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Apr 2019

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Method for Primary Epithelial Cell Culture from the Rat Choroid Plexus
大鼠脉络丛原代上皮细胞培养方法   

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Abstract

The choroid plexus consists of a network of secretory epithelial cells localized throughout the lateral, third and fourth ventricles of the brain. Cerebrospinal fluid (CSF) is generated by the choroid plexus and released into the ventricular environment. This biofluid contains an enriched source of proteins, ions, and other signaling molecules for extracellular support of neurons and glial cells within the central nervous system. Given that other cells in the brain also release factors into the CSF, in vitro investigations of choroid plexus function are necessary to isolate processes selectively occurring within and released from this tissue. Here, we describe a protocol to isolate choroid plexus tissue from each of the ventricular locations, and the cell culture conditions required to support growth and maintenance of these epithelial cells. This technique allows for investigations of the functional significance of the choroid plexus, such as for the examination of stimuli promoting the release of growth factors and extracellular vesicles (e.g., exosomes and microvesicles) from ventricle-specific choroid plexus epithelial cells.

Keywords: Choroid plexus (脉络丛), Primary cell culture (原代细胞培养), Cerebrospinal fluid (脑脊液), Epithelial cells (上皮细胞), Extracellular vesicles (胞外囊泡), Rat (大鼠)

Background

The choroid plexus produces cerebrospinal fluid (CSF) and maintains chemical homeostasis in the extracellular fluid of the central nervous system. The choroid plexus is comprised of epithelial cells, connective tissue and blood vessels, and this structure is localized in all ventricular regions, including within the lateral, third and fourth ventricles. At each of these sites, the choroid plexus appears to differ relative to structure, function and factors produced and released into the ventricles (Johanson et al., 2005; Lun et al., 2015; Lallai et al., 2019). This tissue actively produces and secretes various signaling molecules, such as growth hormones, transthyretin and transferrin, into the brain and has been implicated in a wide range of functions related to brain development, aging, nutrient transport, endocrine regulation, and pathogenesis of neurodegenerative disorders. As such, dysfunction in the choroid plexus would potentially alter CSF composition and compromise brain health. As the field progresses, the vital function of factors derived from the choroid plexus will certainly continue to emerge, and these advances may then provide a foundation for novel approaches to treat neuropathology in humans.

Previous in vitro approaches to examine choroid plexus function have derived choroidal epithelial cell culture from rat, porcine, human and immortalized murine cells (Zheng and Zhao, 2002; Monnot and Zheng, 2013; Tenenbaum et al., 2013; Delery and MacLean, 2019). However, several considerations have been noted that limit the usefulness of prior techniques. First, immortalized cell lines do not appear to retain the properties of choroid plexus epithelial cells (CPEC) since they become very susceptible to changes in morphology and property in vitro (Angelow et al., 2004). Along these lines, we have found highly variable expression of the choroid plexus specific marker transthyretin across passages in the cell lines Z310 (obtained from Dr. Wei Zheng, Purdue University) and HCPEpiC (commercially available from ScienCell Research Laboratories), suggesting altered protein expression in the immortalized state compared to primary-derived tissues (Lallai and Fowler, unpublished findings). Next, since the location of the epithelial cells used to derive the cell lines may be from one or multiple ventricular locations, and given that different transcript expression and function is found in choroid plexus tissue among the ventricular locations (Lun et al., 2015; Lallai et al., 2019), the immortalized cell lines may not be representative of the specific subregion of the tissue of interest. Indeed, the prior approaches have focused on choroid plexus dissection solely derived from the lateral and/or fourth ventricular locations (e.g., as opposed to the smaller third ventricle), have combined choroid plexus from multiple locations into one sample for analysis, or have focused on species with larger yields of choroid plexus tissue (e.g., primate, porcine and human).

To overcome these challenges, we developed a modified protocol to derive primary culture of choroidal epithelial cells from rodents, which allows one to discern between choroid plexus tissue from third, lateral and fourth ventricles (Lallai et al., 2019). With this approach, we have been able to investigate the release of factors into the cell culture medium, thereby mimicking the physiological release of factors and vesicles into CSF of the brain. Of note, the current protocol also uses exosome-depleted fetal bovine serum (FBS) to allow for examination of extracellular vesicles released from the choroid plexus cells in culture conditions; this condition has been incorporated since non-depleted FBS has been shown to contain a variety of extracellular vesicles containing bovine-derived proteins, RNA and DNA, which may contaminate analyses and conclusions (Wei et al., 2016; Kornilov et al., 2018). Moreover, the cultures obtained from this protocol provide cells with distinct structural characteristics of epithelial cells and express the choroid plexus specific protein transthyretin (Lallai et al., 2019). In the following sections, we describe the methods to discretely dissect choroid plexus tissue from different ventricles and generate primary culture choroidal epithelial cells from rat. Further, recommendations and troubleshooting tips are also provided.

Materials and Reagents

  1. 0.22 μm filter (Thermo Scientific, catalog number: 595-4520)
  2. Petri dishes (Fisher Scientific, Diameter 100 mm, catalog number: FB0875712)
  3. Clear plastic lab wrap (VWR, catalog number: 46610-056)
  4. Razor blades, straight or double edge (Electron Microscopy Sciences, catalog number: 72003-01)
  5. 1.7 ml Microtubes, sterile (Olympus Plastics, catalog number: 22-281S)
  6. P1000 pipet tip (Fisher Scientific, catalog number: 02-707-124)
  7. Cell culture plates, 96-well (Eppendorf, catalog number: 0030730119)
  8. Adult Rat to derive brain tissue (Wistar, Charles River, catalog number: Crl:WI 003, or another species/vendor is also acceptable)
  9. Superglue (Loctite, catalog number: 1647358)
  10. Laminin (Corning, catalog number: CB-40232)
  11. Collagenase, Type II (Gibco, catalog number: 17101015)
  12. Exosome-depleted FBS (SBI System Biosciences, catalog number: EXO-FBS-250A-1)
  13. 1x TrypLE Express (Gibco, catalog number: 12605-010)
  14. Poly-D-Lysine (PDL) (Corning, catalog number: CB-40210)
  15. Isoflurane (Patterson Veterinary, catalog number: 07-893-1389)
  16. 1x DMEM (Corning, catalog number: 10-017-CV)
  17. 1x DPBS (Gibco, catalog number: 14040-117)
  18. Glucose (Fisher Scientific, catalog number: D16-1)
  19. 1x Pen-Strep (Gibco, catalog number: 15140122)
  20. 1x HBSS (Corning, catalog number: 21-022-CV)
  21. Calcium Chloride (Acros Organics, catalog number: 206795000)
  22. Trypan Blue (Gibco, catalog number: 15250-061)
  23. Cytosine arabinoside (AraC) (Sigma-Aldrich, catalog number: C1768-1G)
  24. Cell culture grade sterile water (Corning, catalog number: 25055CV)
  25. Dissecting Medium (see Recipes)
  26. 5x Collagenase, Type II Stock Solution (see Recipes)
  27. 1x Collagenase, Type II Working Solution (see Recipes)
  28. Choroid Plexus epithelial cells (CPEC) medium (see Recipes)

