Name: obtaining primary osteocytes through murine calvarial fractionation of gfp-expressing osteocytes
Uploaded: 2020-06-02T15:00:00
Description: Watch this Scientific Journal Video about Obtaining Primary Osteocytes Through Murine Calvarial Fractionation of GFP-Expressing Osteocytes at JoVE.com

Dette arbejde blev delvist støttet af et JSPS KAKENHI-tilskud fra Japan Society for the Promotion of Science (nr. 19K10397 til H.K. og nr. 18K09862 til I.M.).

Alle dyreprocedurer og dyrepleje blev udført i overensstemmelse med Tohoku University regler og forskrifter.
1. Dissektion af nyfødt dmp1-topas mus calvaria
<ol><li>Til denne protokol skal du bruge 6-127 dage gamle hvalpe af C57BL/6-Tg(Dmp1-Topaz)1Ikal/J-mus. Afliv mus med 5% isofluran inhalation og overfør derefter hvalpene til 70% ethanol.</li>
	<li>Overfør den aflivede hvalp til en ikke-behandlet kulturskål.</li>
	<li>Brug saks og pincet til at tage fat i huden i bunden af kraniet og...

<ol>
	<li>Manolagas, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Birth+and+death+of+bone+cells:+basic+regulatory+mechanisms+and+implications+for+the+pathogenesis+and+treatment+of+osteoporosis.">Birth and death of bone cells: basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis.</a> Endocrine Reviews. 21 (2), 115-137 (2000).</li><li>Bonewald, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteocytes+as+Dynamic+Multifunctional+Cells.">Osteocytes as Dynamic Multifunctional Cells.</a> Annals of the New York Academy of Sciences. 1116 (1), 281-290 (2007).</li><li>Bonewald, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanosensation+and+Transduction+in+Osteocytes.">Mechanosensation and Transduction in Osteocytes.</a> BoneKEy osteovision. 3 (10), 7-15 (2006).</li><li>Feng, J. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+DMP1+causes+rickets+and+osteomalacia+and+identifies+a+role+for+osteocytes+in+mineral+metabolism.">Loss of DMP1 causes rickets and osteomalacia and identifies a role for osteocytes in mineral metabolism.</a> Nature Genetics. 38 (11), 1310-1315 (2006).</li><li>Lane, N. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucocorticoid-Treated+Mice+Have+Localized+Changes+in+Trabecular+Bone+Material+Properties+and+Osteocyte+Lacunar+Size+That+Are+Not+Observed+in+Placebo-Treated+or+Estrogen-Deficient+Mice.">Glucocorticoid-Treated Mice Have Localized Changes in Trabecular Bone Material Properties and Osteocyte Lacunar Size That Are Not Observed in Placebo-Treated or Estrogen-Deficient Mice.</a> Journal of Bone and Mineral Research. 21 (3), 466-476 (2005).</li><li>Dallas, S. L., Prideaux, M., Bonewald, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Osteocyte:+An+Endocrine+Cell+...+and+More.">The Osteocyte: An Endocrine Cell ... and More.</a> Endocrine Reviews. 34 (5), 658-690 (2013).</li><li>van der Plas, A., Nijweide, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+purification+of+osteocytes.">Isolation and purification of osteocytes.</a> Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 7 (4), 389-396 (1992).</li><li>Westbroek, I., De Rooij, K. E., Nijweide, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteocyte-specific+monoclonal+antibody+MAb+OB7.3+is+directed+against+Phex+protein.">Osteocyte-specific monoclonal antibody MAb OB7.3 is directed against Phex protein.</a> Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 17 (5), 845-853 (2002).</li><li>Gu, G., Nars, M., Hentunen, T. A., Metsikk&#246;, K., V&#228;&#228;n&#228;nen, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+primary+osteocytes+express+functional+gap+junctions+in+vitro.">Isolated primary osteocytes express functional gap junctions in vitro.</a> Cell and Tissue Research. 323 (2), 263-271 (2006).</li><li>Kramer, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteocyte+Wnt/&#946;-Catenin+Signaling+Is+Required+for+Normal+Bone+Homeostasis.">Osteocyte Wnt/&#946;-Catenin Signaling Is Required for Normal Bone Homeostasis.</a> Molecular and Cellular Biology. 30 (12), 3071-3085 (2010).</li><li>Kato, Y., Windle, J. J., Koop, B. A., Mundy, G. R., Bonewald, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+an+Osteocyte-like+Cell+Line,+MLO-Y4.">Establishment of an Osteocyte-like Cell Line, MLO-Y4.