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Jan 2016
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Long-term in vitro Culture of Cryptosporidium parvum
长期体外培养小隐孢子虫   

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Abstract

Continuous in vitro growth of Cryptosporidium parvum has proved difficult and conventional in vitro culture techniques result in short-term (2-5 days) growth of the parasite resulting in thin-walled oocysts that fail to propagate using in vitro cultures, and do not produce an active infection using immunosuppressed or immunodeficient mouse models (Arrowood, 2002). Here we describe the use of hollow fiber bioreactors (HFB) that simulate in vivo conditions by providing oxygen and nutrients to host intestinal cells from the basal surface and permit the establishment of a low redox, high nutrient environment on the apical surface. When inoculated with 105 C. parvum (Iowa isolate) oocysts the bioreactor produced 108 oocysts per ml (20 ml extra-capillary volume) after 14 days, and was maintained for over 2 years. In vivo infectivity studies using a TCR-α-immune deficient mouse model showed that oocysts produced from the bioreactor at 6, 12 and 18 months were indistinguishable from the parent Iowa isolate used to initiate the culture. HFB produced oocysts had similar percent excystation profiles to the parent Iowa isolate.

Keywords: Parasitology (寄生虫学), Cell culture (细胞培养), Protozoans (原生动物), Cryptosporidia (隐孢子虫), Cryptosporidium parvum (小隐孢子虫), Hollow fiber bioreactor (中空纤维生物反应器)

Background

Cryptosporidium parvum is an intracellular obligate parasite of the intestinal tract of man and other mammals resulting in an acute diarrhea. The disease is self-limiting in immunocompetent individuals, however, in immunocompromised adults and young children, the disease can be life-threatening (Kotloff, 2017). It is amongst the top three diagnosed enteric diseases of children in economically low resource countries (Kotloff et al., 2013; Sow et al., 2016), and is estimated to account for 9% of child deaths globally (Bhutta and Black, 2013; Checkley et al., 2015). In countries with a high burden of pediatric diarrheal disease, it has been shown that there is a correspondingly high incidence of malnutrition, stunting, and impaired cognitive functions (Lang and MA-LED Network Investigators, 2015). Despite the significant health risks and global distribution of this disease, there is no consistently effective therapy for the most-at-risk population. Recent advances in manipulating the parasite genome (Vinayak et al., 2015) and chemotherapeutic profiling (Arnold et al., 2017; Love et al., 2017; Manjunatha et al., 2017) are an encouraging sign that this bleak situation will change. Culture of the parasite has concentrated on developing a 3D culture system using adult murine colon cells (Baydoun et al., 2017) or novel bioengineered human intestinal cells (DeCicco RePass et al., 2017) which has produced novel insights into parasite invasion and significantly extended the length of culture time compared to the 2D culture method. However, these techniques are limited in terms of parasite numbers obtained. The 3D models overcome the major obstacle associated with conventional 2D culture methods where host cells receive nutrients and oxygen from the apical surface (except for those systems that use porous membrane inserts like the Costar Transwell system); this is contrary to the in vivo situation where the enterocytes receive nutrients and oxygen from the basal surface and the apical surface faces the lumen of the gut. However, current intestinal implant models fail to provide the low oxygen environment present inside the gut lumen which restricts the long-term growth of the parasite. The use of hollow fiber technology allows the creation of the biphasic environment present in the gut and overcomes many problems associated with the long-term culture of C. parvum (Morada et al., 2016). This protocol describes the establishment of hollow fiber bioreactors that can be used to simulate in vivo conditions by providing oxygen and nutrients to the basal surface of host intestinal cells that are attached to the outside of the hollow fibers (Figure 1). The environment inside the reactor is adjusted to mimic the lumen of the gut hence the apical surface of the intestinal cells are established in a low redox, high nutrient environment, that favors high growth rates and long-term maintenance of C. parvum. The use of this method provides 108-109 oocysts which can be used for molecular and biochemical studies and has the advantage of avoiding the use of harsh chemicals such as K-dichromate, which is used as a long-term storage medium at (4 °C) and has the advantage of sanitizing the oocysts; and chlorine currently used as both a sanitizer and to enhance excystation of oocysts obtained from animal sources (Arrowood, 2008).


Figure 1. Hollow Fiber Bioreactor. A. Schematic representation of the HFB; B. HFB setup.

Materials and Reagents

  1. BrandTechTM BRANDTM reagent reservoirs (BrandTech Scientific, catalog number: 703459 )
  2. Aluminum foil 18 in x 500 ft (Sigma-Aldrich, catalog number: Z185159-1EA )
  3. BD syringe with various tips, 60 ml (BD, catalog number: 309653 )
  4. BD Diagnostic Systems SYR only Luer-LokTM 10 ml 200PK RX (Thermo Fisher Scientific, catalog number: B302995)
    Manufacturer: BD, catalog number: 302995 .
  5. BD Disposable syringes with Luer-LokTM Tips, 3 ml (BD, catalog number: 309657 )
  6. FisherbrandTM sterile alcohol prep pads (Fisher Scientific, catalog number: 22-363-750 )
  7. BD VacutainerTM general use syringe needles (BD, catalog number: 305180 )
  8. FisherbrandTM borosilicate glass disposable serological pipets with regular tip, standard length, 5 ml (Fisher Scientific, catalog number: 13-678-27E )
  9. FisherbrandTM borosilicate glass disposable serological pipets with regular tip, standard length, 10 ml (Fisher Scientific, catalog number: 13-678-27F )
  10. FisherbrandTM borosilicate glass disposable serological pipets with regular tip, short length, 25 ml (Fisher Scientific, catalog number: 13-678-36D )
  11. EMD MilliporeTM MillexTM-GP sterile syringe filters with PES membrane (Fisher Scientific, catalog number: SLGP033RS)
    Manufacturer: Merck, catalog number: SLGP033RS .
  12. CorningTM polyethylene terephthalate (PET) centrifuge tubes, 15 ml (Corning, catalog number: 430055 )
  13. CELLSTARTM Greiner Bio-OneTM TC treated cell culture flasks, 250 ml (Greiner Bio One International, catalog number: 658175 )
  14. FisherbrandTM premium microcentrifuge tubes, 1.5 ml (Fisher Scientific, catalog number: 05-408-129 )
  15. FisherbrandTM low-retention pipet tips–filtered, 10 μl (Fisher Scientific, catalog number: 02-717-158 )
  16. FisherbrandTM low-retention pipet tips–filtered, 20 μl (Fisher Scientific, catalog number: 02-717-161 )
  17. FisherbrandTM low-retention pipet tips–filtered, 200 μl (Fisher Scientific, catalog number: 02-717-165 )
  18. QuibitTM assay tubes (Thermo Fisher Scientific, InvitrogenTM, catalog number: Q32856 )
  19. MicroAmpTM optical 96-well reaction plate with barcode (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4306737 )
  20. MicroAmpTM optical adhesive film (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4311971 )
  21. Filters, bottle top 1 L (Thermo Fisher Scientific, catalog number: 597-4520 )
  22. DynabeadsTM MPCTM-S magnetic particle concentrator (Thermo Fisher Scientific, catalog number: A13346 )
  23. DynabeadsTM MPCTM-6 magnetic particle concentrator (Thermo Fisher Scientific, catalog number: 12002D )
  24. DynabeadsTM L10 tubes (Thermo Fisher Scientific, catalog number: 74003 )
  25. VWR® 8-425 screw thread vials (VWR, Avantor, catalog number: 66020-950 )
  26. Cap screw top black, with PTFE/silicone pre-slit septa, 10 mm, packed in a clean environment (PerkinElmer, catalog number: N9306052 )
  27. C. parvum 18S-rRNA primers: Cp18S-995F: 5’-TAGAGATTGGAGGTTCCT-3’ and Cp18S-1206R: 5’-CTCCACCAACTAAGAACGCC-3’ (Thermo Fisher Scientific, Waltham, MA, USA)
  28. Human 18S-rRNA primers: Hs18S-F1373: 5’-CCGATAACGAACGAGACACTCTGG-3’ and Hs18S-R1561: 5’-TAGGGTAGGCACACGCTGAGCC-3’ (Thermo Fisher Scientific, Waltham, MA, USA)
  29. Quant-iTTM Qubit RNA BR assay kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: Q10211 )
  30. RNeasy mini kit, 250 (QIAGEN, catalog number: 74106 )
  31. RNase-free DNase set, 50 (QIAGEN, catalog number: 79254 )
  32. iScriptTM RT-qPCR sample preparation reagent (Bio-Rad Laboratories, catalog number: 1708898 )
  33. Luna® Universal one step RT-qPCR kit protocol (New England BioLabs, catalog number: E3005 )
  34. Sterile phosphate buffered saline (Thermo Fisher Scientific, GibcoTM, catalog number: 10010049 )
  35. Minimum Essential Medium (MEM) with L-glutamine and phenol red, without HEPES (Thermo Fisher Scientific, GibcoTM, catalog number: 11095-114 )
  36. Horse Serum, New Zealand origin (Thermo Fisher Scientific, GibcoTM, catalog number: 16050122 )
  37. UltraPureTM DNase/RNase-free distilled water (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10977015 )
  38. Ethanol, 70% (Sigma-Aldrich, catalog number: 1009744000)
    Manufacturer: Merck, catalog number: 100974 .
  39. Roche DiagnosticsTM glucose test strips for Accutrend® Plus meters (Fisher Scientific, catalog number: 22-045-871)
    Manufacturer: Roche Diagnostics, catalog number: 11447475160
  40. Crypt-a-GloTM reagent only kit (Waterborne, catalog number: A400FLR-20 )
  41. Sporo-GloTM (Waterborne, catalog number: A600FLR-20X
  42. Trypsin-EDTA (0.25%), phenol red (Thermo Fisher-Scientific, GibcoTM, catalog number: 25200056
  43. L-glutamine (Sigma-Aldrich, catalog number: G3126 )
  44. HEPES (Sigma-Aldrich, catalog number: H3375 )
  45. D-glucose (Sigma-Aldrich, catalog number: G8270 )
  46. Ascorbic acid (Sigma-Aldrich, catalog number: A0278 )
  47. P-Aminobenzoic acid (Sigma-Aldrich, catalog number: A9878 )
  48. Calcium pantothenate (Sigma-Aldrich, catalog number: C8731 )
  49. Folic acid (Sigma-Aldrich, catalog number: F7876 )
  50. Heparin (Sigma-Aldrich, catalog number: H3393 )
  51. Antibiotic/antimycotic 100x (Thermo Fisher Scientific, catalog number: 15240062 )
  52. Taurodeoxycholate (Sigma-Aldrich, catalog number: T0557 )
  53. Thioglycolic acid (MP Biomedicals, catalog number: 102933 )
  54. Mannitol (Sigma-Aldrich, catalog number: M4125 )
  55. Glutathione (Sigma-Aldrich, catalog number: G6013 )
  56. Taurine (Sigma-Aldrich, catalog number: T8691 )
  57. Betaine hydrochloride (Sigma-Aldrich, catalog number: B3501 )
  58. Cysteine (Sigma-Aldrich, catalog number: C7352 )
  59. Oleic acid (Sigma-Aldrich, catalog number: O1257-10MG )
  60. Cholesterol (Sigma-Aldrich, catalog number: C4951-30MG )
  61. DynabeadsTM anti-Cryptosporidium (Thermo Fisher Scientific, catalog number: 73011 )
  62. Nitrogen Medical Grade 99.998% purity (TW Smith, Brooklyn, NY)
  63. Carbon dioxide, Bone Dry, 99.998% (TW Smith, Brooklyn, NY)
  64. ECS medium mix (see Recipes)
  65. ICS medium mix (see Recipes)