Equipment

  1. Rodent guillotine (Harvard Apparatus, catalog number: 73-1918)
  2. Dissecting brain matrix for rat, coronal plane (Ted Pella, catalog number: 15007)
  3. Dissecting tools
    1. Small scissors (Roboz Surgical, catalog number: RS-5840)
    2. Fine tip forceps (Roboz Surgical, catalog number: RS-4960)
    3. Tissue forceps (Roboz Surgical, catalog number: RS-8102)
    4. Microdissection scissors (Roboz Surgical, catalog number: RS-5671)
    5. Micro Bone Rongeur (Roboz Surgical, catalog number: RS-8306)
  4. Hemocytometer (Fisher Scientific, catalog number: 0267151B)
  5. Tally counter (VWR, catalog number: 23609-102)
  6. Anesthesia machine for isoflurane (E-Z Systems, EZ-150C Classic Vaporizer Machine)
  7. Isoflurane chamber for rat (E-Z Systems, EZ-178 Mouse/Rat Sure-Seal Induction Chamber)
  8. Dissecting microscope (Objective for 2-5x magnification, Omano, Stereo Zoom Microscope OM 113-1LP)
  9. Refrigerated centrifuge (To accommodate 1.7 ml microtubes, Eppendorf, model: 5215R)
  10. Bead bath for incubation at 37 °C (Fisher Scientific Isotemp Digital-Control Water Bath, model: 205)
  11. Inverted fluorescent microscope (Objectives for 4x, 10x, and 20x magnification, Life Sciences, Leica DM4000 B LED)
  12. Cell culture CO2 incubator (Thermo Scientific, Series 8000 Water-Jacketed CO2 Incubators)
  13. Biosafety hood (NUAIRE, Biological Safety Cabinet Class II Type A/B3)

Procedure

  1. Preparation
    Prepare Poly-D-Lysine (PDL) and laminin-coated cell culture plasticware one day prior to dissection:
    1. To prepare cell culture plasticware, thaw the appropriate volumes of PDL and laminin. The amount of PDL and laminin varies according to the size of the well. It’s essential that the liquid film covers the entire surface of the bottom and between 2-4 mm of the side walls. For example, in a 96-well cell culture plate, apply 0.05 to 0.1 ml of solution with a micropipette to fill each well 2-4 mm up the sides of the wells.
    2. Dilute PDL 1:50 with sterile cell culture grade water and dilute laminin 1:50 with DMEM. Sterile water should be filtered, for instance with a Thermo Scientific Barnstead GenPure water purification system.
    3. Use a micropipette to add 0.05 to 0.1 ml of PDL to the cell culture plate and then incubate at 37 °C for at least 4 h to allow the PDL to coat the surface.
    4. After 4 h, aspirate the PDL solution with a vacuum and then rinse the wells 3 times with sterile cell culture grade water.
    5. Add the laminin solution with a micropipette to the plate to coat the surface, wrap in saran wrap and place at 4 °C overnight. Keep the wells filled with laminin until just prior to plating with cell mixture.

  2. Choroid plexus tissue collection from rat brain
    1. To facilitate processing, set up the biosafety hood with pipets, TrypLE Express, 1x Collagenase, Type II Working Solution, HBSS, CPEC medium, and tools prior to starting dissections.
    2. Place rat in isoflurane induction chamber preloaded with 3% isoflurane/oxygen mixture to anesthetize the rat. Maintain the level of isoflurane until the animal is fully anesthetized. This can be evidenced by pinching the foot and tail; there should be no reaction from the rat, which will verify that the animal is under full anesthesia prior to proceeding. Thereafter, quickly decapitate the rat with the rodent guillotine. Hold the head in your hand and cut the skin on the top of the head longitudinally along the top of the skull to the tip of the nose. Separate the skin laterally to expose the skull. Use microbone rongeurs to remove the skull from around the brain, starting at the cerebellum and moving anterior toward the olfactory bulbs. The brain removal and subsequent dissection should occur as quickly as possible to prevent tissue degradation. For best results, complete the dissections within 1 h or less. Moreover, ensure that the brain tissue and samples are submerged in dissecting medium (Recipe 1), including when storing in microtubes on ice following dissection.
    3. Transfer the brain to the chilled rat brain matrix in a Petri dish (Figure 1A), which has been placed on wet ice for 5 min before proceeding. Rinse brain with chilled dissecting medium. During the subsequent phases the tissue will be continuously wet with chilled dissecting medium to maintain tissue integrity and ensure maximum cell yield.
    4. The brain can now be cut along the coronal plane with a straight edge blade at two separate locations: (1) at the level of the septum (Anterior-Posterior (AP) ~1.00) and (2) at the level of the raphe (~AP -5.5) [see coordinate references in Paxinos and Watson (1997)] (Figure 1B).
    5. Transfer the posterior brain portion, containing the 4th ventricle, to a new, sterile Petri dish containing a small drop of superglue. Orient the brain chunk onto the superglue with the raphe brain side facing downward, and the cerebellum and brain stem pointing upwards (Figure 1C). Add chilled dissecting medium to the Petri dish to completely cover the brain chunk.
    6. Transfer the middle brain portion, containing the lateral and 3rd ventricles, to another sterile Petri dish and superglue the brain chunk on the ventral surface (e.g., with the cortex facing upwards) (Figure 1D). Add chilled dissecting medium to the Petri dish to completely cover the brain chunk.
    7. During the next steps, place the dish under a dissecting microscope to ensure accurate dissection technique (Figure 1E).


      Figure 1. Preparation of the brain for dissection. A. After removing the brain, place into chilled brain matrix. B. Insert two straight edge razor blades into the brain matrix to create three chunks: anterior [containing the olfactory bulbs to the level of the septum (approximately AP–~1.0)], posterior [containing the brainstem, cerebellum and midbrain to the level of the raphe (approximately AP–~5.5)], and (3) central portion. C. Glue the posterior chunk onto the bottom of the Petri dish with the brainstem (black arrow) and cerebellum (white arrow) facing upwards. D. Glue the central chunk onto the bottom of the Petri dish with the cortex facing upwards. E. Pour chilled dissecting medium into the Petri dish to submerge the brain chunks and then place under the dissecting microscope for visualization.

    8. Delicately separated the cerebellum from the brain stem using forceps to allow for visualization of the fourth ventricle. The choroid plexus will be visualized within this opening as a light red/pink string of floating tissue (Figure 2A). Use the forceps to gently remove the choroid plexus. Verify that brain tissue is not attached by microscopic inspection; the visualization of the choroid plexus (e.g., light red/pink string-like appearance) will be distinct from brain tissue (e.g., white/grey tissue) (Figure 2B). If any brain tissue is attached to the choroid plexus, carefully remove the tissue with forceps from the target choroid plexus tissue. It is better to collect less tissue and be sure that the choroid plexus sample is pure, rather than to collect extra tissue areas that may contaminate the cell populations in culture. Place the sample in a 1.7 ml microtube with dissecting medium, close the cap, and store on ice.


      Figure 2. Dissection of the fourth ventricle choroid plexus. A. Use fine tip forceps to gently separate the brainstem from the cerebellum. This will reveal the fourth ventricle cavity and will expose the internal choroid plexus tissue. B. Gently pull the choroid plexus tissue to remove it from the fourth ventricle. It will appear stringy and red/light pink in color (white arrow).

    9. For the dissections of the lateral and third ventricle choroid plexus, use a straight edge blade to gently cut the corpus callosum along the longitudinal fissure (Figure 3A). However, do not make a complete cut all the way down to the bottom of the Petri dish; make a partial cut through the corpus callosum to reveal the 3rd ventricle. If the cut goes too deep, the 3rd ventricle choroid plexus tissue will be difficult to visualize and isolate from the brain tissue.
    10. Gently pull the cortex and the hippocampus laterally to either side with forceps to expose the dorsal third ventricle choroid plexus along the midline (Figure 3B), which will appear as a light red/pink floating tissue folded in the center of this area. Remove the choroid plexus tissue by gently pulling it out (Figure 3C), verify that brain tissue is not attached (Figure 3D), place the sample in a 1.7 ml microtube with dissecting medium, and store on ice. When removing the tissue, ensure that you do not pull choroid plexus from other locations by using a precise dissection technique (Figures 4A-4F). Depending on the location of the coronal cut relative to other brain structures, the lateral ventricle choroid plexus may be confused for the dorsal third ventricle choroid plexus tissue. Choroid plexus from the dorsal third ventricle will be the smallest size of tissue collected, since it is only localized on the midline, just above the habenular region. Thus, if a long string of choroid plexus is pulled when isolating from the ventricular region, this is more likely choroid plexus tissue from the lateral ventricles, which would have been exposed if the midline was cut too deeply in Step B9 (see Figures 6A-6B below). Therefore, if it is critical for your studies to maintain the distinction between ventricular locations, the sample may not be used due to ambiguity of localization.


