Isolering af Myoepitelial celler fra voksne murine lacrimal og submandibulære kirtler
Summary
Den tåre kirtel (LG) har to celletyper, der udtrykker α-glat muskel aktin (αsma): myoepithelial celler (mecs) og pericytter. Mecs er af ektodermal oprindelse, findes i mange kirtel væv, mens pericytter er vaskulære glatte muskelceller af endodermale oprindelse. Denne protokol isolerer Mec’er og pericytter fra murine LGs.
Abstract
Den tåre kirtel (LG) er en eksokrin tubuloacinar kirtel, der udskiller et vandigt lag af tåre film. LG epitelial træet består af acinar, duktalt epitelial og myoepitel celler (mecs). Mecs Express Alpha glatte muskel aktin (αsma) og har en kontraktile funktion. De findes i flere kirtel organer og er af ektodermal oprindelse. Desuden, LG indeholder SMA + vaskulære glatte muskelceller af endodermale oprindelse kaldet pericytter: kontraktile celler, der omsluttes overfladen af vaskulære rør. En ny protokol giver os mulighed for at isolere både MECs og pericytter fra voksne murine LGs og submandibulære kirtler (SMGs). Protokollen er baseret på den genetiske mærkning af MECs og pericytter ved hjælp af SMACreErt2/+: Rosa26-tdtomatofl/fl musestamme, efterfulgt af forberedelsen af LG enkelt cellesuspension til fluorescens aktiveret celle sortering (FACS ). Protokollen giver mulighed for adskillelse af disse to cellepopulationer af forskellig oprindelse baseret på ekspression af EpCAM-cellens adhæsions molekyle, mens pericytterne ikke udtrykker EpCAM. Isolerede celler kan anvendes til celle dyrkning eller analyse af genekspression.
Introduction
Myoepithelial celler (MECs) er til stede i mange eksokrine kirtler herunder lacrimal, spyt, harderian, sved, prostata, og mammary. MECs er en unik celletype, der kombinerer en epitelial og en glat muskel fænotype. Mecs Express α-glatte muskel actin (SMA) og har en kontraktile funktion1,2. Ud over mecs, den tåre kirtel (LG) og submandibulær kirtel (SMG) indeholder SMA + vaskulære celler kaldet pericytter, som er celler af endodermale oprindelse, der indhyller overfladen af vaskulære rør3. Selv om mecs og pericytter udtrykke mange markører, SMA er den eneste markør, der ikke udtrykkes i andre LG og SMG celler1,3.
Inden for de sidste 40 år rapporterede flere laboratorier analyser for afkobling af forskellige eksokrine kirtel væv, hvor ikke-enzymatiske og enzymatiske tilgange blev anvendt. I en af de første rapporter, der blev offentliggjort i 1980, beskrev Fritz og medforfattere en protokol til isolering af felin parotideale acini ved hjælp af sekventiel fordøjelse i en kollagenase/trypsin opløsning4. I 1989 justerede Hann og coforfattere denne protokol for acini isolation fra rotte LGs ved hjælp af en blanding af collagenase, hyaluronidase og DNase5. I 1990 offentliggjorde Cripps og kolleger metoden med ikke-enzymatisk afkobling af tåre kirtel acini6. Senere, i 1998, vendte Zoukhri og medforfattere tilbage til en enzymatisk dissociations protokol for at følge op på ca2 +-Imaging på LG og SMG isoleret acini7. Inden for det sidste årti, har forskerne vendt deres fokus på isolering af stamceller/progenitorcellerne fra eksokrine kirtler. Pringle og medforfattere beskrev en protokol i 2011 til isolering af SMG-muse stamceller8. Denne metode var baseret på isolering af stamcelle-holdige salisfærer, som blev opretholdt i kulturen. Forfatterne hævdede, at prolifererende celler, der udtrykte stamcelle-associerede markører kunne isoleres fra disse salikugler8. Shatos og medforfattere offentliggjorde protokollen for progenitorcelle isolation fra uskadede voksne rotte LGs ved hjælp af enzymatisk fordøjelse og indsamling af “befriet” celler9. Senere, i 2015, justerede Ackermann og coforfattere denne procedure for at isolere formodede “murine tåre kirtelceller” (“mlgscs”), som kunne blive formeret som en mono-lags kultur over flere passager10. Ingen af de før nævnte procedurer tillod imidlertid at skelne mellem cellulære undertyper og individuelle populationer af isolerede epitelceller. I 2016 offentliggjorde Gromova og medforfattere en procedure for isolering af LG Stam-/progenitorceller fra voksne murine LGs ved hjælp af FACS11. Denne protokol havde imidlertid ikke til formål at isolere MECs.