Equipment

  1. 1 L Bottles, 33 mm cap (Fisher Scientific, catalog number: 0642114)
    Manufacturer: DWK Life Sciences, catalog number: 61111T1000 .
  2. 125 ml Bottles, 33 mm cap (DWK Life Sciences, catalog number: 219755 )
  3. Roche DiagnosticsTM glucose controls for Accutrend® Plus meters (Fisher Scientific, catalog number: 22-045-733)
    Manufacturer: Roche Diagnostics, catalog number: 05213231160 .
  4. HausserTM LevyTM hemacytometer chamber set (Hausser Scientific, catalog number: 3520 )
  5. FiberCellTM Systems Duet Pump (FiberCell Systems, catalog number: P3202 )
  6. Hollow Fiber Medium Cartridge (FiberCell Systems, catalog number: C2011 )
  7. UVP UV2 Sterilizing PCR Workstation (VWR, Avantor, Bridgeport, NJ 08014, USA)
  8. Class II, Type A, Biohazard Cabinet, model number 10276 (Envirco Cedar Grove, NJ 07009, USA)
  9. Beckman Coulter Microfuge® 16 centrifuge (Beckman Coulter, model: Microfuge® 16 )
  10. EppendorfTM centrifuge 5810R (Eppendorf, model: 5810 R )
  11. Quant Studio 6 Flex 44 (Applied Biosystems, Life Technology, Thermo Fisher Scientific, Waltham, MA, USA)
  12. Thermo Scientific Revco Ultima CO2 incubator (Thermo Fisher Scientific, Waltham, MA, USA)
  13. DynabeadsTM MX mixer base (Thermo Fisher Scientific, catalog number: 15902 )
  14. QubitTM 3.0 fluorometer (Thermo Fisher Scientific, catalog number: Q33216 )
  15. NikonTM Optiphot fluorescence microscope (Nikon, Westbury, NY)
  16. Fisher Scientific Accumet® AR10 pH benchtop meter (Fisher Scientific, catalog number: 13-636-AB150 )
  17. Fisher Scientific dry bath incubator (Fisher Scientific, catalog number: 11-718-2 )
  18. Autoclave (LBR Scientific, Rutherford, NJ) 

Software

  1. Quant Studio RT-PCR software v1.0 (Applied Biosystems, Life Technology, Thermo Fisher Scientific, Waltham, MA, USA)
  2. Sigma Plot v2001 (Systat Software, Inc, San Jose, CA, USA)

Procedure

  1. Cartridge preparation
    1. Sterilization of Reservoir Cap/Bottle Assembly
      1. Insert a 33 ml reservoir cap into the 125 ml bottle. Fit screw cap loosely.
      2. Attach fresh reservoir cap tube (non-sterile), included with the cartridge, to the cap.
      3. Loosely wrap the Luer fittings on the end of the reservoir cap tubing with foil.
      4. Place entire assembly into an autoclave bag and seal.
      5. Autoclave at 121 °C, 15 lb/in2 for 20 min (High Vacuum).
    2. Connection of the reservoir cap to the Flow Path
      1. Remove Fibercell module from its packaging. Check the Luer fittings between the hollow fiber module and the flow path to ensure that they are finger tight.
      2. Remove the foil from one of the Luer fittings on the reservoir cap.
      3. Choose one of the Luer fittings on the flow path. Spray with 70% ethanol and wipe with an alcohol pad.
      4. Remove the Luer cap from the Luer fitting and attach the Luer fitting to the 33 ml reservoir cap. Apply ½ turn counter rotation to the tubing prior to attachment to prevent kinking of the tubing.
      5. Repeat steps to attach the other Luer fitting.
      6. Check fitting and be sure they are tightly attached.

  2. Pre-culture
    When working with adherent cells, the cartridge should be washed with 2-volumes of 125 ml of sterile PBS each for 24 h prior to filling the cartridge with 125 ml of cell culture medium.
    1. Ensure the left and right end port side clamps are open. Aseptically fill 100 ml of sterile PBS into 125 ml bottle attach to cartridge using the 33 mm caps (The flow path and cartridge for medium cartridge holds about 30 ml of media).
    2. Use thumb and forefinger to manually prime the pump until air is pushed out of the tubing then close the left and right end port side clamps. 
    3. Fill a sterile 60 ml syringe with 30 ml sterile PBS and attach to the left extra-capillary space (ECS) port and attach an empty 60 ml syringe to the right ECS port.
    4. Open the clamps on the left and right ECS side ports and push the sterile PBS into the ECS. Tilt cartridge upwards and fill medium taking care to remove all air present in the ECS. If the volume is not sufficient, repeat.
    5. Close the left and right ECS side ports, spray with 70% alcohol and remove the syringes, spray with 70% alcohol and wipe with an alcohol pad prior to attaching clean sterile 3 ml syringes to the ports. Open the left and right side port clamps.
    6. Connect the Fibercell hollow fiber module to the Fibercell pump unit in a 37 °C 5% CO2 incubator. Set a medium flow rate of 5, leave for 24 h.
    7. After 24 h, the left and right end port clamps are closed and the PBS reservoir bottle is aseptically changed for a fresh 125 ml bottle containing 100 ml of sterile PBS and returned to the incubator for 24 h.
    8. Aseptically fill a sterile 125 ml reservoir bottle with 100 ml of MEM media. Leave the reservoir cap loose by ½ turn.
    9. Fill a sterile 60 ml syringe with 30 ml sterile MEM and attach to the left ECS port and attach an empty 60 ml syringe to the right ECS port. 
    10. Open the clamps on the left and right ECS side ports and push the sterile MEM into the ECS. Tilt cartridge upwards and fill medium taking care to remove all air present in the ECS. If the volume is not sufficient, repeat.
    11. Close the left and right ECS side ports, spray with 70% alcohol and remove the syringes, spray with 70% alcohol and wipe with an alcohol pad prior to attaching clean sterile 3 ml syringes to the ports. Open the left and right side port clamps.
    12. Connect the Fibercell hollow fiber module to the Fibercell pump unit in a 37 °C, 5% CO2 incubator. Set a medium flow rate of 5 and leave overnight.
    13. Repeat Steps B8-B12 using MEM plus 10% horse serum.