      Figure 3. Dissection of the third ventricle choroid plexus. A. Use a straight edge razor to gently separate the hemispheres along the sagittal fissure. B. Separate the right and left sides of the hemispheres (containing the cortex and hippocampus) to reveal the dorsal third ventricle choroid plexus, which will appear light pink/red in color (black arrow). C. Coronal view of the brain shows the removal of the dorsal third ventricle choroid plexus. D. The removed choroid plexus (white arrow) will appear structurally characteristic of choroidal tissue.


      Figure 4. Step-by-step dissection of the third ventricle choroid plexus. A. After separating the cortical tissue from each side, the third ventricle choroid plexus can be visualized directly on the midline. The white arrow denotes the location at the top of this choroid plexus tissue, which appears light pink in color. B. The forceps can be gently slid underneath the tissue to dissociate the choroid plexus (arrow) from the surrounding area. C. The choroid plexus tissue should now be movable within the third ventricle area. The white arrow identifies the floating tissue. D. Forceps are used to gently pull the choroid plexus from the ventricular cavity. The white arrow identifies the tissue being gently separated at the top of the brain chunk. E. After removal, the choroid plexus (arrow) should not be attached to any connective tissue. F. Side-view shows the removal of the third ventricle choroid plexus (arrow) from the midline.

    11. Next, to obtain the choroid plexus of the lateral ventricle, move the hippocampus on each side to reveal the lateral ventricles (Figure 5A) and gently pull on the choroid plexus to remove it from this position (Figure 5B). The lateral choroid plexus will appear as a longer string of tissue (Figure 5C). Verify that brain tissue is not attached and that you have correctly discriminated between the lateral ventricle and dorsal third ventricle choroid plexus tissues (Figures 6A-6B). Thereafter, place samples in microtubes with dissecting medium and store on ice.


      Figure 5. Dissection of the lateral ventricle choroid plexus. A. After removal of the third ventricle choroid plexus, gently separate the cortex from the hippocampus (black arrow identifies the hippocampus). This will reveal the lateral ventricle choroid plexus tissue in between these structures (white arrow denoting light pink/red choroidal tissue). B. Gently pull the choroid plexus tissue to remove it from the ventricular cavity. White arrow indicates removed tissue. C. The lateral ventricle choroid plexus will appear a longer and more string-like than the fourth and third ventricle locations.


      Figure 6. Differential characteristics of the lateral and third ventricle choroid plexus allows them to be visually distinguished from one another. A. After removal, the lateral choroid plexus appears long and string-like (black arrow), whereas the third ventricle choroid plexus is smaller in size (white arrow). B. Visualization of the lateral choroid plexus tissue (black arrow) and third ventricle choroid plexus tissue (white arrow). Note the distinguishable differences in the size and appearance.

    12. Spin samples at 300 x g for 3 min. In a biosafety hood, aspirate medium with a micropipette (do not vacuum to avoid aspirating the sample).

  3. Tissue dissociation
    1. Place TrypLE Express in bead bath to warm at 37 °C.
    2. Immediately after aspirating medium in step B12, add 500 μl of 1x Collagenase, Type II with Ca2+ Working Solution (Recipe 3) to each tissue sample and incubate for 15-20 min at 37 °C. Every five minutes tap and flick the tubes hard, about 60 times total to break up the tissue–do NOT vortex. Primary cells are quite sensitive to mechanical stress, and excessive stress will result in increased cell death. You will see the tissue break down and the clear buffer will turn cloudy with small clumps of tissue (Figures 7A-B).
    3. At this stage rinse sample by adding 1 ml of HBSS. Spin tubes at 300 x g for 2-3 min and then aspirate the supernatant with a P1000 pipet tip as follows: first aspirate 200 μl, then an additional 200 μl, and then the final remaining supernatant. Remove as much supernatant as possible, without touching the cell pellet. This approach prevents too much upward draw from the pipet tip and the sample from being aspirated, which may occur with extraction of larger volumes.
    4. To further dissociate the samples, tap the pellet and add 500 μl of TrypLE Express. Incubate the tubes at 37 °C in a bead bath for 10-20 min, tapping the tubes every 5 min. For good viability of the cells, it is important not to over-digest. Therefore, examine the cells in the microtube with an inverted light microscope to verify that the tissue is breaking down into small clumps (Figures 7C-7D).
    5. The process is complete as soon as a suspension with single cells and small clumps (containing less than 10 cells) is achieved.
    6. Next add 500 μl of CPEC medium (Recipe 4). Gently triturate about six times to dissociate the cells using a P1000 pipet tip and pipetting slowly up and quickly down to the first stop on the pipet.
    7. Wait approximately 30 s to allow large clumps of tissue to gravity sediment and then transfer the floating cells in the supernatant to a new microtube. Centrifuge at 300 x g for 3 min. Retain the large clumps of tissue that were previously sedimented at room temperature. If the cell yield is low (see Section D), repeat step C6 by adding CPEC medium and triturating.
    8. After centrifuging, aspirate the supernatant with a P1000 pipet tip and wash the cell pellet with 500 μl of CPEC medium. To prevent over-extraction of the pellet, avoid using the vacuum for fast rinses, but rather use a micropipette, making sure that the end of the tip is on the opposite side of the pellet. Resuspend in fresh 500 μl of CPEC medium and repeat this step twice.
    9. Re-suspend the pellet in a nominal amount of CPEC medium (about 100-200 μl).

  4. Cell counting and plating
    1. Count the cells and determine cell viability (described below in Data analysis). Counting cells is essential to guarantee healthy cells with an efficient proliferation rate, which will guarantee experimental reproducibility and accuracy.
    2. Immediately before use, aspirate the laminin solution out of the cell culture wells, and then rinse the wells 3 times with sterile cell culture grade water. Next, rinse once with 1x DPBS or CPEC medium immediately before adding cells. Be sure to not let the wells dry out.
    3. To plate cells, add appropriate volume of cell mixture to cell culture plasticware accordingly (Table 1):

      Table 1. Guideline for volume of cell mixture based on cell culture plate utilized


    4. Cap the plate and gently swirl the cell culture dish to make sure the cell mixture is evenly dispersed across the surface of the well. Place dish at 37 °C overnight. After 2 days aspirate the CPEC medium and add fresh medium. Continue to change medium every 3 days. This action will clean the well from dead cell debris that are not attached to the surface of the well.
    5. Confluency will be reached after 3 changes of medium (< 10 days). After confluency, cells are ready for investigating the secretion of factors into the medium.
    6. Cultured cells will be stable for another 7 days for experiments.