</a> Journal of Bone and Mineral Research. 12 (12), 2014-2023 (1997).</li><li>Kato, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+an+osteoid+preosteocyte-like+cell+MLO-A5+that+spontaneously+mineralizes+in+culture.">Establishment of an osteoid preosteocyte-like cell MLO-A5 that spontaneously mineralizes in culture.</a> Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 16 (9), 1622-1633 (2001).</li><li>Zhang, C., Bakker, A. D., Klein-Nulend, J., Bravenboer, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+Osteocytes+in+Their+3D+Native+Matrix+Versus+2D+In+Vitro+Models.">Studies on Osteocytes in Their 3D Native Matrix Versus 2D In Vitro Models.</a> Current Osteoporosis Reports. 17 (4), 207-216 (2019).</li><li>Woo, S. M., Rosser, J., Dusevich, V., Kalajzic, I., Bonewald, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+line+IDG-SW3+replicates+osteoblast-to-late-osteocyte+differentiation+in+vitro+and+accelerates+bone+formation+in+vivo.">Cell line IDG-SW3 replicates osteoblast-to-late-osteocyte differentiation in vitro and accelerates bone formation in vivo.</a> Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 26 (11), 2634-2646 (2011).</li><li>Spatz, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Wnt+Inhibitor+Sclerostin+Is+Up-regulated+by+Mechanical+Unloading+in+Osteocytes+in+Vitro.">The Wnt Inhibitor Sclerostin Is Up-regulated by Mechanical Unloading in Osteocytes in Vitro.</a> The Journal of biological chemistry. 290 (27), 16744-16758 (2015).</li><li>Gillet, J. P., Varma, S., Gottesman, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+clinical+relevance+of+cancer+cell+lines.">The clinical relevance of cancer cell lines.</a> Journal of the National Cancer Institute. 105 (7), 452-458 (2013).</li><li>Sandberg, R., Ernberg, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+tumor+characteristic+gene+expression+in+cell+lines+using+a+tissue+similarity+index+(TSI).">Assessment of tumor characteristic gene expression in cell lines using a tissue similarity index (TSI).</a> Proceedings of the National Academy of Sciences of the United States of America. 102 (6), 2052-2057 (2005).</li><li>Kalajzic, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dentin+matrix+protein+1+expression+during+osteoblastic+differentiation,+generation+of+an+osteocyte+GFP-transgene.">Dentin matrix protein 1 expression during osteoblastic differentiation, generation of an osteocyte GFP-transgene.</a> Bone. 35 (1), 74-82 (2004).</li><li>Paic, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+differentially+expressed+genes+between+osteoblasts+and+osteocytes.">Identification of differentially expressed genes between osteoblasts and osteocytes.</a> Bone. 45 (4), 682-692 (2009).</li><li>Nakashima, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+osteocyte+regulation+of+bone+homeostasis+through+RANKL+expression.">Evidence for osteocyte regulation of bone homeostasis through RANKL expression.</a> Nature Medicine. 17 (10), 1231-1234 (2011).</li><li>Kalajzic, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+approaches+to+study+osteocyte+biology.">In vitro and in vivo approaches to study osteocyte biology.</a> Bone. 54 (2), 296-306 (2013).</li><li>Chia, L. Y., Walsh, N. C., Martin, T. J., Sims, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+gene+expression+of+haematopoietic-cell-free+preparations+of+highly+purified+murine+osteocytes.">Isolation and gene expression of haematopoietic-cell-free preparations of highly purified murine osteocytes.</a> Bone. 72, 34-42 (2015).</li><li>Halleux, C., Kramer, I., Allard, C., Kneissel, M., Helfrich, M. H., Ralston, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bone Research Protocols. , 55-66 (2012).</li><li>Bernhardt, A., Wolf, S., Weiser, E., Vater, C., Gelinsky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+to+isolate+primary+human+osteocytes+from+bone.">An improved method to isolate primary human osteocytes from bone.</a> Biomedizinische Technik (Berlin). 65 (1), 107-111 (2020).</li></ol>