  3. Inoculating Cells into the ECS (refer to Video 1)

    Video 1. Procedure for loading either host cells or C. parvum into the HFB

    1. Close the left and right end port slide clamps.
    2. HCT-8 monolayers are trypsinized using Trypsin-EDTA (0.25%), phenol red, washed with MEM and resuspended 1 x 106 in a total volume of 5 ml of ICS mix (see Recipe 2), and placed into a trough.
    3. Spray the left side port Luer connection with 70% ethanol and wipe with an alcohol pad. Remove syringes with an alcohol pad.
    4. Draw up cells using a 10 ml syringe with a blunt end needle.
    5. Following same aseptic procedure attach an empty syringe to the right side port Luer fitting.
    6. If closed, open the side port clamps.
    7. Gently flush the cell suspension through the ECS back and forth between the 2 syringes 3-5 times. This ensures distribution of the cells.
    8. Loosen the reservoir cap ½ turn.
    9. Leave the left end port slide clamp closed. Open the right-hand end port slide clamp. This will allow excess medium to flow into the reservoir bottle.
    10. Close the right side port slide clamp.
    11. Gently depress the plunger of the syringe attached to the left side port until all the medium and cells have been forced into the ECS.
    12. Close the left side port slide clamp and open the right side port slide clamp.
    13. Gently depress the plunger of the syringe attached to the right side port until all the medium and cells have been forced into the ECS.
    14. Close the side port slide clamps. Replace the syringes with new sterile syringes.
    15. Tighten the reservoir cap.
    16. Open the left end port slide clamp.
    17. Place the cartridge into the Fibercell pump inside a 37 °C, 5% CO2 incubator and set flow rate to 5. After 3 days, increase the flow rate to 10.

  4. Addition of C. parvum oocysts
    Fresh MEM plus 10% HS is replaced daily. The pH and glucose concentration of the 48 h and fresh medium are monitored in the ECS using an Accumet® pH meter and an Accutrend Plus® glucose meter. When the glucose drops by 50% in 24 h, the ECS is seeded with 1 x 105 C. parvum oocysts that have been treated with 10% Chlorox®, washed 7x each with 30 ml sterile distilled water and suspended in 10 ml of MEM containing the supplements shown in Recipe 1 (ECS mix). The intra-capillary space (ICS) medium is replaced with that shown in Recipe 2 (ICS mix).
    1. Remove the unit to a sterile hood and slide the L and R end port clamps to closed.
    2. Spray the left and right ECS ports with 70% alcohol using an alcohol pad and remove the syringe from the left ECS port.
    3. Attach the syringe containing the 10 ml of C. parvum oocysts suspended in the ECS mix to the left ECS port.
    4. Attach a clean sterile 10 ml syringe to the right ECS port.
    5. Open the slide clamps to the left and right ECS ports. Push down on the syringe attached to the left port (containing C. parvum oocysts), gently pull up on the right syringe.
    6. When the left syringe is fully depressed, push down on the right syringe to mix the contents of the ECS.
    7. Repeat the procedure (Steps 5 and 6) 3-4 times end up with both the left and right syringe approximately 50% full (5 ml in each).
    8. Close the right ECS port clamp, open the right end port slide clamp. Push down on the syringe attached to left ECS port until the contents have been added to the ECS (Take care not to add air to the cartridge). Slide the left ECS port clamp closed. Open the right ECS slide clamp and push down on the syringe until the contents of the syringe attached to this port have been added (Take care not to add air to the cartridge). Slide the right ECS port clamp closed. When inoculating the cartridge initially the user should experience minimal backpressure, after several months (> 6 months) of continuous culture an increase in backpressure can be experienced presumably due to build-up of the host-cell layer and debris from sloughed cells. When using the 3 ml ECS cartridge after a year of continuous culture, the backpressure is significant and it is not recommended for studies beyond this point. The 20 ml ECS cartridge was maintained for over 2 years without encountering this problem and is the cartridge of choice for long-term studies.
    9. Slide the left end port clamp to open. Spray the left and right ECS ports with 70% alcohol and use an alcohol pad to remove the syringes and replace with clean, sterile 10 ml syringes.
    10. Ensure both left and right end port clamps are closed and replace the reservoir bottle with 125 ml of the complete ICS medium mix (Recipe 2). Open both left and right end ports and return the cartridge to the pump. Set the flow rate to 10.
    11. The pH and glucose in the ICS medium are monitored daily, by removing 5 ml directly from the reservoir bottle. When the glucose concentration falls from 360 mg/dl to approx. 200 mg/dl in 24 h, change the reservoir for a 500 ml bottle of the complete ICS medium mix, containing 460 mg/dl of glucose.
    12. Continue to monitor the pH and glucose, and when the glucose concentration falls to 50% (200-230 mg/dl) in 24 h, change the reservoir for a 1 L bottle of the complete ICS medium mix, containing 500 mg/dl glucose.
    13. The pH and glucose are monitored every 48 h for the duration of the culture as an indicator of the integrity of the host cell layer.

  5. Analysis of C. parvum growth by changes in Cp18S-rRNA
    RNA Preparation:
    1. Centrifuge a 50 μl sample, discard the supernatant and add 100 μl of iScript Buffer (lysis buffer).
    2. Freeze/thaw 6x by immersing in liquid nitrogen for 1 min followed by rapid thaw at 70 °C for 1.5 min.
    3. Centrifuge the lysate for 5 min at 16,162 x g (14,800 rpm).
    4. Carefully transfer the supernatant using a 200 μl pipette (DO NOT TOUCH THE PELLET) to a clean tube.
    5. Add 1 volume of 70% ethanol to the lysate, and mix well by pipetting. Do not centrifuge.
    6. Transfer up to 700 μl of the sample, including any precipitate, to an RNeasy Mini spin column placed in a 2 ml collection tube.
    7. Centrifuge for 15 sec at 8,000 x g (10,412 rpm). Discard flow through.
    8. Add 350 μl of Buffer RW1 to the column. Centrifuge for 15 sec at 8,000 x g (10,412 rpm).
    9. Add 10 μl DNase I stock solution to 70 μl buffer RDD. Mix gently by inverting the tube. Centrifuge for 15 sec at 8,000 x g (10,412 rpm).
    10. Add DNase I incubation mix (80 μl) from Step E9 to the column membrane and place on the benchtop for 15 min
    11. Add 350 μl buffer RW1 to the column. Centrifuge for 15 sec at 8,000 x g (10,412 rpm). Discard flow through.
    12. Add 500 μl buffer RPE to the column. Centrifuge for 15 sec at 8,000 x g (10,412 rpm) to wash the membrane. Discard flow through.
    13. Add 500 μl buffer RPE to the column. Centrifuge for 2 min at 8,000 x g (10,412 rpm). Discard flow through
    14. Place the column in a new 1.5 ml collection tube. Add 30-50 μl of RNase free water directly to the column membrane. Centrifuge for 1 min at 8,000 x g (10,412 rpm) to elute the RNA. Label tubes with sample description, date, and amount of water added.
    15. Either quantitate RNA immediately or store samples at -80 °C.
    16. RNA is quantitated using the broad range Qubit RNA assay kit using a Qubit 3.0 fluorometer according to the manufacturer’s instructions.
    17. Enumerate C. parvum by quantitating Cp18S-rRNA in HFB samples and compare it to a standard prepared using 106, 107 and 108 C. parvum oocysts. Carry out the procedure using 5 ng of RNA with Luna Universal One-Step® RT-qPCR kit (NEB E3005) employing C. parvum 18S-rRNA primers: (Cp18S-995F: 5’-TAGAGATTGGAGGTTCCT-3’ and Cp18S-1206R: 5’-CTCCACCAACTAAGAACGCC-3’) and compare it to human 18S-rRNA primers: (Hs18S-F1373: 5’-CCGATAACGAACGAGACACTCTGG-3’ and Hs18S-R1561: 5’-TAGGGTAGGCACACGCTGAGCC-3’). Perform the procedure according to the manufacturer’s instructions using a Quant Studio 6 Flex 44.
    18. The HFB reproducibly produces 5 x 107 to 108 oocysts per ml after 2 weeks of culture, and this is maintained if not more than 50% of the ECS volume is sampled every 7-10 days. Sampling 90% of the cartridge volume at one time resulted in 4-week recovery time to obtain 108 oocysts per ml.
    19. Stain oocysts with Crypt-o-Glo® according to the manufacturer’s instructions and evaluate using a NikonTM Optiphot fluorescence microscope.
    20. Stain motile stages with Spor-a-Glo® according to the manufacturer’s instructions and evaluate using a NikonTM Optiphot fluorescence microscope.