Data analysis

How to count cells:

  1. To count cells, combine the cell mixture from Step D3 with Trypan Blue at a ratio of 1:1 (i.e., 15 μl cell mixture:15 μl Trypan Blue). Use a pipet to apply 25 μl of the Trypan Blue-treated cell suspension to the hemocytometer. If using a glass hemocytometer, gently fill both chambers underneath the coverslip and allow the cell suspension to evenly draw out due to capillary action.
  2. Using a microscope, focus on the grid lines of the hemocytometer with a 10x objective.
  3. Use a handheld Tally Counter to count the live unstained cells (live cells do not take up Trypan Blue) in one set of 16 squares (Figures 7E-7F). Before starting to count, chose two of the boundary lines (left-top or right-bottom) and be consistent while counting. For example, only count cells that are set within the given square and that are on the right-hand and bottom boundary line. Do not count cells on the left-hand or top boundary lines of a square to prevent double-counting cells that lie on the border of 2 squares.
  4. Move the hemocytometer to the next set of 16 corner squares and continue counting until all 4 sets of 16 corners are counted.
  5. Record the yield.
  6. To determine a viability estimate, add the number of live and dead cell counts together to obtain a total cell count (dead cells will be stained with Trypan Blue) (Figures 7E-7F). Next, divide the live cell count by the total cell count value to obtain percent viability.


    Figure 7. Tissue preparation for primary cell culture of the dorsal third ventricle and lateral ventricle choroid plexus. A-B. Visualization of the dorsal third ventricle (A) and lateral ventricle (B) choroid plexus tissue during the first 2 min in 1x Collagenase, Type II with Ca2+ Working Solution inside the microcentrifuge tubes at 20x magnification. Note the distinguishable differences in size and appearance between the choroid plexus tissue from the two locations. C-D. Visualization of the dorsal third ventricle (C) and lateral ventricle (D) choroid plexus tissue after 15 min in 1x Collagenase, Type II with Ca2+ Working Solution at 40x magnification. Note that small clumps of tissue and single cells are more distinguishable. E-F. Visualization of cells from the dorsal third ventricle (E) and lateral ventricle (F) choroid plexus tissue in Trypan Blue solution on the hemocytometer under 20x magnification. For live cells, the cell membrane is not permeated by the dye, and therefore, the cells appear clear and reflective (e.g., white arrow as one example). In contrast, dead cells acquire the dye and therefore appear darker in color, which is representative of the blue stain (e.g., black arrow as one example).

Notes

  1. Healthy choroid plexus cells are bright, refractile, and clear. The presence of a lot of light blue-colored cells may indicate over-digestion and/or over-trituration.
  2. Expect a yield of about 10,000 cells from the third ventricle choroid plexus and 30,000 cells from both the lateral and the fourth ventricle choroid plexus, per each rat.
  3. Choroid plexus cells from pups are smaller than those of adults, but they dissociate more quickly in 1x Collagenase, Type II Working Solution and need less time in TrypLE Express (much less than 20 min).
  4. To culture primary choroid plexus cells for extended periods of time (>2 weeks), add 20 μM of AraC to prevent the overgrowth of fibroblasts.

Recipes

  1. Dissecting Medium
    1. Combine 1x DPBS with glucose and Pen-Strep to create a solution with a 0.6% glucose and 1x Pen-Strep concentration (for example, to make 1 L of dissecting medium combine 1,000 ml of 1x DPBS, 6 g of glucose, and 10,000 units/ml of Pen-Strep)
    2. Stir until solution is clear
    3. Filter with a 0.22 μm filter by screwing the plastic filter top onto the autoclaved, glass container that the final solution will be stored in. Connect the filter top to a vacuum and turn on the vacuum to filter the solution through. Filtering ensures purity of solutions so that sterility is not compromised
    4. Store at 4 °C
    5. For best results, make fresh dissecting medium within 24 h prior to the dissections
  2. 5x Collagenase, Type II Stock Solution
    1. Dilute Collagenase, Type II in 1x HBSS supplemented with 3 mM CaCl2 to make a solution with a final concentration of 7.5 mg/ml
    2. Filter through a 0.22 μm filter
    3. Aliquot 2 ml of the stock solution into sterile 2 ml tubes to store at -20 °C
    4. Stock solution can be stored for up to 6 months
  3. 1x Collagenase, Type II Working Solution
    Mix 5x Collagenase, Type II Stock Solution with 1x HBSS supplemented with 3 mM CaCl2 in a 1:5 ratio
  4. Choroid Plexus epithelial cells (CPEC) medium
    1. Combine 1x DMEM with FBS and Pen-Strep to achieve a solution with a 10% FBS and 1x Pen-Strep concentration (for example, to make 1 L of CPEC medium combine 900 ml of DMEM with 100 ml of FBS and 10,000 units/ml Pen-Strep)
    2. Filter the solution with a 0.22 μm filter
    3. Store in an autoclaved, glass container at 4 °C

Acknowledgments

This work was supported by the National Institutes of Health (NIH) (NIDA Grant DP1-DA039658) to C.D.F. and the Tobacco-Related Disease Research Program (TRDRP) (Grant T30FT0967) to V.L. Findings derived from this technique have been published by the authors (Lallai et al., 2019) and represent technical modifications of the prior report in mouse tissue (Barkho and Monuki, 2015).

Competing interests

The authors declare no competing interests.

Ethics

All experiments were conducted in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved in protocol AUP-17-206 (approval period 12/6/2017-12/6/2021) by the Institutional Animal Care and Use Committee at the University of California, Irvine.

References

  1. Angelow, S., Zeni, P. and Galla, H. J. (2004). Usefulness and limitation of primary cultured porcine choroid plexus epithelial cells as an in vitro model to study drug transport at the blood-CSF barrier. Adv Drug Deliv Rev 56(12): 1859-1873.
  2. Barkho, B. Z. and Monuki, E. S. (2015). Proliferation of cultured mouse choroid plexus epithelial cells. PLoS One 10(3): e0121738.
  3. Delery, E. C. and MacLean, A. G. (2019). Culture model for non-human primate choroid plexus. Front Cell Neurosci 13:396.
  4. Johanson, C. E., Duncan, J. A., Stopa, E. G. and Baird, A. (2005). Enhanced prospects for drug delivery and brain targeting by the choroid plexus-CSF route. Pharm Res 22(7): 1011-1037.
  5. Kornilov, R., Puhka, M., Mannerström, B., Hiidenmaa, H., Peltoniemi, H., Siljander, P., Seppänen-Kaijansinkko, R. and Kaur, S. (2018). Efficient ultrafiltration-based protocol to deplete extracellular vesicles from fetal bovine serum. J Extracell Vesicles 7(1): 1422674.
  6. Lallai, V., Grimes, N., Fowler, J. P., Sequeira, P. A., Cartagena, P., Limon, A., Coutts, M., Monuki, E. S., Bunney, W., Demuro, A. and Fowler, C. D. (2019). Nicotine acts on cholinergic signaling mechanisms to directly modulate choroid plexus function. eNeuro 6(2).
  7. Lun, M. P., Johnson, M. B., Broadbelt, K. G., Watanabe, M., Kang, Y. J., Chau, K. F., Springel, M. W., Malesz, A., Sousa, A. M., Pletikos, M., Adelita, T., Calicchio, M. L., Zhang, Y., Holtzman, M. J., Lidov, H. G., Sestan, N., Steen, H., Monuki, E. S. and Lehtinen, M. K. (2015). Spatially heterogeneous choroid plexus transcriptomes encode positional identity and contribute to regional CSF production. J Neurosci 35(12): 4903-4916.
  8. Monnot, A. D. and Zheng, W. (2013). Culture of choroid plexus epithelial cells and in vitro model of blood-CSF barrier. Methods Mol Biol 945: 13-29.
  9. Paxinos, G. and Watson, C. (1997). The rat brain in stereotaxic coordinates. 3rd edition. San Diego: Academic Press.
  10. Tenenbaum, T., Steinmann, U., Friedrich, C., Berger, J., Schwerk, C. and Schroten, H. (2013). Culture models to study leukocyte trafficking across the choroid plexus. Fluids Barriers CNS 10(1): 1.
  11. Wei, Z., Batagov, A. O., Carter, D. R. and Krichevsky, A. M. (2016). Fetal bovine serum RNA interferes with the cell culture derived extracellular RNA. Sci Rep 6: 31175.
  12. Zheng, W. and Zhao, Q. (2002). The blood-CSF barrier in culture. Development of a primary culture and transepithelial transport model from choroidal epithelial cells. Methods Mol Biol 188: 99-114.

