞厚的上皮屏障和衬在供给心肌和脑的血管的紧密内皮屏障上的运输通过内吞过程发生,该内吞过程将这些极化细胞的一侧与另一侧连接。该过程称为转胞吞作用(Garcia-Castillo et al。>,2017)。通过受体介导的内吞作用和穿过消化道和呼吸道粘膜的囊泡运输的免疫球蛋白的吸收和分泌最着名的是这一过程。对转胞吞作用的兴趣也受到利用该途径在紧密上皮和内皮屏障上递送治疗性肽和蛋白质的潜力的刺激(Thuenauer 等人,>,2017)。

转胞吞作用是一种活跃的(ATP驱动的)过程。在一些情况下,通过细胞间紧密连接在细胞周围被动扩散可以发生大分子跨越紧密上皮和内皮屏障的转运(Fung 等,>,2018)。但除了这种小的细胞间渗漏水平外,细胞周围的大分子的生理学上有意义的细胞旁转运仅在细胞间紧密连接被拆除时发生 - 通常在病理条件下。对于大溶质的非特异性和低水平的跨细胞转运也可能通过液相内吞作用和货物错误分选到转胞吞囊泡而不是晚期内体/溶酶体途径中发生,其接收由非受体内化的大部分货物。介导机制。因此,任何针对健康上皮细胞或内皮屏障体外>或体内>的转胞吞作用的测定需要控制这些混杂的非特异性途径。

我们最近发现,可以利用跨上皮屏障的鞘糖脂的内源分选来在体内口服递送治疗性肽>(Garcia-Castillo 等,>,2018)。在这里,我们描述了使用在transwell过滤器上生长的培养的极化上皮细胞的体外>测定,我们用它来开发技术,为此目的共同选择这些内源性脂质分选机制。用于利用糖脂分选的测试化合物被修饰为含有生物素残基以允许生物化学捕获和用于检测的荧光团。 Transwell插入物由内腔组成,内腔由可渗透的聚碳酸酯膜支撑物制成,其中细胞接种(图1)。 3-7天后,上皮细胞组装成单细胞厚的单层,细胞间密封紧密连接。因此,transwell的内腔暴露于上皮单层的顶膜并模拟腔表面并且可以容纳200μl的溶液。外腔室 - 可渗透支撑物下方的腔室 - 暴露于单层的基底外侧表面并模拟上皮屏障的浆膜表面。该室容纳1ml基底外侧溶液。通过标准直流电生理学或使用基于交流电的机器EVOM测量的对小离子传输(TER-跨上皮电阻)的被动抗性常规地测量单层到具有密封的功能性紧密连接的“紧密”上皮屏障的组装。将测试化合物添加到顶室中,并允许测定进行以允许跨越屏障转运,然后使用链霉抗生物素蛋白包被的珠子对基底外侧室进行转胞样分析物取样(图2)。


图1. MDCK-II transwell插入物和转胞吞作用测定的示意图


图2.从基底外侧培养基中捕获和读取生物素化肽 - 融合体(改编自Garcia-Castillo的图1A et al。>,2018。知识共享署名许可
使用该测定可以实现中皮摩尔灵敏度。我们的基本方法可用于测量进入转胞吞途径的其他分子的转运 - 例如结合Fcγ受体FcRn的IgG,其在两个方向上交叉穿过极化上皮单层(Nelms 等。 >,2017)。我们的测定的强度是直接,定量和灵敏地测量转化的化合物的量的能力。细胞之间的非特异性细胞旁渗漏与通过细胞的真实转运在几个方面受到控制,例如通过使用不能参与转胞吞途径的分析物(在我们的例子中,报告肽缺乏与糖脂载体的融合,或通过融合到糖脂)具有不能进入转胞吞途径的神经酰胺结构,或通过4°C温度阻滞抑制内吞作用或转胞吞作用,使用Dyngo通过化学抑制内吞作用,以及通过siRNA敲除负责转胞吞作用的基因,如Garcia-Castillo 所述>等人>(2018)。