  6. Tips to aid reproducibility
    1. Keep the inlet and outlet lines to the reservoir bottle behind the line used to prime the pump, this prevents kinking of the lines and reduces the chance of contamination when handling the cartridge.
    2. Use an alcohol pad to wrap around fittings when changing the syringes prevents contamination.
    3. If the glucose concentration of the reservoir bottle does not drop significantly after 48 h, replenish the host cells by adding 106 HCT-8 cells to the extra-capillary space.
    4. To achieve the consistent growth of C. parvum, it is important to maintain a low redox balance of the extra-capillary medium.
      Note: To achieve this, we prepared the redox mix using sterile distilled water that is boiled and purged with 0.4 μm filtered N2 gas. When cooled to ambient temperature, thiols are dissolved under an N2 atmosphere.
    5. Removal of 50% (10 ml) of the culture is possible by displacing the extra-capillary medium with an equal volume of fresh ECS medium. When displacing the cells depress the syringe gently (10 sec with a 10 ml syringe) so as not to cause turbulence. It is advised that the culture not be disturbed for 10-14 days post-sampling to enable repopulation of the culture.

Data analysis

  1. Growth profile of total parasite stages from the HFB
    1. Samples (0.5 ml) were removed from the HFB and RNA isolated and analyzed by qRT-PCR using specific primers for C. parvum 18S-rRNA (Cp18S-995F: 5’-TAGAGATTGGAGGTTCCT-3’ and Cp18S-1206R: 5’-CTCCACCAACTAAGAACGCC-3’) and compared with HCT-8 18S-rRNA (Hs18S-F1373: 5’-CCGATAACGAACGAGACACTCTGG-3’ and Hs18S-R1561: 5’-TAGGGTAGGCACACGCTGAGCC-3’) to enumerate parasite number as described by Zhang and Zhu (2015). Parasite numbers increased from 5 x 103/ml on day one to 5 x 108/ml by Day 15 (Figure 2). Using Dynabeads to purify oocysts from the HFB sample we have obtained 109 oocysts from 10 ml of culture volume.


      Figure 2. C. parvum growth curve in the HFB

    2. C. parvum numbers are shown as Log of total parasite stages per ml. Parasite numbers were determined by qRT-PCR using primers for the Cp-18SrRNA as described in the procedure and the ΔCT values determined from a standard using 103, 104 and 105 oocysts (Zhang and Zhu, 2015). The culture was inoculated on day 1 with 5 x 103 oocysts/ml (105). Results are presented as triplicate counts ± SEM.
    3. Oocysts were stained with a monoclonal antibody to the Cryptosporidium oocyst wall protein conjugated to a fluorescent probe (Crypt-a-Glo®) and examined using a fluorescence microscope with an excitation wavelength of 410-485 nm and an emission wavelength of 515 nm (Figure 3A). Motile extracellular stages were stained using a polyclonal antibody conjugated to a red fluorescent probe (Sporo-Glo®) and examined using a fluorescent microscope with an excitation wavelength of 535-550 nm and an emission wavelength of 580 nm (Figure 3B).


      Figure 3. Microscopy of parasites collected from the HFB. A. Oocysts from the HFB stained using Crypt-a-Glo®; B and C. Motile stages from the HFB stained using Sporo-Glo®. Scale bars = 5 μm.

  2. In vivo Infectivity of HFB produced C. parvum
    Oocysts collected from the HFB culture were used to infect TCR-α-deficient mice to demonstrate the maintenance of virulence factors. Parasites cultured in 2D culture flasks for 48 h fail to cause an infection in either immunosuppressed or immune-deficient mouse models. Oocysts were collected from the HFB and purified using Dyna beads® according to the manufacturer’s instructions. Groups of three TCR--deficient mice on a C57BL/6 background (Jackson Laboratories, Bar Harbor, ME, USA) were orally dosed with 106 C. parvum oocyts (0.1 ml in PBS) from the HFB, and compared with 106 oocysts of the parent Iowa isolate (positive control) and mice dosed with 0.1 ml PBS (negative control). There is a statistical difference in weight gain of the C. parvum infected mice from 10 days onward compared to negative controls dosed with PBS (Figure 4A), indicative of an active C. parvum infection. Fecal pellets were collected and softened in PBS prior to smearing on microscope slides and staining by the modified cold Kinyoun acid-fast technique and enumerated microscopically at 400x using a confocal microscope (Nikon, Westbury, NY, USA). Oocysts were detected after 7-10 days and shedding increased daily up to 25 days (Figure 4B).


    Figure 4. In vivo analysis of C. parvum oocysts from the HFB. A. Mouse model C. parvum infection. Triplicate groups of mice were infected with 0.1 ml by oral gavage containing 106 C. parvum oocysts suspended in PBS collected from the HFB , Six-week old C. parvum Iowa isolate (same source as used to initiate the culture) , and compared to controls gavaged with 0.1 ml PBS . Mice were weighed daily. B. Oocyst shedding by HFB cultured mouse model infection. Mouse feces were softened with 0.1 ml distilled water and smeared onto a glass slide, stained using a modified cold Kinyoun acid-fast technique and oocysts counted microscopically at 400x magnification.

  3. Excystation of oocysts collected from the HFB
    The excystation rate of C. parvum oocysts from the HFB was compared to control Iowa isolate oocysts as an index of oocyst viability. C. parvum oocysts were incubated in PBS, pH 7.4 containing 150 mg of Na taurocholate and 50 mg of trypsin at 37 °C for 30 min and dual stained using Crypt-a-Glo® and Sporo-Glo®. The percent of excysted oocysts was determined by counting oocysts and sporozoites using a hemacytometer using a fluorescent microscope (Nikon Optiphot) at 400x magnification. Crypt-a-Glo® stained oocysts were visualized using an excitation wavelength of 410-485 nm and an emission wavelength of 515 nm; Sporo-Glo® stained sporozoites (spz) were visualized using an excitation wavelength of 535-550 nm and an emission wavelength of 580 nm. The results show that approx. 80% of the oocysts collected from the HFB culture system excyst in 30 min, which is similar to the data obtained with the C. parvum parent Iowa isolate. Oocysts from both the HFB culture and the Iowa isolate stored at 4 °C demonstrated a gradual decrease in percent excystation with time of storage, falling to approx. 40% after 6 months (Figure 5).


    Figure 5. Excystation of C. parvum oocysts. Oocysts were excysted in PBS, pH 7.4 containing 150 mg of Na taurocholate and 50 mg of trypsin at 37 °C for 30 min. The percentage of excysted oocysts determined by dual staining with Crypt-a-Glo® and Sporo-Glo® and counting using an Improved Neubauer Hemacytometer. The percent excystation is calculated from the following formula: spz count/4 x oocyst count x 100%.

  4. Changes in glucose concentrations of HFB
    The glucose concentration of the intra-capillary and extra-capillary space was measured as an indicator of host cell growth (Figure 6). When the medium glucose concentration falls to 50% of its starting concentration, the new medium must be replenished to prevent loss of host cells. The extra-capillary glucose concentration remains constant at approximately 275 mg/dl (Figure 6, dashed line).


    Figure 6. Glucose concentration of intra-capillary space medium. The glucose concentration of the medium providing nutrients to the host cells is monitored daily. The starting concentration is 350 mg/dl in a 125 ml flask which is changed to 425 mg/dl/500 ml flask on day 4. When the glucose concentration drops to 210-225 mg/dl in 48 h, (typically by Day 10) the glucose is increased to 475-500 mg/dl in a 1 L flask. The glucose concentration typically falls to 50% in 48 h, hence the medium is replenished on a Monday, Wednesday, Friday schedule thereafter. The glucose in the extra-capillary space, which contains the parasites, remains constant at 275 mg/dl.

Recipes

  1. ECS medium mix

    Components are added and the medium is filter sterilized
    *Prepared using nitrogen degassed water and stored in 1 ml aliquots under a nitrogen gas phase at -20 °C
    #Stock kept at 4 °C
  2. ICS medium mix

    Components are added and the medium is filter sterilized

Acknowledgments

We would like to thank Dr’s Saul Tzipori and Sangun Lee (Cummings School of Veterinary Medicine, Tufts University, N. Grafton, MA, USA) for helpful advice with the in vivo animal studies. Dr’s Louis Weiss and Leslie Gunther-Cummins (Departments of Pathology and Medicine, and Analytical Imaging Facility, Albert Einstein College of Medicine, Bronx, NY, USA) for electron microscopy. This work was funded by the Bill and Melinda Gates Foundation (NY). The protocol is adapted from Morada et al. (2016). The authors declare no conflicts of interest or competing interests.