简介

[摘要 ] 脉络膜丛由分泌性上皮细胞网络组成,遍布整个大脑的侧脑室,第三脑室和第四脑室。脉络丛会产生脑脊液(CSF),并释放到心室环境中。这种生物流体含有丰富的蛋白质,离子和其他信号分子来源,可为中枢神经系统内的神经元和神经胶质细胞提供细胞外支持。考虑到大脑中的其他细胞也会将因子释放到脑脊液中,因此进行了体外研究 脉络丛功能的丧失对于分离在该组织内选择性发生并从其释放的过程是必要的。在这里,我们描述了从每个心室位置分离脉络丛组织的协议,以及支持这些上皮细胞生长和维持所需的细胞培养条件。该技术允许研究脉络丛的功能重要性,例如检查促进从心室特异性脉络丛上皮细胞释放生长因子和细胞外囊泡(例如,外泌体和微囊泡)的刺激物。

[背景 ] 脉络丛可产生脑脊液(CSF),并在中枢神经系统的细胞外液中维持化学稳态。脉络丛由上皮细胞,结缔组织和血管组成,并且该结构位于所有心室区域,包括外侧,第三和第四心室内。在这些部位的每一个处,脉络丛似乎相对于产生和释放到心室中的结构,功能和因子而言是不同的(Johanson 等,2005; Lun 等,2015; Lallai 等,2019)。该组织活跃地产生并分泌各种信号分子,例如生长激素,运甲状腺素蛋白和运铁蛋白到大脑中,并参与了与大脑发育,衰老,营养物质运输,内分泌调节和神经退行性疾病的发病机理有关的多种功能。 。这样,功能障碍在脉络丛中瓦特OULD潜在地改变CSF组合物和妥协脑健康。随着该领域的发展,源自脉络丛的因子的重要功能必将继续出现,这些进展可能为治疗人类神经病理学的新方法提供基础。

先前用于检查脉络丛功能的体外方法已从大鼠,猪,人和永生化的鼠细胞衍生出脉络膜上皮细胞培养物(Zheng和Zhao,2002; Monnot and Zheng,2013; Tenenbaum 等人,2013; Delery和MacLean,2019 )。然而,已经注意到一些考虑因素限制了现有技术的实用性。首先,永生化细胞系似乎不保留脉络丛上皮细胞(的性质CPEC) ,因为它们变得非常容易受到变化小号在形态和性质在体外(Angelow 等人,2004) 。沿着这些思路,我们发现跨细胞系Z310(可从普渡大学的郑正博士获得)和HCPEpiC (可从ScienCell Research Laboratories 商购)的细胞传代的脉络丛特异性标记运甲状腺素蛋白高度可变的表达,提示蛋白质表达改变与原始来源的组织相比处于永生状态(Lallai 和Fowler,未发表的发现)。接下来,由于用于衍生细胞系的上皮细胞的位置可能来自一个或多个心室位置,并且鉴于不同的转录表达,在心室位置之间的脉络丛组织中发现了一种功能(Lun et al。,2015 ; Lallai 。等人,2019) ,所述永生化细胞系可能不具有代表性的特定的子区域的所述组织ó ?F兴趣。实际上,先前的方法集中于仅从外侧和/或第四脑室位置(例如,与较小的第三脑室相对)获得的脉络膜丛解剖,已经将来自多个位置的脉络丛合并成一个样本用于分析,或者已经聚焦对脉络丛组织产量较高的物种(例如灵长类,猪和人)。

为了克服自身的挑战,我们开发了一种经修改协议脉络膜上皮细胞的派生原代培养来自啮齿动物,它允许一个从第三,横向和第四脑室脉络丛组织之间辨别(Lallai 等人,2019) 。通过这种方法,我们已经能够研究因子向细胞培养基中的释放,从而模拟了因子和囊泡向大脑CSF的生理释放。值得注意的是,当前的协议还使用了外泌体耗尽的胎牛血清(FBS),以便在培养条件下检查从脉络丛细胞释放的细胞外囊泡。由于已证明未耗尽的FBS包含多种细胞外囊泡,其中包含牛衍生的蛋白质,RNA和DNA,可能会污染分析和结论(Wei 等人,2016; Kornilov 等人,2018) )。此外,从该协议中获得的培养物为细胞提供了上皮细胞的独特结构特征,并表达了脉络丛特异性蛋白运甲状腺素蛋白(Lallai et al。,2019)。在以下各节中,我们描述了从不同心室离散解剖脉络丛组织并从大鼠产生原代培养脉络膜上皮细胞的方法。此外,还提供了建议和故障排除技巧。

关键字:脉络丛, 原代细胞培养, 脑脊液, 上皮细胞, 胞外囊泡, 大鼠

材料和试剂


 


0.22 μ米过滤器(热科学,目录号:595-4520)
培养皿(Fisher Scientific,直径100 毫米,目录号:FB0875712)
清除p 拉斯蒂克升AB 瓦特说唱(VWR,目录号:46610-056)
直或双刃剃须刀(电子显微镜科学,目录号:72003-01)
1.7毫升无菌无菌微管(Olympus Plastics,目录号:22-281S)
P1000移液器吸头(Fisher Scientific,目录号:02-707-124)
细胞培养板,96 - 井仪(Eppendorf,目录号:0030730119)
成年大鼠获取脑组织(Wistar ,Charles River,目录号:Crl:WI 003,或其他物种/供应商也可接受)
强力胶(乐泰,目录号:1647358)
层粘连蛋白(康宁,目录号:CB-40232)
II型胶原酶(Gibco ,目录号:17101015)
耗尽外来体的FBS(SBI System Biosciences,目录号:EXO-FBS-250A-1)
1x TrypLE Express(Gibco ,目录号:12605-010)
Poly-D-赖氨酸(PDL)(Corning ,目录号:CB-40210)
异氟烷(帕特森兽医,目录号:07-893-1389)
1个DMEM(Corning,目录号:10-017-CV)
1x DPBS(Gibco ,目录号:14040-117)
葡萄糖(Fisher Scientific,目录号:D16-1)
1x Pen-Strep(Gibco ,货号:15140122)
1个HBSS (Corning,目录号:21-022-CV)
氯化钙(Acros Organics,目录号:206795000)
台盼蓝(Gibco ,目录号15250-061)
胞嘧啶阿拉伯糖苷(AraC )(Sigma-Aldrich,目录号:C1768-1G)
细胞培养级无菌水(Corning,目录号:25055CV)
解剖培养基(请参见食谱)
5x胶原酶,II型原液(请参阅食谱)
1x胶原酶,II型工作溶液(请参阅食谱)
脉络膜丛上皮细胞(CPEC)培养基(请参阅食谱)
 


设备


 


1. 啮齿类动物断头台(哈佛仪器,目录号:73-1918)      


2. 解剖大鼠,冠状面的脑基质(       Ted Pella,货号:15007)


3. 迪ssecting工具      


小剪刀(Roboz Surgical,目录号:RS-5840)
细尖镊子(Roboz Surgical,目录号:RS-4960)
组织钳(Roboz Surgical,目录号:RS-8102)
显微解剖剪刀(Roboz Surgical,目录号:RS-5671)
Micro B one R ongeur (Roboz Surgical,目录号:RS-8306)
4. 血细胞计数器(Fisher Scientific,目录号:0267151B)      