关键字:跨细胞转运, 内吞分选, 鞘脂糖, 上皮屏障, 药物运输

材料和试剂

注意:>

  1. 根据制造商的建议存储材料和试剂。  >
  2. 如Garcia-Castillo等,2018中所述使用荧光肽和糖鞘脂 - 肽融合物。该测定法需要荧光团和生物素的共存附着于您想要测量的化合物(参见图3)。含有与神经节苷脂GM1连接的生物素和荧光团的肽融合物使用(Garcia-Castillo等,2018)中描述的专门方法在内部合成。前体肽由New England Peptide(Gardner,MA,USA)合成,神经节苷脂购自Sandro Sonnino博士(意大利米兰)。然而,理论上,任何含有荧光团和生物素的大分子(肽,蛋白质,核酸或化学物质)都可用于该测定。示例化合物如下所示。这里,低壳氨基酸描绘了D-异构体。由于甘氨酸(显示为上壳G)不具有手性,因此该肽基本上不含L-氨基酸,其在体内易于降解。>


    图3.用于转胞吞试验的肽 - 脂质融合构建体的结构 

  1. 移液器吸头(USA Scientific,目录号:1126-7810,1120-8810,1121-3810)
  2. 0.4μm孔径Transwell ®刀片(Corning,目录号:3413)
    注意:Transwell > ®> 板保持在室温下。>
  3. 96孔分析板(无盖,黑色平底,未处理,聚苯乙烯)(Corning,Costar ®,目录号:3916)
  4. 铝箔(Fisher Scientific,Fisherbrand TM ,目录号:S05356A)
  5. MDCK-II细胞(来自美国典型培养物保藏中心的亲本系列)(ATCC,目录号:CCL-34)(来自Steven Claypool博士的礼物)
  6. 不含脂肪酸的牛血清白蛋白(Sigma-Aldrich,目录号:A6003-10G)(冻干粉,基本上不含脂肪酸)
  7. Pierce TM 链霉抗生物素蛋白磁珠(赛默飞世尔科技,目录号:88817)
    注意:根据制造商的建议,珠子的储存温度为4°C。>
  8. Formamide(Sigma-Aldrich,目录号:F9037-100ML)
  9. 二甲基甲酰胺(DMF)(Sigma-Aldrich,目录号:227056)
  10. 生物素(Sigma-Aldrich,目录号:B4501-1G)
  11. DMEM 4.5 g / L D-glucose(Corning,目录号:10-013-CVR)
  12. Pen / Strep,100x(Thermo Fisher Scientific,Gibco TM ,目录号:15140148)
  13. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM ,目录号:A3160502)
  14. Tris base(Sigma-Aldrich,目录号:T1503-1KG)
  15. NaCl(Sigma-Aldrich,目录号:S9625-10KG)
  16. 吐温20(西格玛奥德里奇,目录号:P2287-500ML)
  17. 0.25%胰蛋白酶-EDTA(Thermo Fisher Scientific,Gibco TM ,目录号:25200072)
  18. PBS(Thermo Fisher Scientific,Gibco TM )
  19. EDTA(Sigma-Aldrich,目录号:E4884-500G)
  20. MDCK-II完全生长培养基(见食谱)
  21. 基础设施解决方案(见食谱)
  22. 顶端解决方案(见食谱)
  23. TBS 10x(见食谱)
  24. 1x TBS-T(见食谱)
  25. 0.5 M EDTA pH 8.0(参见食谱)
  26. 洗脱缓冲液(见食谱)

设备

  1. 便携式移液器® XP移液器控制器(Drummond Scientific,型号:4-000-101)
  2. Incubator Forma Scientific CO 2 水套式培养箱(Thermo Fisher Scientific,型号:Forma TM 系列II,目录号:3110)
  3. 离心机(Eppendorf,型号:5415C)
  4. EVOM2 Epithelial Voltohmmeter(世界精密仪器,型号:EVOM2)
  5. TECAN Spark多模式酶标仪(Tecan Trading,型号:Spark ®)
  6. MagneSphere ®技术磁分离支架(Promega,目录号:Z5342)
  7. 超声波仪(不锈钢AquaSonic超声波清洗机,VWR,型号:50T)

软件

  1. GraphPad Prism 7
    GraphPad软件7825 Fay Avenue,Suite 230 La Jolla,CA 92037 USA( https://www.graphpad .com / scientific-software / prism / )