References

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  3. Arrowood, M. J. (2008). In vitro cultivation. In: Cryptosporidium and Cryptosporidosis. Fayer, R. and Xiao, L. (Eds.). CRC Press, Boca Raton: 499-525.
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  5. Bhutta, Z. A. and Black, R. E. (2013). Global maternal, newborn, and child health--so near and yet so far. N Engl J Med 369(23): 2226-2235.
  6. Checkley, W., White, A. C., Jr., Jaganath, D., Arrowood, M. J., Chalmers, R. M., Chen, X. M., Fayer, R., Griffiths, J. K., Guerrant, R. L., Hedstrom, L., Huston, C. D., Kotloff, K. L., Kang, G., Mead, J. R., Miller, M., Petri, W. A., Jr., Priest, J. W., Roos, D. S., Striepen, B., Thompson, R. C., Ward, H. D., Van Voorhis, W. A., Xiao, L., Zhu, G. and Houpt, E. R. (2015). A review of the global burden, novel diagnostics, therapeutics, and vaccine targets for Cryptosporidium. Lancet Infect Dis 15(1): 85-94.
  7. DeCicco RePass, M. A., Chen, Y., Lin, Y., Zhou, W., Kaplan, D. L. and Ward, H. D. (2017). Novel bioengineered three-dimensional human intestinal model for long-term infection of Cryptosporidium parvum. Infect Immun 85(3): e00731-16.
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  11. Love, M. S., Beasley, F. C., Jumani, R. S., Wright, T. M., Chatterjee, A. K., Huston, C. D., Schultz, P. G. and McNamara, C. W. (2017). A high-throughput phenotypic screen identifies clofazimine as a potential treatment for cryptosporidiosis. PLoS Negl Trop Dis 11(2): e0005373.
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  15. Vinayak, S., Pawlowic, M. C., Sateriale, A., Brooks, C. F., Studstill, C. J., Bar-Peled, Y., Cipriano, M. J. and Striepen, B. (2015). Genetic modification of the diarrhoeal pathogen Cryptosporidium parvum. Nature 523(7561): 477-480.
  16. Zhang, H. and Zhu, G. (2015). Quantitative RT-PCR assay for high-throughput screening (HTS) of drugs against the growth of Cryptosporidium parvum in vitro. Front Microbiol 6: 991.

简介

Cryptosporidium parvum 的连续体外生长已证明是困难的,并且常规体外培养技术导致短期(2-5天)生长寄生虫导致薄壁卵囊不能使用体外培养物繁殖,并且不使用免疫抑制或免疫缺陷小鼠模型产生活跃感染(Arrowood,2002)。在这里,我们描述了中空纤维生物反应器(HFB)的使用,通过提供氧气和营养物质从基础表面宿主肠细胞模拟体内条件,并允许建立低氧化还原,高营养环境顶面。当接种10 5 C时。 parvum (爱荷华州分离物)卵囊生物反应器在14天后每ml产生10个 8 卵囊(20ml额外毛细血管体积),并保持2年以上。使用TCR-α免疫缺陷小鼠模型的体内感染性研究显示,在6,12和18个月时从生物反应器产生的卵囊与用于启动培养的亲本Iowa分离物无法区分。 HFB产生的卵囊具有与亲本爱荷华分离物类似的百分比分析。

【背景】 Cryptosporidium parvum 是人和其他哺乳动物肠道的细胞内专性寄生虫,导致急性腹泻。该疾病在免疫功能正常的个体中是自限性的,然而,在免疫功能低下的成人和幼儿中,该疾病可能危及生命(Kotloff,2017)。它是经济资源低的国家中三种被诊断出的儿童肠道疾病之一(Kotloff et al。,2013; Sow et al。,2016),估计是占全球儿童死亡人数的9%(Bhutta和Black,2013; Checkley et al。,2015)。在儿童腹泻病负担高的国家,已经表明营养不良,发育迟缓和认知功能受损的发病率相应较高(Lang和MA-LED Network Investigators,2015)。尽管这种疾病具有显着的健康风险和全球分布,但对于风险最高的人群而言,没有一贯有效的治疗方法。操作寄生虫基因组的最新进展(Vinayak et al。,2015)和化学治疗分析(Arnold et al。,2017; Love et al。,2017; Manjunatha et al。,2017)是一个令人鼓舞的迹象,表明这种黯淡的局面将会发生变化。寄生虫的培养集中在使用成年小鼠结肠细胞(Baydoun et al。,2017)或新型生物工程人肠细胞(DeCicco RePass 等。)开发3D培养系统。 >,2017)与2D培养方法相比,它对寄生虫入侵产生了新的见解并显着延长了培养时间。然而,这些技术在获得的寄生虫数量方面受到限制。 3D模型克服了与传统2D培养方法相关的主要障碍,其中宿主细胞从顶端表面接收营养物和氧气(除了那些使用多孔膜插入物的系统,如Costar Transwell系统);这与体内情况相反,其中肠细胞从基底表面接收营养物和氧,并且顶端表面面向肠腔。然而,目前的肠道植入物模型不能提供肠腔内存在的低氧环境,这限制了寄生虫的长期生长。中空纤维技术的使用允许产生肠道中存在的双相环境并克服与 C的长期培养相关的许多问题。 parvum (Morada et al。,2016)。该协议描述了中空纤维生物反应器的建立,该生物反应器可用于通过向附着于中空纤维外部的宿主肠细胞的基底表面提供氧气和营养来模拟体内条件(图1)。调节反应器内的环境以模拟肠腔,因此肠细胞的顶端表面在低氧化还原,高营养环境中建立,这有利于高生长速率和 C的长期维持。孢子虫。使用该方法可提供10个 8 -10 9 卵囊,可用于分子和生物化学研究,并具有避免使用诸如K-等刺激性化学品的优点。重铬酸盐,在(4°C)下用作长期储存介质,具有消毒卵囊的优点;氯和氯目前既用作消毒剂又用于增强从动物来源获得的卵囊的外渗(Arrowood,2008)。
图1.中空纤维生物反应器。 A.HFB的示意图; B. HFB设置。