5. 理货计数器(VWR,目录号:23609-102)      


6. 异氟醚麻醉机(EZ Systems,EZ-150C经典型蒸发器机)      


7. 大鼠异氟烷腔(EZ Systems,EZ-178小鼠/大鼠Sure-Seal密封感应腔)      


8. 解剖MI croscope(O b jective为2-5x倍率,Omano ,立体声放大显微镜OM 113-1LP)      


9. 冷冻离心机(? ? 容纳1.7毫升微管,的Eppendorf,型号:52 15R)      


10. 于37孵育的珠浴    °C(Fisher Scientific Isote mp 数字控制水浴锅,型号:205)


11. 倒置荧光显微镜(用于4倍,10倍和20倍放大率的目标,生命科学,Leica DM4000 B LED)   


12. 细胞培养CO 2 培养箱(Thermo Scientific ,8000系列带水套的CO 2 培养箱)   


13. 生物安全罩(NUAIRE,II级生物安全柜A / B3)   


 


程序


 


制备
解剖前一天,准备聚D-赖氨酸(PDL)和层粘连蛋白包被的细胞培养塑料器皿:


要准备细胞培养塑料器皿,请解冻适量的PDL和层粘连蛋白。PDL和层粘连蛋白的量根据孔的大小而变化。至关重要的是,液膜要覆盖底部的整个表面以及侧壁2-4毫米之间。例如,在96孔细胞培养板中,用微量移液器将0.05到0.1 ml溶液加到每个孔的2-4毫米处。
用无菌细胞培养级水稀释PDL 1:50,并用DMEM稀释层粘连蛋白1:50。无菌水应进行过滤,例如使用Thermo Scientific Barnstead GenPure 净水系统进行过滤。
使用微量移液器向细胞培养板中添加0.05至0.1 ml的PDL,然后在37°C孵育至少4 h,以使PDL覆盖表面。
4小时后,用真空抽吸PDL溶液,然后用无菌细胞培养级水冲洗孔3次。
用微量移液器将层粘连蛋白溶液添加到板上以涂满表面,包裹在萨兰包装中并在4°C下放置过夜。保持孔中充满层粘连蛋白,直到即将细胞混合物铺板之前。
 


从大鼠大脑收集脉络丛组织
为了便于处理,在开始解剖前,用移液器,TrypLE Express,1x胶原酶,II型工作溶液,HBSS,CPEC培养基和工具建立生物安全罩。
将大鼠放入预装有3%异氟醚/氧气混合物的异氟烷诱导室中以麻醉大鼠。保持异氟烷水平,直到动物完全麻醉。这可以通过捏脚和尾巴来证明。大鼠不应有任何反应,这将在进行之前验证动物是否处于完全麻醉状态。此后,用啮齿动物断头台迅速给大鼠断头。用手握住头部,沿颅骨顶部至鼻尖纵向切割头部顶部的皮肤。横向分离皮肤以露出头骨。使用米ICRO b 一个? ongeurs 从大脑除去周围颅骨,起始于小脑和移动anterio 朝向嗅球河 脑切除和随后的解剖应尽快发生,以防止组织退化。为了获得最佳结果,请在1小时以内完成解剖。此外,确保脑组织和样品浸没在解剖培养基中(配方1),包括解剖后存放在冰上的微管中时。
将大脑转移到皮氏培养皿中冷却的大鼠脑基质中(图1A),该皿已在湿冰上放置5分钟,然后继续。用冷的解剖培养基冲洗大脑。在随后的阶段中,组织将被冷却的解剖培养基连续润湿,以保持组织完整性并确保最大的细胞产量。
现在可以使用直刃刀片在两个单独的位置沿冠状平面切割大脑:(1)在隔膜水平(前-后(AP)?1.00 ) 和(2)在鼻沟水平(( ?AP -5.5 )[参见坐标中引用Paxinos和Watson(1997)](图1B)。
将包含第4 个心室的后脑部分转移到新的无菌培养皿中,该培养皿中含有一小滴强力胶。将脑块定向在强力胶上,使脑脊神经的一面朝下,小脑和脑干朝上(图1C)。在培养皿中加入冷却的解剖培养基,以完全覆盖大脑块。
将包含侧脑室和第三脑室的中脑部分转移到另一个无菌培养皿中,并在腹侧表面(例如,皮层朝上)上盖上大脑块(图1D)。在培养皿中加入冷却的解剖培养基,以完全覆盖大脑块。
在接下来的步骤中,将培养皿置于解剖显微镜下以确保精确的解剖技术(图1E)。
 






图1.准备解剖的大脑。A.取出大脑后,放入冰冷的脑基质中。B.将两个直刃剃须刀片插入脑基质以形成三个块:前部[包含嗅球至隔膜的水平(大约AP–?1.0 )],后部[包含脑干,小脑和中脑的水平(大约AP–?5.5 )],和(3)中心部分。C.胶水后块到的底部P 与脑干(黑色箭头)和小脑(白色箭头)ETRI菜朝上。D.胶中央组块到的底部P 与皮质朝上ETRI菜。E.倒入冷却的解剖介质进入P ETRI盘以浸没解剖显微镜可视化下的脑组块,然后地方。


 


使用镊子将小脑与脑干精确分离,以显示第四脑室。脉络丛将在该开口内显示为淡红色/粉红色的漂浮组织(图2A)。使用镊子轻轻地去除脉络丛。通过显微镜检查确认未附着脑组织;脉络丛的可视化(例如,浅红色/粉红色样的外观)将不同于脑组织(例如,白色/灰色组织)(图2B )。如果任何脑组织附着在脉络丛上,请用镊子小心地从目标脉络丛组织中取出组织。最好收集较少的组织并确保脉络丛样本是纯净的,而不是收集可能污染培养中细胞群体的多余组织区域。将样品放入带有解剖培养基的1.7 ml 微管中,盖上盖子,并保存在冰上。
 






图2。第四脑室脉络丛的解剖。A.用细尖镊子将脑干与小脑轻轻分开。这将揭示第四脑室腔并暴露内部脉络丛组织。B.轻轻拉动脉络丛组织,将其从第四脑室中取出。它将显示为红色和浅粉红色(白色箭头)。


对于侧脑室脉络丛和第三脑室脉络丛的解剖,请使用直刃刀片沿纵裂轻轻切开the体(图3A)。但是,请不要完全切开P etri碟的底部;切开call体,露出第三脑室。如果切得太深,第三脑室脉络丛组织将难以可视化并与脑组织隔离。
轻轻地用镊子将皮层和海马从侧面拉至任一侧,以沿中线暴露背侧第三脑室脉络丛(图3B),这将显示为淡红色/粉红色的漂浮组织在该区域的中心折叠。轻轻拉出脉络丛组织(图3C),确认未附着脑组织(图3D),将样品放入带有解剖培养基的1.7 ml 微管中,并保存在冰上。当移除组织,确保不通过使用精确的解剖技术(图拉从其他地点脉络丛小号4A- 4 F)。根据冠状切口相对于其他脑部结构的位置,侧脑室脉络丛可能会混淆背侧第三脑室脉络丛组织。来自背侧第三脑室的脉络丛将是收集的最小组织,因为它仅位于中线,正好在唇状区域上方。因此,如果在从心室区域分离时拉出一长串脉络神经丛,则更可能是侧脑室的脉络丛组织,如果在S tep B 9(s ee图s 6A- 6 B)。因此,如果对您的研究而言至关重要的是保持心室位置之间的区别,则由于定位不明确,可能无法使用该样本。
 