程序

  1. 第1天:电镀和极化电池
    1. 胰蛋白酶化
      1. 从用于传代的原种细胞培养瓶中吸出MDCK-II生长培养基。 
      2. 对于T-75烧瓶的MDCK-II细胞,加入5-10ml温热的无菌PBS。轻轻倾斜/旋转烧瓶以清洗细胞。删除PBS。
      3. 对于T-75烧瓶,加入3ml 0.25%胰蛋白酶-EDTA。倾斜/旋转烧瓶以确保细胞表面均匀涂覆。
      4. 在加湿条件下返回具有5%CO 2 的37℃培养箱5-10分钟。
      5. 在具有相衬的光学显微镜下观察。如果细胞被正确分离,它们将呈圆形并悬浮在悬浮状态。
      6. 将细胞悬液收集在锥形管中并上下吸移细胞6-8次。不应观察到团块。细胞悬液应该是均匀的混合物。 
    2. 将MDCK-II细胞以200μl的200,000个细胞密度添加到完全生长培养基中(参见配方)至Transwell ®插入物的顶端(12孔0.4μm孔径Transwell ® -65 mm)。此外,将1ml完全生长培养基加入基底外侧室。
      1. 为(未处理的)对照制备两个Transwell ®插入物,为报告肽制备2个Transwell ®插入物,并且每个糖鞘脂 - 肽融合制备2个Transwell ® 。测试 
      2. 每种条件都作为生物学复制品进行测试(每种条件下即>,2 Transwell ®插入物)。实验必须始终包括未处理和报道肽控制。
    3. 将板孵育2-3天以允许在37℃下在5%CO 2 的加湿条件下极化。

  2. 第3天:测试鞘糖脂化合物的制备
    1. 将含有冻干化合物的试管以13,000rpm(~13,800 x g >)离心3分钟,使材料沉淀到管底部。
    2. 加入1份体积的DMF(实际体积取决于储备浓度 - 对于我们的化合物,这是~60μl),超声处理30-60秒,然后约5秒涡旋。
      注意:  >
      1. 超声波在浴室超声波仪(VWR,AquaSonic,型号50T)中完成。>
      2. 您希望达到~50-200μM范围内的高终浓度(在含有约100μg冻干肽[MW~2,103]的试管中,加入50μlDMF和100μl水以获得原液溶液在~50-200μM范围内)。使用NanoDrop测定所有测试化合物的浓度(对于我们研究中使用的Alexa Flour 488,我们使用495 nm处的吸光度。)>
      3. 此时解决方案可能会出现混浊和橙红色。>
    3. 加入2份H 2 O(例如120μl水,使最终体积为180μl),然后快速5秒涡旋,以达到H 2中的最终33%DMF O。
      注意:  >
      1. 溶液应为亮绿色且无颗粒状。如果是橙色,则添加更多DMF或H > 2 > O,直到它变为绿色。 (我们观察到的颜色变化是附着在报告肽上的Alexa Flour 488)  >
      2. 此时,该原液将用于最终稀释成“Apical”培养基以添加到细胞中。>
    4. 为了制备这种Apical测试溶液,将适当体积的原液稀释到Apical Solution(参见配方)中,使其达到0.1μM的终浓度。
      注意:目标是使脂质与dfBSA的比例最终为1:1。>

  3. MDCKII细胞中的转胞吞作用
    1. 使用EVOM上皮伏安计在2-3天后检查MDCK-II Transwell ®插入物的电阻,以测量紧密连接的完整性。
      注意:用于转胞吞作用实验的MDCK-II单层的可接受电阻在250Ω-300Ω范围内。>
    2. 准备根尖和基底外侧解决方案(见食谱)。
    3. 使用无血清DMEM洗涤transwells 2次。在相应的腔室中用顶端(200μl)和基底外侧(1 ml)溶液替换培养基。 
    4. 使细胞在37℃/ 5%CO 2 细胞培养箱中平衡15分钟。
    5. 用含有0.1μM报告肽或0.1μM鞘糖脂融合物的200μl顶端溶液替换顶端腔。
      注意:报告肽和鞘糖脂融合体在33%DMF / 66%H 2 O中的范围为30μM至150μM。>
    6. 在37℃/ 5%CO 2 细胞培养箱中孵育3小时。
    7. 为了量化转胞肽和脂质 - 肽融合,在预先标记的Eppendorf管中收集基底外侧培养基并进行链霉抗生物素蛋白下拉试验。 
    8. 为了计算表观渗透系数(PAPP,cm / sec),还在预先标记的Eppendorf管中收集顶室溶液。使用范围从0nM到200nM的标准曲线在3小时后内插肽室中的肽或脂质 - 肽融合物的浓度。