Den første isolerede osteocyt var fra en kyllingecalvaria7 isoleret ved anvendelse af (OB7.3) eller den aviante variant af PHEX; Denne metode er imidlertid begrænset af tilgængeligheden af brugbare antistoffer, da osteocytspecifikke antistoffer, der også er speciespecifikke, skal fremstilles. Forskere brugte en anden modifikation af den sekventielle enzymatiske proces for at opnå osteocytter fra mus og rotte lange knogler; Den rapporterede renhed af disse kulturer blev fastsat til ca. 70%<sup...

Osteocytter er terminalt differentierede celler fra osteoblastiske forfædre, der blev indlejret i deres udskillede matrix1. De er de mest rigelige og længstlevende celler blandt knoglecellepopulationer. De bor inden for lakuner og har en karakteristisk stellatmorfologi med dendritter, der strækker sig gennem kanaler kaldet canaliculi, der danner et omfattende netværk af kommunikation og metabolisk udveksling med deres omgivende miljø og knogleoverfladen2. Osteocytter koreograferer både osteoblaster og osteoklaster roller i knoglemodellering, de er de primære mekanosensoriske celler, der giver tilpasning til mekanisk belastning3, er involveret i fosfathomeostase4 og knoglematrixmineralisering5, og sammen med lacunocanalicular systemet fungerer de som et endokrin organ, der signalerer fjernt væv6.
Osteocytter er placeret i en mineraliseret matrix, hvilket begrænser tilgængeligheden og gør deres isolering udfordrende, hvilket forhindrer in vitro-undersøgelse. En af de første isolationsmetoder beskrev isolerede osteocytter fra kyllingecalvaria ved anvendelse af et osteocytspecifikt monoklonalt antistof (OB7.3)7, som senere blev kendt for at være den aviære variant af PHEX (PHosphate-regulerende gen med homologi til endopeptidaser på X-kromosomet)8. Andre forskere brugte sekventiel fordøjelse af rotte 9 eller mus 10 lange knogler for at opnå osteocytrige fraktioner, hvor osteocytrenhed blev rapporteret at være omkring70%9. Begrænsninger af denne procedure omfatter den suboptimale renhed af kulturer, der er forurenet med andre celletyper udover osteocytter, og at osteocytter potentielt kan overgros af andre celler i kultur, da osteocytter har mistet evnen til at dele sig. Disse udfordringer begrænser anvendeligheden af langsigtede kulturer.
For at overvinde disse begrænsninger blev forskellige osteocytcellelinjer udviklet. MLO-Y4-cellelinjen11 og MLO-A5-cellelinjen12 er især de mest undersøgte cellelinjer, som er nyttige til undersøgelse af osteocytter i det tidlige stadium; De er dog mindre nyttige til at studere moden osteocytsignalering, da de udtrykker lave niveauer af sclerostin og FGF2313, som begge er modne osteocytmarkører. Andre cellelinjer, herunder IDG-SW3 14 og Ocy45415, udtrykker høje niveauer af sclerostin og FGF23 og er nyttige til at studere det sene osteocytstadium. Cellelinjer viser sig at være nyttige forskningsværktøjer; Ikke desto mindre kommer de ikke uden begrænsninger, da de ikke fuldt ud repræsenterer biologien i den primære celle. Forskellige cellelinjer repræsenterer forskellige udviklingsstadier af osteocytmodenhedsspektret, og cellelinjer repræsenterer ikke heterogeniteten af primære osteocytter16,17.
For at opnå rene kulturer af primære osteocytter udnyttede forskere cre-musemodellen, hvor 8-kb dmp1-promotoren bruges til at drive grønt fluorescerende protein (GFP) ekspression i osteocytter18,19. Dual transgene mus (pOB-Col 2.3- GFP-cyan og DMP1-GFP-topas) af Paic et al.19 og dmp1-topas transgene mus af Nakashima et al.20 er blevet brugt til at opnå osteocytpopulationer. I hvilke de anvendte sekventiel fordøjelse og FACS af osteocytter, der udtrykker GFP for at erhverve kulturer af primære osteocytter19,20. Retningen af cre i 10-kb dmp1 reportermusen Ai9, som aktiverer tdTomato-proteinet, viste sig at være til stede i osteocytter, osteoblaster, muskler og celler i knoglemarven. 8-kb dmp1-promotoren havde det samme ekspressionsmønster, men kun en andel af osteoblaster og knoglemarvsceller udtrykte proteinet, hvilket indikerer, at 8-kb dmp1-promotoren er mere specifik21. På trods af dette bør resultater opnået ved anvendelse af 8-kb dmp1-promotoren fortolkes med forsigtighed, og genekspressionsprofiler bør rutinemæssigt udføres ved hjælp af osteocyt vs. osteoblastspecifikke markører for at sikre, at den opnåede population er af høj nok renhed.
Osteoklastmarkører OSCAR og Dcstamp blev fundet i hæmatopoietiske ikke-depleterede vs. udtømte osteocytpopulationer, dette fund førte forfatterne til at konkludere, at fordøjelser opnået ved fraktionering af 8-kb dmp1-topas neonatal calvaria og GFP-sortering er forurenet med hæmatopoietiske celler. Forurening med hæmatopoietiske celler kunne have været afbødet ved at stramme GFP-sorteringsporten, da GFP-positive hæmatopoietiske celler havde en rimelig lavere GFP-intensitet end GFP-positive mesenkymale celler (osteocytter)22.
Metoderne til undersøgelse af osteocytter in vitro har bidraget til den seneste rigdom af information om osteocytbiologi. Imidlertid forbliver osteocytisolering en arbejdskrævende og langvarig procedure med lave celleudbytter. Den beskrevne metode til knoglefordøjelse ved anvendelse af collagenase og EDTA, ofte op til fraktion 8,23, tager flere timer, hvor osteocyt levedygtighed beskattes. Forskere har rapporteret at bruge fraktioner (2-20125) til cellesortering 20 og har vist, at ekspressionsprofilen for gener forbundet med osteocytter versus osteoblaster bekræfter succesen med at isolere rene osteocytpopulationer20. I denne artikel beskriver vi processen med at opnå fraktioner (2-20125), og vi sammenligner udbyttet af osteocytter fra hver fraktion startende med fraktion 1 til 8 for at bestemme afkastet af osteocytter i hver fraktion. Vi beskriver også dissektion af nyfødt dmp1-topas mus calvaria og calvarial fordøjelse ved hjælp af collagenase og ethylendiamintetraeddikesyre (EDTA) samt forberedelse af celler til FACS.