图3.第三脑室脉络丛的解剖。答:用一把直刃剃刀沿着矢状裂隙轻轻分开半球。B.分开半球的右侧和左侧(包含皮质和海马体)以显示背侧第三心室脉络丛,其颜色将显示为浅粉红色/红色(黑色箭头)。C.大脑冠状位视图显示了背侧第三脑室脉络丛的切除。D.去除的脉络丛(白色箭头)将显示脉络膜组织的结构特征。


 






图4.第三脑室脉络丛的逐步解剖。A.从两侧分离皮??质组织后,可以直接在中线上看到第三脑室脉络膜丛。白色箭头表示该脉络丛神经组织顶部的位置,看起来呈浅粉红色。B.镊子可以在组织下方轻轻滑动,以使脉络丛(箭头)与周围区域分离。C.脉络丛组织现在应该可以在第三脑室区域内移动。白色箭头标识浮动组织。D.用镊子轻轻地将脉络丛从心室腔中拉出。白色箭头表示在脑块顶部逐渐分离的组织。E.取出后,脉络丛(箭头)不应附着在任何结缔组织上。F.侧视图显示从中线取下第三脑室脉络丛(箭头)。


 


接下来,要获得侧脑室的脉络膜丛,请在每侧移动海马以露出侧脑室(图5A),然后轻轻拉动脉络膜丛使其从该位置移开(图5B)。脉络膜外侧丛将表现为较长的组织带(图5C)。确认未附着脑组织,并且正确区分了侧脑室和背侧第三脑室脉络丛组织(图s 6A- 6 B)。之后,将样品放入带有解剖培养基的微管中,并保存在冰上。
 






图5.侧脑室脉络丛的解剖。A.去除第三脑室脉络丛后,将皮层与海马体轻轻分开(黑色箭头标识海马体)。这将揭示这些结构之间的侧脑室脉络丛神经组织(白色箭头表示浅粉红色/红色脉络膜组织)。B.轻轻拉动脉络丛组织,将其从心室腔中取出。白色箭头表示已取出的组织。C.侧脑室脉络丛会比第四个和第三个脑室位置更长,更像字符串。


 






图6.侧脑室脉络丛和第三脑室脉络丛的差异特征使它们在视觉上能够彼此区分开。A.去除后,外侧脉络膜丛长而呈线状(黑色箭头),而第三脑室脉络丛的大小较小(白色箭头)。B.外侧脉络丛神经组织(黑色箭头)和第三脑室脉络丛神经组织(白色箭头)的可视化。注意大小和外观上的明显区别。


 


以300 xg 旋转样品3分钟。在生物安全罩中,用微量移液器吸出培养基(请勿抽真空,以免吸出样品)。
 


组织解离
将TrypLE Express放入珠浴中,以加热至37°C。
在步骤B12吸介质后,立即加入500 μ 升1X胶原酶,II型的用Ca 2+ 工作溶液(配方3)到每个组织样本在37℃下孵育15-20分钟。每五分钟轻拍并轻拂试管一次,总计约60次以破坏组织–不要涡旋。原代细胞对机械应力非常敏感,过度的应力会导致细胞死亡增加。您会看到组织破裂,透明的缓冲液会随着小块组织变浑浊(图s 7A-B)。
在此阶段,通过添加1 ml HBSS冲洗样品。旋管,在300 ×g下2-3分钟,然后吸用P1000吸管尖上清液如下:第一抽吸200 μ 升,再追加200 μ 升,然后最终剩余的上清液。去除尽可能多的上清液,而不接触细胞沉淀。这种方法可以防止从移液器吸头上吸取过多的东西,并且避免吸出样品,这可能会在抽取较大体积时发生。
为了进一步解离的S amples,点击沉淀,加入500 μ 升的的TrypLE 快车。将试管在珠浴中于37°C孵育10-20分钟,每5分钟敲击一次试管。对于细胞的良好可行性,这是小鬼ortant不以OV ER- 消化。因此,用倒置光学显微镜检查微管中的细胞,以验证组织正在分解成小块(图s 7C- 7 D)。
一旦实现了具有单细胞和小团块(包含少于10个细胞)的悬浮液,该过程就完成了。
接着加入500 μ 升CPEC培养基(配方4)。轻轻研磨约六次,以使用P1000移液器吸头将细胞解离,然后缓慢向上,向下快速移液至移液器的第一个停止位。
等待约30秒钟,使大块组织沉淀下来,然后将上清液中的漂浮细胞转移到新的微管中。以300 xg离心3分钟。保留先前在室温下沉淀的大块组织。如果细胞产量较低(请参阅D 部分),请添加CPEC培养基并研磨,重复步骤C 6 。
离心后,吸用P1000吸管尖上清,以500洗涤细胞沉淀μ 升CPEC培养基。为防止沉淀物过度提取,请避免使用真空进行快速漂洗,而应使用微量移液器,确保尖端的末端在沉淀物的另一侧。重悬在新鲜的500 μ 升CPEC培养基中,并重复该步骤两次。
重悬在CPEC介质的标称量的沉淀(约100-200 μ 升)。
 


细胞计数和电镀
计数细胞,并测定细胞活力(在数据下面描述一个nalysis)。细胞计数对于确保健康细胞具有有效的增殖速率至关重要,这将保证实验的可重复性和准确性。
在即将使用之前,将层粘连蛋白溶液从细胞培养孔中吸出,然后用无菌细胞培养级水冲洗孔3次。接下来,在添加细胞之前,立即用1x DPBS或CPEC培养基冲洗一次。确保不要让孔变干。
要铺板细胞,相应地向细胞培养塑料器皿中添加适当体积的细胞混合物(表1):
 


? 能够1.准则为细胞混合物的体积比,以细胞培养板用于


细胞培养板大小


细胞混合物的体积


96 - 孔板中,PDL-涂覆层粘连蛋白


200 μ 升/孔(10000个细胞/孔)


96 - 孔平板


100 μ 升/孔


24 - 孔板


0.5毫升/孔


12 - 孔平板


1毫升/孔


6 - 孔板


2毫升/孔


 


                            盖好板并轻轻旋转细胞培养皿,以确保细胞混合物均匀分散在整个孔表面。将盘子放在37°C下过夜。2天后,抽吸CPEC培养基并添加新鲜培养基。每3天继续更换培养基。该操作将清除孔中未附着在孔表面的死细胞碎片。
后3变化汇合将达到? 介质(的< 10天)。汇合后,细胞已准备好调查因子向培养基中的分泌。
培养的细胞将再稳定7天进行实验。




数据一nalysis


 


如何计算细胞:


计数细胞,将细胞混合物从步骤D3与台盼蓝以1比组合:1(即,15 μ 升细胞混合物:15 μ 升台盼蓝)。使用移液管25施加μ 升的台盼蓝处理的细胞悬浮液的血球。如果使用玻璃血细胞计数器,则轻轻地在盖玻片下填充两个腔室,并由于毛细作用使细胞悬浮液均匀地抽出。
使用显微镜,以10倍物镜聚焦在血细胞计数器的网格线上。
使用手持式计数器计数活的未染色的细胞(活细胞不占用台盼蓝)在一组的16个平方(图小号7E- 7 F)。在开始计数之前,请选择两条边界线(左上或右下),并在计数时保持一致。例如,仅对设置在给定正方形内且在右侧和底部边界线上的像元进行计数。请勿在正方形的左侧或顶部边界线上计数像元,以防止重复计算位于2个正方形边界上的像元。
将血细胞计数器移至下一组16个角点并继续计数,直到对所有4组16个角点进行计数。
记录产量。
为了确定可行性的估计,添加的活的和死的细胞数进行计数在一起以获得总细胞计数(图(死细胞将与台盼蓝染色)小号7E- 7 F)。接下来,将活细胞计数除以总细胞计数值,以获得存活率百分比。
 