  4. 基底外侧培养基样品的链霉抗生物素蛋白下拉分析
    1. 用TBS-T洗涤链霉抗生物素蛋白磁珠3次。
      注意:>
      1. 珠子永远不会被涡旋。通过轻柔倒置将它们放入储备管中。>
      2. 每个样品需要10微升珠子。>
      示例:> 对于12个样品,将120μl链霉抗生物素蛋白珠放入Eppendorf管中,并使用磁力架用1 ml TBS-T洗涤3次。 >
    2. 在50μlTBS中重悬10μl珠子,然后加入1ml基底外侧样品中 示例:> 对于12个样品,通过添加600μlTBS重悬120μl链霉抗生物素蛋白珠。然后,向每个基底外侧样品中加入50μl链霉抗生物素蛋白珠。>
    3. 将链球菌抗生物素蛋白珠的基底外侧样品在4°C孵育过夜,并用箔覆盖头顶旋转。

  5. 第4天:基底外侧样品的洗脱和读数
    1. 使用磁架收集珠子并用TBS-T洗涤3次。
      注意:每次洗涤均使用1 ml TBS-T进行,并通过反转将珠粒混合。>
    2. 通过添加220μl洗脱缓冲液(参见配方)并煮沸,从珠中洗脱结合的肽或脂质 - 肽融合物。
      注意:>
      1. 反转珠子以确保它们处于溶液中。>
      2. <珠>在珠子溶液中,在65℃下在加热块中煮沸2分钟。>
    3. 煮沸后,用磁架收集珠子。&nbsp;
    4. 在黑色96孔板上吸取100μl每种样品×2(技术重复)。
    5. 在TECAN Spark酶标仪上读取荧光,用于Alexa-488通道和每种化合物的标准曲线。&nbsp;
      1. 设置:

      2. 样本标准曲线(图4)


        图4.用于计算经转化的化合物的量的肽(蓝色)和脂质 - 肽融合(红色)的样品标准曲线。 绘制的是3次技术重复的平均值±SEM。

      3. 所测试的每种化合物的基底外侧标准曲线在0 pM至1,000 pM的洗脱缓冲液中进行。
        1. 在洗脱缓冲液中制备1μM肽和脂质 - 肽原液。
        2. 制备1ml 10nM溶液(10μl1μM+990μl洗脱缓冲液)。
        3. 用洗脱缓冲液进行连续稀释,从1,000 pM开始:
          1,000 pM =100μl10nM +900μl洗脱缓冲液
          500 pM =500μl1,000pM +500μl洗脱缓冲液
          250 pM =500μl500pM +500μl洗脱缓冲液
          125 pM =500μl250pM +500μl洗脱缓冲液
          62.5 pM =500μl125pM +500μl洗脱缓冲液
          31.5 pM =500μl62.5pM +500μl洗脱缓冲液
          15.6 pM =500μl31.5pM +500μl洗脱缓冲液
          0 pM =洗脱缓冲液

数据分析

GraphPad Prism软件用于计算脂质 - 肽融合的浓度。如上所述,每种测试化合物的标准曲线用于将荧光强度与已知浓度的肽或脂质 - 肽融合相关联。最初使用495nm处的吸光度来计算储备浓度。有关绘制和插入标准曲线的详细说明,请参阅 GraphPad软件网站。
为了计算表观渗透系数(PAPP,cm / sec),使用范围从0nM到200nM的标准曲线来内插在连续孵育3小时后留在顶室中的肽或脂质 - 肽溶液的浓度。

基于随时间推移的基底外侧隔室中脂质 - 肽融合的出现率,计算在transwell中生长的细胞单层的表观渗透系数(PAPP,cm / sec):
Papp(cm / sec)= [VD /(A×MD)]×(DMR / Dt)
Papp(cm / sec)= [cm 3 /(cm 2 x mol)] x(mol / sec)

VD =顶端(供体)体积(cm 3 )(例如>,0.2 ml = 0.2 cm 3 )
MD =顶端(供体)量(mol)
A =顶端(供体)室的膜表面积(cm 2 )(即>,transwell表面积= 0.33 cm 2 在此方案中)
DMR / Dt =随时间(秒)转移到基底外侧(接收器)隔室的化合物(mol)的量。

笔记

  1. 该方案也在我们的实验室中常规用于人肠T84细胞和人结肠Caco-2细胞。
  2. 必须使用EVOM测量确认紧密连接完整性。
  3. 为了溶解报告肽和鞘糖脂 - 报告肽融合物,首先加入DMF并超声处理30-60秒,然后加水。
  4. 在无血清DMEM中进行测定。&nbsp;
  5. 使用化合物与脱脂-BSA的摩尔比1:1。
  6. 在制备化合物溶液时,将溶液保持在37°C,切勿将稀释的脂质置于冰上,因为它们可能形成胶束。&nbsp;
  7. 对于细胞旁渗漏的典型对照是4℃温度阻滞以阻止内吞作用/转胞吞作用机制。
  8. 我们在我们的实验室中使用该测定法来测试基因沉默(使用esiRNA)和小分子药物的效果(如Garcia-Castillo中描述的Dyngo-4a >,2018)。&nbsp;
  9. 使用该测定法,我们进行了含有神经酰胺部分修饰的鞘糖脂 - 肽融合体的结构 - 功能研究。此外,使用该测定法,我们测试了肠降血糖素激素GLP-1(胰高血糖素样肽1)的转胞吞作用。