For nylig har vi vist, at vi er i stand til at isolere SMA + celler fra 3 uger gamle SMA-GFP mus12. Men på dette tidspunkt har vi ikke adskilt forskellige populationer af SMA + celler. Her etablerede vi en ny procedure for direkte isolation af differentierede Mec’er og pericytter fra voksne LGs og SMGs.
Protocol
Representative Results
Discussion
Dette manuskript beskrev en protokol af MEC og pericyt isolation fra LG og SMG. Denne procedure var baseret på genetiske mærkning af SMA, den eneste pålidelige biomarkør af MECs og pericytter.
Det haster med at udvikle denne protokol var motiveret af den næsten totale fravær af litteratur, der fremhæver isoleringen af MECs fra murine LGs og SMGs. Selv om genetiske mærkning tidligere blev brugt, ved hjælp af SMA-GFP mus til at isolere SMA + celler fra unge tre-ugers gamle LGs<sup class…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Vi takker Dr. Ivo kalajzic for at give os med SMACreErt2 mus stamme, Takeshi umazume for mus hale og genotyping, Mark Shelley for at erhverve professionelle billeder til figur 2. Vi takker også Scripps Rådet for videnskabelige redaktører og Mark Shelley for videnskabelig engelsk redigering. Vi er taknemmelige for den Scripps Research Institute flow cytometry kerne for hjælp med celle sortering og til Dr. Robin Willenbring for flere diskussioner/rådgivning om FACS dataanalyse.
Dette arbejde blev støttet af nationale institutter for sundhed, National Eye Institute tilskud 5 R01 EY026202 og 1 R01 EY028983 til H.P.M.
Materials
| Biosafety Cabinet | SterilCard Baker | 19669.1 | Class II type A/B3 |
| 10 ml Disposable serological pipets | VWR | 89130-910 | Manufactured from polystyrene and are supplied sterile and plugged |
| 10 mL Disposable serological pipets | VWR | 89130-908 | Manufactured from polystyrene and are supplied sterile and plugged |
| 15 mL High-clarity polypropylene conical tubes | Falcon | 352196 | |
| 25 mL Disposable serological pipets | VWR | 89130-900 | Manufactured from polystyrene and are supplied sterile and plugged |
| 5 mL FACS round-bottom tubes | Fisher Scientific, Falcon | 14-959-11A | |
| 50 mL High-clarity polypropylene conical tubes | Falcon | 352070 | |
| Antibiotic-antimycotic | Invitrogen | 15240-062 | |
| Appropriate filter and non-filter tips | Any available | Any available | |
| BD Insulin Syringes | Becton Dickinson | 328468 | with BD Ultra-Fine needle ½ mL 8 mm 31G |
| BD Syringes 10 mL | Becton Dickinson | 309604 | Sterile |
| Brilliant Violet 421 anti mouse CD326 (EpCAM) | Biolegend | 118225 | Monoclonal Antibody (G8.8) |
| CaCl2 1M solution | BioVision | B1010 | sterile |
| Cell culture dishes 35 mm | Corning | 430165 | Non-pyrogenic, sterile |
| Collagenase Type I | Wortington | LS004194 | |
| Corn oil | Any avaliable | Any avaliable | From grocery store |
| Corning cell strainer size 70 μm | Sigma-Aldrich | CLS431751-50EA | |
| Digital Stirrer PC-410D | Corning | Item# UX-84302-50 | |
| Dispase II | Sigma-Aldrich | D4693-1G | |
| Dissecting scissors, curved blunt | McKesson Argent | 487350 | Metzenbaum 5-1/2 Inch surgical grade stainless steel non-sterile finger ring handle |
| DNase I | Akron Biotech, catalog number | AK37778-0050 | |
| Dulbecco’s Modified Eagle’s Medium – low glucose (DMEM) | Sigma-Aldrich | D5546-500ML | with 1000mg/L glucose and sodium bicarbonate, without L-glutamine |
| Dulbecco’s Modified Eagle’s Medium/F12 (DMEM/F12) | Millipore | DF-042-B | without HEPES, L-glutamine |