图7.背第三脑室和侧脑室脉络丛的原代细胞培养的组织准备。AB。最初的2分钟内,在1x胶原酶II型中,在离心管内以Ca 2+ 工作液加Ca 2+ 工作液以放大的20x 倍率观察背侧第三脑室(A)和侧脑室(B)脉络丛组织。请注意,脉络丛神经组织在两个位置之间在大小和外观上存在明显的差异。光盘。在1x胶原酶II型中用Ca 2+ 工作溶液在40x 放大倍数下观察15分钟后,观察背侧第三脑室(C)和侧脑室(D)脉络丛神经组织。请注意,较小的组织块和单个细胞更易于区分。英孚 在20倍放大率下,在血细胞计数器上观察锥虫蓝解决方案中背侧第三脑室(E)和侧脑室(F)脉络丛神经组织中的细胞。对于活细胞,细胞膜不被染料渗透,因此,细胞看起来清晰且反射(例如,白色箭头为一个例子)。相反,死细胞获得该染料,因此看起来颜色更深,这代表蓝色污点(例如,黑色箭头为一个示例)。


 


笔记


 


健康的脉络丛细胞明亮,可折射且清晰。许多浅蓝色细胞的存在可能表明过度消化和/或过度研磨。
预计每只大鼠第三脑室脉络丛的产量约为10,000个细胞,外侧和第四脑室脉络丛的产量约为30,000个细胞。
幼崽的脉络丛细胞比成人小,但它们在1x胶原酶,II型工作溶液中解离的速度更快,并且在TrypLE Express中需要的时间更少(少于20分钟)。
培养初级脉络丛细胞的时间(> 2周)长时间,加入20 μ 中号的阿糖胞苷,以防止成纤维细胞的过度生长。
 


菜谱


 


解剖培养基
将1x DPBS与葡萄糖和Pen-Strep混合以创建具有0.6%葡萄糖和1x Pen-Strep浓度的溶液(例如,使1升解剖培养基合并1,000 ml 1x DPBS,6 g葡萄糖和10 ml ,00 0 单位/ ml 的Pen-Strep )
搅拌直至溶液澄清
用0.22滤波器μ 米过滤器通过螺纹连接的塑料过滤器顶部上的高压灭菌,玻璃容器,最终的解决方案将被存储在。连接所述过滤器顶部至真空并打开真空过滤通过该溶液。过滤可确保溶液的纯度,从而不影响无菌性
储存在4°C
为了获得最佳结果,请在解剖前24小时内制作新鲜的解剖培养基
5x胶原酶,II型原液
在1x HBSS中稀释II型胶原蛋白酶,并补充3 mM CaCl 2 制成最终浓度为7.5 mg / ml的溶液
通过0.22滤波器μ 米过滤器
将2 ml储备溶液分装到2 ml无菌试管中,以储存在-20°C
储备液最多可保存6个月
1x胶原酶,II型工作溶液
以1:5的比例混合5x胶原酶,II型储备液和1x HBSS以及3mM CaCl 2的混合物


脉络膜上皮细胞(CPEC)
结合1X DMEM与FBS和青霉素-链霉素,以实现用10%FBS和1x青霉素-链霉素的浓度(对考试的溶液PLE,使1升CPEC介质的结合900毫升DMEM的用100ml的FBS 和10 ,000单位/ ml Pen-Strep )
过滤与0.22溶液μ 米音响滤波器
存放在4°C的高压灭菌玻璃容器中
 


致谢


 


这项工作得到了CDF 的国立卫生研究院(NIH)(NIDA Grant DP1-DA039658)和VL 的烟草相关疾病研究计划(TRDRP)(Grant T30FT0967 )的支持。作者已经发表了从这项技术中得出的发现(Lallai et al。,2019),并代表了先前对小鼠组织的报道的技术修改(Barkho and Monuki,2015)。


 


利益争夺


 


作者宣称没有利益冲突。


 


伦理


 


所有experi 发言:是在严格按照NIH指南实验室动物的护理和使用进行了与在协议AUP-17-206被批准(许可期12/6 / 2017-12 /2021分之6)b y中的机构动物加州大学尔湾分校护理与使用委员会。


 


参考文献


 


Angelow ,S.,Zeni ,P。和Galla ,HJ(2004)。原代培养的猪脉络丛神经上皮细胞作为体外模型研究在血脑脊液屏障处的药物转运的有用性和局限性。进阶药物Deliv 版本56(12):1859至1873年。
Barkho ,BZ和Monuki ,ES(2015)。培养的小鼠脉络丛上皮细胞的增殖。PLoS One 10(3):e0121738。
Delery ,EC和MacLean,AG(2019)。非人类灵长类动物脉络丛的培养模型。前沿细胞神经科学13:396。
Johanson ,CE,Duncan,JA,Stopa ,EG和Baird,A.(2005年)。脉络丛-CSF途径提高了药物递送和脑靶向的前景。药物研究22(7):1011-1037。
科尔尼洛夫,R.,Puhka ,M.,Mannerstr ? 米,B.,Hiidenmaa ,H.,Peltoniemi ,H.,Siljander ,P.,塞普? NEN-Kaijansinkko ,R。和Kaur联系,S。(2018)。基于超滤的高效协议,可从胎牛血清中清除细胞外囊泡。? Extracell 囊泡7(1):1422674。
Lallai ,五,格兰姆斯,N.,福勒,JP,西奎拉,PA,卡塔赫纳,P.,利蒙,A.,库茨,M.,Monuki ,ES,Bunney ,W.,Demuro ,A和福勒,CD (2019)。尼古丁作用于胆碱能信号传导机制,直接调节脉络丛功能。eNeuro 6(2)。
伦,国会议员,约翰逊,MB,Broadbelt ,KG,渡边,M。,康,YJ,周,KF,Springel ,MW,Malesz ,A。索萨,AM,普莱蒂科斯,M。,阿德利塔,T。,卡里基奥, ML,Zhang,Y.,Holtzman ,MJ,Lidov ,HG,Sestan ,N.,Steen,H.,Monuki ,ES和Lehtinen ,MK(2015)。空间异质性脉络丛丛转录组编码位置同一性并有助于区域CSF产生。? 神经科学35(12):4903-4916。
Monnot ,AD和Zheng,W.(2013)。脉络丛上皮细胞的培养和血脑脊液屏障的体外模型。方法分子生物学945:13-29。
Paxinos ,G。和Watson,C。(1997)。鼠脑处于立体定位坐标。3 次版本。圣地亚哥:学术出版社。
特南鲍姆,T.,斯氏,U.,弗里德里希,C.,伯杰,J.,Schwerk ,C。和Schroten ,H.(2013)。培养模型研究跨脉络丛的白细胞运输。流体屏障CNS 10(1):1。
Wei,Z.,Batagov ,AO,Carter,DR和Krichevsky ,AM(2016)。胎牛血清RNA干扰细胞培养衍生的细胞外RNA。科学代表6:31175。
Zheng W.和Zhao Q.(2002)。培养中的血脑脊液屏障。脉络膜上皮细胞的原代培养和上皮运输模型的发展。方法分子生物学188:99-114。
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Copyright: © 2020 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Lallai, V., Ahmed, A. and Fowler, C. D. (2020). Method for Primary Epithelial Cell Culture from the Rat Choroid Plexus. Bio-protocol 10(4): e3532. DOI: 10.21769/BioProtoc.3532.
  2. Lallai, V., Grimes, N., Fowler, J. P., Sequeira, P. A., Cartagena, P., Limon, A., Coutts, M., Monuki, E. S., Bunney, W., Demuro, A. and Fowler, C. D. (2019). Nicotine acts on cholinergic signaling mechanisms to directly modulate choroid plexus function. eNeuro 6(2).
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