食谱

  1. MDCK-II完全成长媒体
    DMEM
    10%FBS
    1x笔/ Strep
    储存在4°C
  2. 基础外侧溶液(40毫升)
    40毫升DMEM
    400毫克脱脂BSA(df-BSA)(终浓度:1%[w / v])
    在测定当天准备新鲜
  3. 顶端溶液(40毫升)
    40毫升DMEM
    24.6μl基底外侧溶液(配方2,df-BSA终浓度:0.1μM)
    在测定当天准备新鲜
  4. TBS 10x(1升)
    24克Tris base
    88克NaCl
    溶于900毫升蒸馏水和pH 7.4
    加入蒸馏水至终体积为1L
    在室温下储存
  5. 1x TBS-T(1升)
    100毫升10倍TBS
    1毫升吐温20
    900毫升蒸馏水
    在室温下储存
  6. 0.5 M EDTA pH 8.0(1 L)
    186.1 g Na 2 EDTA至800 ml MilliQ H 2 O
    加入NaOH达到pH 8.0
    添加MilliQ H 2 O至1升
  7. 洗脱缓冲液(95%甲酰胺,10 mM EDTA,0.4 mg / ml生物素)(100 ml)
    95毫升100%甲酰胺
    2 ml 0.5 M EDTA,pH 8.0
    40毫克生物素
    3毫升MilliQ H 2 O

致谢

该项目由NIH F-32 DK111072-01支持M.D.G-C; DK084424,DK048106,DK104868,以及从Novo Nordisk到W.I.L的无限制创新补助金; DK104868到D.C.我们承认哈佛消化疾病中心DK034854。我们感谢Lencer和Chinnapen实验室的所有成员进行了有益的讨论。

利益争夺

作者声明没有利益冲突或竞争利益。

参考

  1. Fung,K.Y。Y.,Fairn,G。D. and Lee,W。L.(2018)。 上皮细胞和内皮细胞的跨细胞囊泡运输:挑战和机遇。 交通> 19(1):5-18。&nbsp;
  2. Garcia-Castillo,M。D.,Chinnapen,D。J. and Lencer,W。I.(2017)。 穿过极化上皮细胞的膜运输。 Cold Spring Harb Perspect Biol > 9(9):a027912。&nbsp;
  3. Garcia-Castillo,MD,Chinnapen,DJ,Te Welscher,YM,Gonzalez,RJ,Softic,S.,Pacheco,M.,Mrsny,RJ,Kahn,CR,von Andrian,UH,Lau,J.,Pentelute,BL和Lencer,WI(2018年)。 通过利用鞘糖脂的内源性分选来对治疗性肽进行粘膜吸收。 Elife > 7:e34469。
  4. Nelms,B.,Dalomba,N。F.和Lencer,W。(2017)。 有针对性的RNAi筛选可识别影响受体介导的转胞吞作用不同阶段的因素。 J Cell Biol > 216(2):511-525。&nbsp;
  5. Thuenauer,R.,Muller,S.K。和Romer,W。(2017)。 药物输送中蛋白质和脂质受体介导的转胞吞作用的途径。 专家 Opin Drug Deliv > 14(3):341-351。
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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 Garcia-Castillo 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. Garcia-Castillo, M. D., Lencer, W. I. and Chinnapen, D. J. (2018). Transcytosis Assay for Transport of Glycosphingolipids across MDCK-II Cells. Bio-protocol 8(20): e3049. DOI: 10.21769/BioProtoc.3049.
  2. Garcia-Castillo, M. D., Chinnapen, D. J., Te Welscher, Y. M., Gonzalez, R. J., Softic, S., Pacheco, M., Mrsny, R. J., Kahn, C. R., von Andrian, U. H., Lau, J., Pentelute, B. L. and Lencer, W. I. (2018). Mucosal absorption of therapeutic peptides by harnessing the endogenous sorting of glycosphingolipids. Elife 7: e34469.
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