| Easypet 3 pipette controller | Eppendorf | 4430000018 | with 2 membrane filters 0.45 µm, 0.1 – 100 mL |
| Ethanol | Sigma-Aldrich | E7023-500ML | |
| Ethylenediaminetetraacetic acid (EDTA) | Sigma-Aldrich | E6758 | |
| Fisher Vortex Genie 2 | Fisher Scientific | 12-812 | |
| FlowJo version 10 | Any available | Any available | |
| Fluorescence binocular microscope Axioplan2 | Carl Zeiss | ID# 094207 | |
| Ghost Red 780 Viability Dye | Tonbo Biosciences | 13-0865-T100 | |
| GlutaMAX Supplement | ThermoFisher Scientific, Gibco | 35050061 | |
| Glycerol 99% | Sigma-Aldrich | G-5516 | |
| Hand tally counter | Heathrow Scientific | HEA6594 | |
| Hank's Balanced Salt Solution (HBSS) | Sigma Millipore | H6648-500ML | Modified, with sodium bicarbonate, without calcium chloride, magnesium sulphate, phenol red. |
| Hank's Balanced Salt Solution (HBSS) | ThermoFisher Scientific | 14025092 | With calcium, magnesium, no phenol red. |
| Hausser Bright-Line Phase Hemocytometer | Fisher Scientific | 02-671-51B | 02-671-51B |
| HEPES 1M solution | ThermoFisher Scientific, Gibco | 15630-080 | Dilute 1/10 in ddH20 |
| HyClone Fetal Bovine Serum (FBS) | Fisher Scientific | SH3007002E | |
| Hydrochloric Acid (HCl), 5N Volumetric Solution | JT Baker | 5618-03 | To adjust Tris buffer pH |
| Innova 4230 Refrigerated Benchtop Incubator | New Brunswick Scientific | SKU#: | Shaker; 37 °C, 5% CO2 in air |
| Iris scissors | Aurora Surgical | AS12-021 | Pointed tips, delicate, curved, 9 cm, ring handle |
| Isoflurane Inhalation Anesthetic | Southern Anesthesia Surgical (SAS) | PIR001325-EA | |
| MgCl2 1M solution | Sigma-Aldrich | 63069-100ML | |
| Microcentrifuge tubes 1.5 mL | ThermoFisher Scientific | 3451 | Clear, graduated, sterile |
| Microsoft Power Point | Any available | Any available | |
| NaCl powder | Sigma-Aldrich | S-3014 | |
| Nalgene 25 mm Syringe Filters | Fisher Scientific | 724-2020 | |
| Pen Strep | Gibco | 15140-122 | |
| pH 510 series Benchtop Meter | Oakton | SKU: BZA630092 | |
| Phosphate buffered saline (PBS) | ThermoFisher Scientific | 10010023 | pH 7.4 |
| Pure Ethanol 200 Proof | Pharmco-Aaper | 111000200 | |
| Red blood cell lysis buffer 10x | BioVision | 5831-100 | |
| Roto-torque Heavy Duty Rotator | Cole Parmer | MPN: 7637-01 | |
| Safe-lock round bottom Eppendorf tubes 2 mL | Eppendorf Biopur | 22600044 | PCR inhibitor, pyrogen and RNAse-free |
| Scissors | Office Depot | 375667 | |
| Sorting flow cytometer MoFlo Astrios EQ | Beckman Coulter | B25982 | With Summit 6.3 software |
| Sorvall Legend Micro 17R Microcentrifuge | Thermo Scientific | 75002441 | All centrifugation performed at RT |
| Sorvall RT7 Plus Benchtop Refrigerated Centrifuge | Thermo Scientific | ID# 21550 | RTH-750 Rotor. All centrifugation performed at RT |
| Stemi SV6 stereo dissecting microscope | Carl Zeiss | 455054SV6 | With transmitted light base |
| Tamoxifen | Millipore Sigma | T5648-1G | |
| Trizma base powder | Sigma-Aldrich | T1503 | |
| Trypan blue solution | Millipore Sigma | T8154 | |
| Two Dumont tweezers #5 | World Precision Instruments | 500342 | 11 cm, Straight, 0.1 x 0.06 mm tips |
| Upright microscope | Any available | Any available | With transmitted light base |
| Vacuum filtration systems, standard line | VWR | 10040-436 | |
| Variable volume micropipettes | Any available | Any available |
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