<table>
	<tbody>
		<tr>
			<td>BD FACSDiva software</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Data aquisition and analysis</td>
		</tr>
		<tr>
			<td>BD Falcon Tube</td>
			<td>BD Biosciences</td>
			<td>352235</td>
			<td>12 x 75 mm Tube with Cell Strainer Cap, 35 &#956;m nylon mesh.</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich, MO, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Wako, Osaka, Japan</td>
			<td>034-22363</td>
			<td>0.2% (w/v), crude collagenase mix sourced from C. histolyticum.</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Dojindo, Kumamoto, Japan</td>
			<td></td>
			<td>5mM EDTA prepared with 0.1% BSA</td>
		</tr>
		<tr>
			<td>FACSAriaTM II</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Biowest, Nuaill&#233;, France</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isolation buffer</td>
			<td></td>
			<td></td>
			<td>70mM NaCl, 10mM NaHCO, 60mM sorbitol, 3mM K2HPO4, 1mM CaCl2, 0.1% (w/v) BSA, 0.5% (w/v) glucose and 25 mM HEPES</td>
		</tr>
		<tr>
			<td>Millex Sterile Filter Unit</td>
			<td>Merck Millipore, Ireland</td>
			<td>SLGV033RS</td>
			<td>0.22&#956;m</td>
		</tr>
		<tr>
			<td>Nylon cell strainer</td>
			<td>FALCON, NY, USA</td>
			<td></td>
			<td>40&#956;m</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Life Technologies, NY, USA</td>
			<td></td>
			<td>0.5% x10. diluted to x1 in PBS</td>
		</tr>
		<tr>
			<td>&#945;-MEM</td>
			<td>Wako, Osaka, Japan</td>
			<td></td>
			<td>Containing 10% fetal bovine serum, 100 IU/ml penicillin G, and 100 &#956;g/ml streptomycin</td>
		</tr>
	</tbody>
</table>

obtaining primary osteocytes through murine calvarial fractionation of gfp-expressing osteocytes