关键字:寄生虫学, 细胞培养, 原生动物, 隐孢子虫, 小隐孢子虫, 中空纤维生物反应器

材料和试剂

  1. BrandTech TM BRAND TM 试剂库(BrandTech Scientific,目录号:703459)
  2. 铝箔18英寸x 500英尺(Sigma-Aldrich,目录号:Z185159-1EA)
  3. BD注射器带各种吸头,60 ml(BD,目录号:309653)
  4. BD诊断系统SYR仅Luer-Lok TM 10 ml 200PK RX(赛默飞世尔科技,目录号:B302995)
    制造商:BD,目录号:302995。
  5. BD一次性注射器,带Luer-Lok TM 吸头,3 ml(BD,目录号:309657)
  6. Fisherbrand TM 无菌酒精预备垫(Fisher Scientific,目录号:22-363-750)
  7. BD Vacutainer TM 一般使用注射器针头(BD,目录号:305180)
  8. Fisherbrand TM 硼硅酸盐玻璃一次性血清移液管,标准长度,标准长度,5 ml(Fisher Scientific,目录号:13-678-27E)
  9. Fisherbrand TM 硼硅酸盐玻璃一次性血清移液管,标准长度,标准长度,10 ml(Fisher Scientific,目录号:13-678-27F)
  10. Fisherbrand TM 硼硅酸盐玻璃一次性血清移液管,常规尖端,短长度,25 ml(Fisher Scientific,目录号:13-678-36D)
  11. EMD Millipore TM Millex TM -GP PES膜无菌注射器过滤器(Fisher Scientific,目录号:SLGP033RS)
    制造商:Merck,目录号:SLGP033RS。
  12. 康宁 TM 聚对苯二甲酸乙二醇酯(PET)离心管,15 ml(Corning,目录号:430055)
  13. CELLSTAR TM Greiner Bio-One TM TC处理细胞培养瓶,250 ml(Greiner Bio One International,目录号:658175)
  14. Fisherbrand TM 优质微量离心管,1.5 ml(Fisher Scientific,目录号:05-408-129)
  15. Fisherbrand TM 低保留移液管吸头 - 过滤,10μl(Fisher Scientific,目录号:02-717-158)
  16. Fisherbrand TM 低保留移液器吸头 - 过滤,20μl(Fisher Scientific,目录号:02-717-161)
  17. Fisherbrand TM 低保留移液管吸头 - 过滤,200μl(Fisher Scientific,目录号:02-717-165)
  18. Quibit TM 测定管(Thermo Fisher Scientific,Invitrogen TM ,目录号:Q32856)
  19. 具有条形码的MicroAmp TM 光学96孔反应板(Thermo Fisher Scientific,Applied Biosystems TM ,目录号:4306737)
  20. MicroAmp TM 光学胶膜(Thermo Fisher Scientific,Applied Biosystems TM ,目录号:4311971)
  21. 过滤器,瓶顶1升(Thermo Fisher Scientific,目录号:597-4520)
  22. Dynabeads TM MPC TM -S磁粉颗粒浓缩器(赛默飞世尔科技,目录号:A13346)
  23. Dynabeads TM MPC TM -6磁粉颗粒浓缩器(赛默飞世尔科技,目录号:12002D)
  24. Dynabeads TM L10管(赛默飞世尔科技,目录号:74003)
  25. VWR ® 8-425螺纹样品瓶(VWR,Avantor,目录号:66020-950)
  26. 顶盖黑色,带PTFE /硅胶预切口隔垫,10 mm,在干净的环境中包装(PerkinElmer,目录号:N9306052)
  27. ℃。 parvum 18S-rRNA引物:Cp18S-995F:5'-TAGAGATTGGAGGTTCCT-3'和Cp18S-1206R:5'-CTCCACCAACTAAGAACGCC-3'(Thermo Fisher Scientific,Waltham,MA,USA)
  28. 人18S-rRNA引物:Hs18S-F1373:5'-CCGATAACGAACGAGACACTCTGG-3'和Hs18S-R1561:5'-TAGGGTAGGCACACGCTGAGCC-3'(Thermo Fisher Scientific,Waltham,MA,USA)
  29. Quant-iT TM Qubit RNA BR检测试剂盒(Thermo Fisher Scientific,Invitrogen TM ,目录号:Q10211)
  30. RNeasy迷你套件,250(QIAGEN,目录号:74106)
  31. RNase-free DNase set,50(QIAGEN,目录号:79254)
  32. iScript TM RT-qPCR样品制备试剂(Bio-Rad Laboratories,目录号:1708898)
  33. Luna ®通用一步法RT-qPCR试剂盒方案(New England BioLabs,目录号:E3005)
  34. 无菌磷酸盐缓冲盐水(Thermo Fisher Scientific,Gibco TM ,目录号:10010049)
  35. 含有L-谷氨酰胺和酚红的最低必需培养基(MEM),不含HEPES(Thermo Fisher Scientific,Gibco TM ,目录号:11095-114)
  36. 马血清,新西兰血清(赛默飞世尔科技,Gibco TM ,目录号:16050122)
  37. UltraPure TM DNase / RNase-free蒸馏水(Thermo Fisher Scientific,Invitrogen TM ,目录号:10977015)
  38. 乙醇,70%(Sigma-Aldrich,目录号:1009744000)
    制造商:Merck,产品目录号:100974。
  39. 用于Accutrend ® Plus仪表的罗氏诊断 TM 葡萄糖试纸(Fisher Scientific,目录号:22-045-871)
    制造商:Roche Diagnostics,目录号:11447475160
  40. Crypt-a-Glo TM 试剂盒(水性,目录号:A400FLR-20)
  41. Sporo-Glo TM (水性,目录号:A600FLR-20X)&nbsp;
  42. L-谷氨酰胺(Sigma-Aldrich,目录号:G3126)
  43. HEPES(Sigma-Aldrich,目录号:H3375)
  44. D-葡萄糖(Sigma-Aldrich,目录号:G8270)
  45. 抗坏血酸(Sigma-Aldrich,目录号:A0278)
  46. 对氨基苯甲酸(Sigma-Aldrich,目录号:A9878)
  47. 泛酸钙(Sigma-Aldrich,目录号:C8731)
  48. 叶酸(Sigma-Aldrich,目录号:F7876)
  49. 肝素(Sigma-Aldrich,目录号:H3393)
  50. 抗生素/抗真菌药100x(赛默飞世尔科技,目录号:15240062)
  51. 牛磺酸脱氧胆酸盐(Sigma-Aldrich,目录号:T0557)
  52. 巯基乙酸(MP Biomedicals,目录号:102933)
  53. 甘露醇(Sigma-Aldrich,目录号:M4125)
  54. 谷胱甘肽(Sigma-Aldrich,目录号:G6013)
  55. 牛磺酸(Sigma-Aldrich,目录号:T8691)
  56. 甜菜碱盐酸盐(Sigma-Aldrich,目录号:B3501)
  57. 半胱氨酸(Sigma-Aldrich,目录号:C7352)
  58. 油酸(Sigma-Aldrich,目录号:O1257-10MG)
  59. 胆固醇(Sigma-Aldrich,目录号:C4951-30MG)
  60. Dynabeads TM anti- Cryptosporidium (赛默飞世尔科技,目录号:73011)
  61. 氮医用级99.998%纯度(TW Smith,Brooklyn,NY)
  62. 二氧化碳,骨干,99.998%(TW Smith,Brooklyn,NY)
  63. ECS中等混合(见食谱)
  64. ICS中等混合(见食谱)
  65. 胰蛋白酶-EDTA(0.25%),酚红(Thermo Fisher-Scientific,Gibco TM ,目录号:25200056)&nbsp;

设备

  1. 1 L瓶,33 mm盖(Fisher Scientific,目录号:0642114)
    制造商:DWK Life Sciences,目录号:61111T1000。
  2. 125毫升瓶,33毫米盖(DWK生命科学,目录号:219755)
  3. 罗氏诊断 TM 葡萄糖控制用于Accutrend ® Plus仪表(Fisher Scientific,目录号:22-045-733)
    制造商:Roche Diagnostics,目录号:05213231160。
  4. Hausser TM Levy TM 血细胞计数器室组(Hausser Scientific,目录号:3520)
  5. FiberCell TM Systems Duet Pump(FiberCell Systems,目录号:P3202)
  6. 中空纤维介质盒(FiberCell Systems,目录号:C2011)
  7. UVP UV2灭菌PCR工作站(VWR,Avantor,Bridgeport,NJ 08014,USA)
  8. II级,A型,生物危害柜,型号10276(Envirco Cedar Grove,NJ 07009,USA)
  9. Beckman Coulter Microfuge ® 16离心机(Beckman Coulter,型号:Microfuge ® 16)
  10. Eppendorf TM 离心机5810R(Eppendorf,型号:5810 R)
  11. Quant Studio 6 Flex 44(Applied Biosystems,Life Technology,Thermo Fisher Scientific,Waltham,MA,USA)
  12. Thermo Scientific Revco Ultima CO 2 培养箱(Thermo Fisher Scientific,Waltham,MA,USA)
  13. Dynabeads TM MX混合器基础(赛默飞世尔科技,目录号:15902)
  14. Qubit TM 3.0荧光计(赛默飞世尔科技,目录号:Q33216)
  15. Nikon TM Optiphot荧光显微镜(Nikon,Westbury,NY)
  16. Fisher Scientific Accumet ® AR10 pH台式仪表(Fisher Scientific,目录号:13-636-AB150)
  17. Fisher Scientific干浴培养箱(Fisher Scientific,目录号:11-718-2)
  18. 高压灭菌器(LBR Scientific,Rutherford,NJ)&nbsp;

软件

  1. Quant Studio RT-PCR软件v1.0(Applied Biosystems,Life Technology,Thermo Fisher Scientific,Waltham,MA,USA)
  2. Sigma Plot v2001(Systat Software,Inc,San Jose,CA,USA)

程序

  1. 墨盒准备
    1. 储罐盖/瓶组件的灭菌
      1. 将33毫升储液罐盖插入125毫升瓶中。松散地安装螺帽。
      2. 将墨盒附带的新储液器盖管(非无菌)连接到盖子上。
      3. 用箔片将Luer接头松散地包裹在储液器盖管的末端。
      4. 将整个组件放入高压灭菌袋中并密封。
      5. 在121℃,15磅/英寸 2 高压灭菌20分钟(高真空)。
    2. 将储液罐盖连接到流路
      1. 从包装中取出Fibercell模块。检查中空纤维模块和流路之间的鲁尔接头,确保它们用手指拧紧。
      2. 从储液器盖上的一个鲁尔接头上取下箔片。
      3. 选择流路上的其中一个鲁尔接头。用70%乙醇喷洒并用酒精垫擦拭。
      4. 从Luer接头上取下Luer盖子,将Luer接头连接到33 ml储液器盖上。在连接之前,将½转反向旋转到管道上,以防止管道扭结。
      5. 重复步骤以连接其他Luer配件。
      6. 检查配件并确保它们紧密连接。

  2. 预培养
    当使用粘附细胞时,应在用125ml细胞培养基填充盒之前,用2体积的125ml无菌PBS洗涤盒24小时。
    1. 确保左右端口侧夹具打开。无菌地将100毫升无菌PBS装入125毫升瓶中,使用33毫米盖子将其连接到药筒上(流动路径和用于中型药筒的药筒容纳约30毫升培养基)。
    2. 使用拇指和食指手动灌注泵,直到空气被推出管道然后关闭左右端口侧夹。&nbsp;
    3. 用无菌PBS填充无菌60 ml注射器并连接到左侧毛细管外空间(ECS)端口,并将空的60 ml注射器连接到正确的ECS端口。
    4. 打开左侧和右侧ECS侧端口上的夹子,将无菌PBS推入ECS。向上倾斜墨盒并填充介质,注意去除ECS中存在的所有空气。如果音量不够,请重复。
    5. 关闭左侧和右侧ECS侧端口,喷洒70%酒精并取出注射器,喷洒70%酒精并用酒精垫擦拭,然后将干净的无菌3 ml注射器连接到端口。打开左侧和右侧端口夹。
    6. 将Fibercell中空纤维模块连接到37°C 5%CO 2 培养箱中的Fibercell泵装置。设定中等流速为5,离开24小时。
    7. 24小时后,关闭左右端口夹具,无菌地更换PBS储液瓶,用于装有100ml无菌PBS的新鲜125ml瓶子,并返回培养箱24小时。
    8. 用100ml MEM培养基无菌填充无菌125ml储液瓶。将储液罐盖松开½圈。
    9. 用无菌MEM填充无菌60 ml注射器并连接到左侧ECS端口,并将空的60 ml注射器连接到正确的ECS端口。&nbsp;
    10. 打开左侧和右侧ECS侧端口上的夹子,将无菌MEM推入ECS。向上倾斜墨盒并填充介质,注意去除ECS中存在的所有空气。如果音量不足,请重复
    11. 关闭左侧和右侧ECS侧端口,喷洒70%酒精并取出注射器,喷洒70%酒精并用酒精垫擦拭,然后将干净的无菌3 ml注射器连接到端口。打开左侧和右侧端口夹。
    12. 将Fibercell中空纤维模块连接到37°C,5%CO 2 培养箱中的Fibercell泵装置。设定中等流速为5并过夜。
    13. 使用MEM加10%马血清重复步骤B8-B12。

  3. 将细胞接种到ECS中(参见视频1)

    视频
    1. 关闭左右端口滑动夹。
    2. 使用胰蛋白酶-EDTA(0.25%),酚红,用MEM洗涤HCT-8单层胰蛋白酶,并在总体积为5ml的ICS混合物中重悬浮1×10 6 (参见方法2),并放入槽中。
    3. 用70%乙醇喷涂左侧端口Luer连接,并用酒精垫擦拭。用酒精垫取下注射器。
    4. 使用带有钝端针的10 ml注射器吸取细胞。
    5. 按照相同的无菌程序,将空注射器连接到右侧端口Luer配件上。
    6. 如果关闭,请打开侧面端口夹。
    7. 通过ECS在2个注射器之间来回轻轻冲洗细胞悬液3-5次。这确保了细胞的分布。
    8. 松开储液罐盖½转。
    9. 让左端口滑动夹关闭。打开右端口滑动夹。这将允许过量的介质流入储液瓶。
    10. 关闭右侧端口滑动夹。
    11. 轻轻按下连接在左侧端口的注射器柱塞,直到所有介质和细胞都被压入ECS。
    12. 关闭左侧端口滑动夹并打开右侧端口滑动夹。
    13. 轻轻按下连接在右侧端口的注射器柱塞,直到所有介质和细胞都被压入ECS。
    14. 关闭侧面滑动夹。用新的无菌注射器更换注射器。
    15. 拧紧油箱盖。
    16. 打开左端口滑动夹。
    17. 将药筒放入37°C,5%CO 2 培养箱内的Fibercell泵中,并将流速设置为5. 3天后,将流速增加到10。

  4. 添加 C. parvum 卵囊
    每天更换新鲜MEM和10%HS。使用Accumet ® pH计和Accutrend Plus ®血糖仪在ECS中监测48小时和新鲜培养基的pH和葡萄糖浓度。当葡萄糖在24小时内下降50%时,ECS接种1×10 5 C.用10%Chlorox ®处理过的卵囊卵囊,每次用30ml无菌蒸馏水洗涤7次,并悬浮于含有配方1(ECS混合物)中所示补充剂的10ml MEM中。毛细血管内空间(ICS)培养基被替换为配方2(ICS混合物)中所示的培养基。
    1. 将设备拆至无菌罩并将L和R端口夹滑动至关闭状态。
    2. 使用酒精垫用70%酒精喷洒左右ECS端口,然后从左侧ECS端口取下注射器。
    3. 连接含有10 ml C的注射器。 parvum 卵囊悬浮在ECS混合物中的左侧ECS端口。
    4. 将干净的无菌10 ml注射器连接到正确的ECS端口。
    5. 打开左侧和右侧ECS端口的滑动夹。向下推动连接到左侧端口的注射器(包含 C。parvum 卵囊),轻轻向上拉右侧注射器。
    6. 当左注射器完全按下时,向下按右注射器以混合ECS的内容物。
    7. 重复该过程(步骤5和6)3-4次,最后左右注射器大约50%满(每个5毫升)。
    8. 关闭右侧ECS端口夹,打开右端口滑动夹。向下推动连接到左ECS端口的注射器,直到内容物已添加到ECS中(注意不要向盒中添加空气)。将左ECS端口夹滑动关闭。打开右侧ECS滑动夹并向下按压注射器,直至添加到此端口的注射器内容物(注意不要向墨盒中添加空气)。滑动右侧ECS端口夹闭合。当最初接种药筒时,使用者应经历最小的背压,在连续培养几个月(> 6个月)之后,可能经历由于宿主细胞层的积聚和来自脱落细胞的碎片而经历的背压增加。在连续培养一年后使用3 ml ECS滤芯时,背压很明显,不建议用于超出此点的研究。 20毫升ECS滤芯保持2年以上没有遇到这个问题,是长期研究的首选滤芯。
    9. 滑动左端口夹以打开。用70%酒精喷洒左右ECS端口,并使用酒精垫取下注射器,并用干净无菌的10 ml注射器更换。
    10. 确保关闭左右端口夹具,并用125 ml完整的ICS培养基混合物替换储液瓶(配方2)。打开左右端口并将墨盒返回泵。将流速设为10.
    11. 通过直接从储液瓶中取出5ml,每天监测ICS培养基中的pH和葡萄糖。当葡萄糖浓度从360mg / dl下降到约。在24小时内200mg / dl,更换储液器以获得500ml瓶子的完整ICS培养基混合物,其含有460mg / dl葡萄糖。
    12. 继续监测pH和葡萄糖,当葡萄糖浓度在24小时内降至50%(200-230 mg / dl)时,更换储液罐以获得1 L瓶含有500 mg / dl葡萄糖的完整ICS培养基混合液。
    13. 在培养期间每48小时监测pH和葡萄糖,作为宿主细胞层完整性的指示。

  5. 分析 C。通过 Cp 18S-rRNA的变化实现细胞生长 RNA准备:
    1. 离心50μl样品,弃去上清液,加入100μliScriptBuffer(裂解缓冲液)。
    2. 通过在液氮中浸泡1分钟冻结/解冻6x,然后在70℃下快速解冻1.5分钟。
    3. 将裂解物在16,162 x g (14,800rpm)下离心5分钟。
    4. 小心地将上清液用200μl移液管(请勿触摸PELLET)转移到干净的试管中。
    5. 向裂解物中加入1体积的70%乙醇,并通过移液充分混合。不要离心。
    6. 将最多700μl的样品(包括任何沉淀物)转移到置于2 ml收集管中的RNeasy Mini离心柱中。
    7. 在8,000 x g (10,412rpm)下离心15秒。丢弃流量。
    8. 向色谱柱中添加350μlBufferRW1。在8,000 x g (10,412 rpm)下离心15秒。
    9. 将10μlDNaseI储备溶液加入70μl缓冲液RDD中。通过倒置管轻轻混合。在8,000 x g (10,412 rpm)下离心15秒。
    10. 将步骤E9中的DNase I孵育混合物(80μl)添加到柱膜中并放置在台面上15分钟
    11. 将350μl缓冲液RW1添加到色谱柱中。在8,000 x g (10,412rpm)下离心15秒。丢弃流量。
    12. 向色谱柱中加入500μl缓冲液RPE。在8,000 x g (10,412rpm)下离心15秒以洗涤膜。丢弃流量。
    13. 向色谱柱中加入500μl缓冲液RPE。在8,000 x g (10,412rpm)下离心2分钟。通过
      放弃流程
    14. 将色谱柱放入新的1.5 ml收集管中。将30-50μl不含RNase的水直接加入柱膜中。在8,000 x g (10,412 rpm)下离心1分钟以洗脱RNA。标记管,样品描述,日期和添加的水量。
    15. 立即定量RNA或将样品保存在-80°C。
    16. 根据制造商的说明,使用Qubit 3.0荧光计,使用广泛的Qubit RNA测定试剂盒对RNA进行定量。
    17. 枚举 C.通过在HFB样品中定量 Cp 18S-rRNA并将其与使用10 6 ,10 7 和10制备的标准进行比较,得到parvum 8 C. parvum 卵囊。使用含有 C的Luna Universal One-Step ® RT-qPCR试剂盒(NEB E3005),使用5 ng RNA进行该程序。 parvum 18S-rRNA引物:(Cp18S-995F:5'-TAGAGATTGGAGGTTCCT-3'和Cp18S-1206R:5'-CTCCACCAACAGAGAACGCC-3')并将其与人18S-rRNA引物进行比较:(Hs18S-F1373: 5'-CCGATAACGAACGAGACACTCTGG-3'和Hs18S-R1561:5'-TAGGGTAGGCACACGCTGAGCC-3')。使用Quant Studio 6 Flex 44按照制造商的说明执行此过程。
    18. 在培养2周后,HFB可重复地每毫升产生5×10 6个 7 至10 8 卵囊,如果不超过50%的ECS体积被采样,则保持这一点。每7-10天一次。一次取样90%的药筒体积导致4周的恢复时间,以获得每毫升10 8 卵囊。
    19. 根据制造商的说明用Crypt-o-Glo ®染色卵囊,并使用Nikon TM Optiphot荧光显微镜进行评估。
    20. 根据制造商的说明用Spor-a-Glo ®染色运动阶段,并使用Nikon TM Optiphot荧光显微镜进行评估。