Osteocytten, der engang blev anset for at være en passiv beboer i knoglen givet backstage-funktionen til at registrere mekanisk belastning, bringes nu i rampelyset og har vist sig at have flere hovedfunktioner som aktivt at ændre den ekstracellulære matrix og danne et endokrin organ med det lacunocanalicular system, der omslutter det og sender beskeder til fjerne steder. På grund af de metoder, der gjorde det muligt at teste osteocytten in vitro fra isolering af primære osteocytter til osteocytlignende cellelinjer, oplever osteocytter nu en rungende interesse og en bølge af viden om struktur og funktion. Mange aspekter af osteocytbiologien og interaktion med andre molekylære komponenter er endnu ikke opdaget. I denne protokol beskriver vi detaljeret den effektive isolering af primære osteocytter fra dmp1-topas neonatal musecalvaria, som udtrykker det grønne fluorescerende protein i osteocytter gennem cellefraktionering og efterfølgende erhvervelse af kulturer af primære osteocytter af FACS.

Formålet med denne protokol er at demonstrere processen med at opnå kulturer af primære osteocytter fra dmp1-topas neonatal musecalvaria gennem en fraktioneringsproces under anvendelse af collagenase til nedbrydning af kollagenmatrixen og EDTA til calciumchelation, hvorefter celler fremstilles til FACS for at adskille osteocytter fra andre cellepopulationer.
Metoder til opnåelse af primære osteocytter fra neonatal musecalvaria beskriver ofte brugen af fraktioner (1\u20128) til sortering<s...

Watch this Scientific Journal Video about Obtaining Primary Osteocytes Through Murine Calvarial Fractionation of GFP-Expressing Osteocytes at JoVE.com

Obtaining Primary Osteocytes Through Murine Calvarial Fractionation of GFP-Expressing Osteocytes

Denne protokol beskriver dissektion af neonatal dmp1-topas musecalvaria og isolering af osteocytter, der udtrykker det grønne fluorescerende protein gennem cellefordøjelse og fraktionering, ud over osteocytpræparation til fluorescensaktiveret cellesortering (FACS).

opnåelse af primære osteocytter gennem murin calvarial fraktionering af GFP-ekspressive osteocytter

The osteocyte, once thought to be a passive resident of the bone given the backstage function of sensing mechanical loading, is now brought to the ...

Primary osteocyte isolation is a lengthy procedure that often leaves out in low cell areas. This protocol provides efficient methods for isolating high numbers of primary GFP-expressing osteocytes. This protocol reduces the handling time and allows a higher number of osteocytes to be obtained in a single procedure with minimal contamination from hematopoietic or mesenchymal cells.For the isolation of GFP-expressing osteocytes, place euthanized, postnatal day six to seven, transgenic mouse pups into a non-treated culture dish, and use scissors and tweezers to grasp the skin at the base of one's skull. Use the scissors to make an initial incision in the lifted skin, followed by cuts along both sides of the skull in front of the ears. Remove the skin to expose the calvaria and holding the head from the nasal bridge, cut the calvaria along the lamboid suture and along the edges of the parietal bones to the frontal part of the tissue.Separate the calvaria from the underlying brain tissue and place the bones concave side up in PBS. To obtain fraction one, pool up to five calvaria per 50-milliliter tube in five milliliters of freshly prepared two-milligram per milliliter collagenase, and incubate the tissues at 37 degrees Celsius and 300 revolutions per minute for 20 minutes. At the end of the incubation, discard the digest from fraction one and wash the calvaria with five milliliters of PBS.Replace the wash with five milliliters of five-millimolar EDTA supplemented with one milligram per milliliter of bovine serum albumin in PBS for 15 minutes at 37 degrees Celsius with shaking. At the end of the incubation, transfer the digest to a 50-milliliter conical tube and wash the calveria with five milliliters of PBS. Then transfer the wash solution to the digest.To obtain fraction two, place the digest for centrifugation and discard the supernatant. Re-suspend the pellet in eight milliliters of alpha minimum essential medium containing 10%fetal bovine serum, or FBS, and antibiotics, and seed the cells onto a 10-centimeter culture dish. Then place the fraction two cells into the cell culture incubator.After collecting and culturing fractions three and four as just demonstrated. Incubate the calveria in five milliliters of five-millimolar EDTA supplemented with one milligram per milliliter of bovine serum albumin in PBS for 15 minutes at 37 degrees Celsius with shaking. At the end of the incubation, collect and culture fraction five as demonstrated for fractions two, three, and four.Immediately after fractionation, collect the cells for sorting. Alternatively, collect the cells after 24 hours of culture by aspirating the medium and gently wash the cells two times with 10 milliliters of PBS per dish per wash. After the second wash, treat the cells with five milliliters of 0.5%tripsin EDTA in PBS per plate.After five minutes at 37 degrees Celsius, add five milliliters of complete alpha minimum essential medium to each plate and gently detach the cells with pipetting. To combine the cells in each fraction, filter the cell suspensions through a 40-micron cell strainer into a single 50-milliliter conical centrifuge tube. After washing the cells with 10 milliliters of PBS, add them to the tube.Filter the cells through a strainer and collect the cells by centrifugation. Once the cells are pelleted down, re-suspend the pellet in complete minimal essential medium to a one times 10 to the seventh cells per milliliter of medium concentration and filter the cells through a 35-micron nylon mesh capped tube. To test the efficiency of this method, fractions were separately sorted and cultured for 24 hours to determine the percentage and yield of osteocytes from each fraction.The density of the osteocytes in fractions two to five were higher than that observed for fraction one and decreased remarkably in fractions six, seven, and eight. Although the percentage of osteocytes obtained between all of the fractions is not statistically significant, the density of the osteocytes differs dramatically between fractions. Here the gating strategy for isolating GFP-positive osteocytes from their GFP-negative counterparts by fluorescence-activated cell sorting is shown.After sorting, osteocytes can be analyzed for the expression of osteocyte markers of interest. In this analysis, the osteocyte marker genes were expressed at higher levels in the isolated GFP-positive osteocytes compared to presort fraction two, which is known as a high osteoblast fraction. Here the morphology of a GFP-positive osteocyte retaining a stellate shape with dendrites extending from the cell body after 24 hours of culture on a plastic culture dish, post sort, can be observed.Pay attention when aspirating the digests as the calveria will be reduced to minute pellets. Remember to use a small tip aspirator to avoid cell loss. Osteocytes have been enigmatic because they are entombed in bone.This technique has paved the way to studying osteocyte molecular interactions and their control over osteoclasts and osteoblasts.

Research

JoVE Journal

Biology

Obtaining Eggs from Xenopus laevis Females

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

Anvendelse af retinsyre hentes osteocytter kulturer fra Primary Mouse Osteoblaster

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell ...

Whole Cell Electrophysiology of Primary Cultured Murine Enterochromaffin Cells

Hele cellen Elektrofysiologi primær kulturperler Murine Enterochromaffin celler

Enterochromaffin (EC) cells in the gastrointestinal (GI) epithelium constitute the largest subpopulation of enteroendocrine cells. As specialized sensory ...

Generation of Murine Primary Colon Epithelial Monolayers from Intestinal Crypts

Generation af Murine Primary Colon Epitel Monolayers fra Tarmkrypter

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in ...

Afsløring af inflammatorisk gigt: Udforskning af funktioner af synovialceller

Isolation and Culture of Primary Synovial Macrophages and Fibroblasts from Murine Arthritis Tissue

Forfatter Spotlight: Isolering og dyrkning af primære synoviale makrofager og fibroblaster fra musegigtvæv  

Isolering og dyrkning af primære synoviale makrofager og fibroblaster fra murine arthritisvæv

Rheumatoid arthritis is an autoimmune disease that leads to chronic inflammation of joints. Synovial macrophages and synovial fibroblasts have central ...