有助于重现性的提示。
  1. 将入口和出口管线保持在用于灌注泵的管线后面的储液瓶,这可以防止管线扭结并减少处理滤芯时污染的可能性。
  2. 更换注射器时,请使用酒精垫缠绕配件,以防止污染。
  3. 如果48小时后储液瓶的葡萄糖浓度没有显着下降,则通过在毛细管外空间添加10个 6 HCT-8细胞来补充宿主细胞。
  4. 实现 C的持续增长。 parvum ,重要的是保持毛细管外培养基的低氧化还原平衡。
    注意:为实现这一目的,我们使用无菌蒸馏水制备氧化还原混合物,煮沸并用0.4μm过滤的N 2 吹扫加油站。当冷却至环境温度时,硫醇在N 2 气氛下溶解。
  5. 通过用等体积的新鲜ECS培养基置换额外毛细管培养基,可以除去50%(10ml)培养物。当移位细胞时,轻轻地压下注射器(10ml注射器10秒),以免引起湍流。建议在采样后10-14天不要扰乱培养物以使培养物重新繁殖。

数据分析

  1. 来自HFB的总寄生虫阶段的生长曲线
    1. 从HFB中取出样品(0.5ml)并分离RNA并使用针对 C的特异性引物通过qRT-PCR分析。 parvum 18S-rRNA(Cp18S-995F:5'-TAGAGATTGGAGGTTCCT-3'和Cp18S-1206R:5'-CTCCACCAACAGAGAACGCC-3')并与HCT-8 18S-rRNA(Hs18S-F1373:5'-)进行比较如Zhang和Zhu(2015)所述,CCGATAACGAACGAGACACTCTGG-3'和Hs18S-R1561:5'-TAGGGTAGGCACACGCTGAGCC-3')计数寄生虫数量。在第15天,寄生虫数量从第1天的5×10 3 / ml增加至5×10 5 /μl/ ml / ml(图2)。使用Dynabeads从HFB样品中纯化卵囊,我们从10 ml培养体积中获得了10个 9 卵囊。


      图2. C. HFB中的parvum 增长曲线

    2. ℃。 parvum 数字显示为每毫升总寄生虫阶段的对数。寄生虫数量通过qRT-PCR测定,使用如程序中所述的Cp-18SrRNA引物和使用10 3 ,10 4 和10 5 卵囊(Zhang和Zhu,2015)。在第1天用5×10 3 卵囊/ ml(10 5 )接种培养物。结果表示为一式三份计数±SEM。
    3. 用缀合荧光探针(Crypt-a-Glo ®)的 Cryptosporidium 卵囊壁蛋白的单克隆抗体染色卵囊,并使用具有激发波长的荧光显微镜检查410-485nm,发射波长515nm(图3A)。使用与红色荧光探针(Sporo-Glo )缀合的多克隆抗体对运动细胞外阶段进行染色,并使用激发波长为535-550nm且发射波长为580nm的荧光显微镜进行检查。 (图3B)。


      图3.从HFB收集的寄生虫的显微镜检查。 A.使用Crypt-a-Glo ®染色的HFB中的卵囊; B和C.使用Sporo-Glo ®染色的HFB的运动阶段。比例尺=5μm。

  2. 体内 HFB产生的感染性 C. parvum
    从HFB培养物收集的卵囊用于感染TCR-α缺陷型小鼠以证明毒力因子的维持。在2D培养瓶中培养48小时的寄生虫不能在免疫抑制或免疫缺陷小鼠模型中引起感染。从HFB收集卵囊并使用Dyna珠子根据制造商的说明纯化。在C57BL / 6背景(Jackson Laboratories,Bar Harbor,ME,USA)上的三组TCR-缺陷小鼠口服给药10 6 C.来自HFB的parvum 卵囊(在PBS中0.1ml),并与亲本Iowa分离物(阳性对照)的10 6 卵囊和给予0.1ml PBS(阴性对照)的小鼠进行比较。 C的体重增加存在统计学差异。与给予PBS的阴性对照相比,从10天开始感染小鼠(图4A),表明活性 C. parvum 感染。收集粪便颗粒并在PBS中软化,然后涂抹在显微镜载玻片上并通过改良的冷Kinyoun酸快速技术染色,并使用共聚焦显微镜(Nikon,Westbury,NY,USA)在400x下显微镜头计数。 7-10天后检测到卵囊,每天脱落最多25天(图4B)。


    图4. 体内 C的分析。来自HFB的parvum 卵囊。 A.小鼠模型 C. parvum 感染。通过含有10μl


    C的口服管饲法将0.1ml小鼠一式三份感染。 parvum 悬浮在PBS中的卵囊从HFB收集,六周大的 C. parvum 爱荷华州孤立(与用于启动文化相同的来源),并与用0.1毫升PBS强化的对照进行比较。每天称重小鼠。 B.通过HFB培养的小鼠模型感染的卵囊脱落。用0.1ml蒸馏水软化小鼠粪便并涂抹在载玻片上,用改良的冷Kinyoun酸快速技术染色,并用放大400倍的显微镜计数卵囊。

  3. 从HFB收集的卵囊的excystation
    C的exorpstation率。将来自HFB的parvum 卵囊与对照爱荷华分离物卵囊进行比较,作为卵囊活力的指标。 ℃。将parvum 卵囊在含有150毫克牛磺胆酸钠和50毫克胰蛋白酶的PBS(pH 7.4)中于37℃温育30分钟,并使用Crypt-a-Glo ®和Sporo进行双重染色-Glo ®。通过使用荧光显微镜(Nikon Optiphot)以400x放大率使用血细胞计数器计数卵囊和子孢子来确定切除的卵囊的百分比。使用410-485nm的激发波长和515nm的发射波长显现Crypt-a-Glo ®染色的卵囊;使用535-550nm的激发波长和580nm的发射波长显现Sporo-Glo ®染色的子孢子(spz)。结果显示约。从HFB培养系统收集的80%的卵囊在30分钟内完成,这与用 C获得的数据相似。 parvum 父母爱荷华州孤立。来自HFB培养物和爱荷华州分离物的卵囊在4℃下储存,表明随着储存时间的延长百分比逐渐降低,降至约。 6个月后40%(图5)。


    图5. C的Excystation。 parvum 卵囊卵囊在含有150毫克牛磺胆酸钠和50毫克胰蛋白酶的PBS(pH 7.4)中于37℃下切除30分钟。通过Crypt-a-Glo ®和Sporo-Glo ®双重染色并使用改进的Neubauer血细胞计数器计数来确定切除的卵囊的百分比。 excystation百分比由以下公式计算:spz count / 4 x oocyst count x 100%。

  4. HFB葡萄糖浓度的变化
    测量毛细血管内和毛细血管外空间的葡萄糖浓度作为宿主细胞生长的指标(图6)。当中等葡萄糖浓度降至其起始浓度的50%时,必须补充新培养基以防止宿主细胞的损失。毛细血管外葡萄糖浓度保持恒定在约275 mg / dl(图6,虚线)。


    图6.毛细血管内空间培养基的葡萄糖浓度每天监测向宿主细胞提供营养的培养基的葡萄糖浓度。在125ml烧瓶中起始浓度为350mg / dl,在第4天变为425mg / dl / 500ml烧瓶。当葡萄糖浓度在48小时内降至210-225mg / dl时(通常在第10天) )在1L烧瓶中葡萄糖增加至475-500mg / dl。葡萄糖浓度通常在48小时内降至50%,因此在此后的周一,周三,周五时间表补充培养基。包含寄生虫的额外毛细血管空间中的葡萄糖保持恒定在275 mg / dl。

食谱

  1. ECS中等混合

    添加组分并对介质进行过滤灭菌。
    *使用氮气脱气水制备,并在-20°C的氮气相下以1 ml等分试样储存。
    #库存保持在4°C
  2. ICS中等混合

    添加组分并将介质过滤灭菌。

致谢

我们要感谢Saul Tzipori博士和Sangun Lee博士(塔夫斯大学卡明斯兽医学院,美国马萨诸塞州N. Grafton)对体内动物研究的有益建议。博士的Louis Weiss和Leslie Gunther-Cummins(病理学和医学系,分析成像设施,Albert Einstein医学院,布朗克斯,纽约,美国)用于电子显微镜检查。这项工作由比尔和梅林达盖茨基金会(纽约)资助。该协议改编自Morada 等人(2016)。作者声明没有利益冲突或竞争利益。

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引用:Yarlett, N. and Morada, M. (2018). Long-term in vitro Culture of Cryptosporidium parvum. Bio-protocol 8(15): e2947. DOI: 10.21769/BioProtoc.2947.
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