Name: isolation, culture, and differentiation of bone marrow stromal cells and osteoclast progenitors from mice
Uploaded: 2018-01-06T14:00:00
Description: Watch this Scientific Journal Video about Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice at JoVE.com

Dette arbejde blev støttet af National Institutes of Health (R01 AR061164-01A1).

Alle eksperimentelle procedurer blev godkendt af det institutionelle Animal Care og brug udvalg på Maine Medical Center Research Institute.
1. indsamling rør forberedelse
<ol><li>Gøre BMSC Kulturmedier ved at supplere Minimum afgørende Medium α (MEM α) med 10% føtal bovint Serum og 1% af Penicillin/Streptomycin.</li>
	<li>Plads 100 µL af BMSC kultur medier i 1,5 mL microcentrifuge rør. 3 knogler vil normalt passer i et enkelt rør så forberede i overensstemmelse hermed.</li>
	<li>S...

<ol>
	<li>Friedenstein, A. J., Latzinik, N. W., Grosheva, A. G., Gorskaya, U. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marrow+microenvironment+transfer+by+heterotopic+transplantation+of+freshly+isolated+and+cultured+cells+in+porous+sponges.">Marrow microenvironment transfer by heterotopic transplantation of freshly isolated and cultured cells in porous sponges.</a> Exp Hematol. 10 (2), 217-227 (1982).</li><li>Chen, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+growth+and+differentiation+of+osteoprogenitors+in+mouse+bone+marrow+stromal+cell+cultures+by+increased+donor+age+and+glucocorticoid+treatment.">Inhibition of growth and differentiation of osteoprogenitors in mouse bone marrow stromal cell cultures by increased donor age and glucocorticoid treatment.</a> Bone. 35 (1), 83-95 (2004).</li><li>Kokabu, S., 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=BMP3+suppresses+osteoblast+differentiation+of+bone+marrow+stromal+cells+via+interaction+with+Acvr2b.">BMP3 suppresses osteoblast differentiation of bone marrow stromal cells via interaction with Acvr2b.</a> Mol Endocrinol. 26 (1), 87-94 (2012).</li><li>Ghali, O., 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=Dexamethasone+in+osteogenic+medium+strongly+induces+adipocyte+differentiation+of+mouse+bone+marrow+stromal+cells+and+increases+osteoblast+differentiation.">Dexamethasone in osteogenic medium strongly induces adipocyte differentiation of mouse bone marrow stromal cells and increases osteoblast differentiation.</a> BMC Cell Biol. 16, 9 (2015).</li><li>Kretlow, J. D., 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=Donor+age+and+cell+passage+affects+differentiation+potential+of+murine+bone+marrow-derived+stem+cells.">Donor age and cell passage affects differentiation potential of murine bone marrow-derived stem cells.</a> BMC Cell Biol. 9, 60 (2008).</li><li>DeMambro, V. 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=Igfbp2+Deletion+in+Ovariectomized+Mice+Enhances+Energy+Expenditure+but+Accelerates+Bone+Loss.">Igfbp2 Deletion in Ovariectomized Mice Enhances Energy Expenditure but Accelerates Bone Loss.</a> Endocrinology. 156 (11), 4129-4140 (2015).</li><li>Maridas, D. 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=IGFBP-4+regulates+adult+skeletal+growth+in+a+sex-specific+manner.">IGFBP-4 regulates adult skeletal growth in a sex-specific manner.</a> J Endocrinol. 233 (1), 131-144 (2017).</li><li>Nadri, S., 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=An+efficient+method+for+isolation+of+murine+bone+marrow+mesenchymal+stem+cells.">An efficient method for isolation of murine bone marrow mesenchymal stem cells.</a> Int J Dev Biol. 51 (8), 723-729 (2007).</li><li>Sreejit, P., Dilip, K. B., Verma, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+mesenchymal+stem+cell+lines+from+murine+bone+marrow.">Generation of mesenchymal stem cell lines from murine bone marrow.</a> Cell Tissue Res. 350 (1), 55-68 (2012).</li><li>Abdallah, B. 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=CD34+defines+an+osteoprogenitor+cell+population+in+mouse+bone+marrow+stromal+cells.">CD34 defines an osteoprogenitor cell population in mouse bone marrow stromal cells.</a> Stem Cell Res. 15 (3), 449-458 (2015).</li><li>Akiyama, K., 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=Characterization+of+bone+marrow+derived+mesenchymal+stem+cells+in+suspension.">Characterization of bone marrow derived mesenchymal stem cells in suspension.</a> Stem Cell Res Ther. 3 (5), 40 (2012).</li><li>Lin, C. S., Ning, H., Lin, G., Lue, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+CD34+truly+a+negative+marker+for+mesenchymal+stromal+cells?.">Is CD34 truly a negative marker for mesenchymal stromal cells?.</a> Cytotherapy. 14 (10), 1159-1163 (2012).</li></ol>

I denne artikel præsenteres to metoder til kultur af BMSCs med deres fordele og begrænsninger. Isolere cellerne fra knoglemarven er en relativt ubesværet proces. Men, at opnå en celle population repræsentative mesenkymale stamceller eller osteoclastic ophav kan være udfordrende på grund af de forskellige cellulære miljø i marv hulrum.
Dyrkning helhed af knoglemarven indholdet giver en tæt repræsentation af i vivo mikromiljø. Alligevel isolerer celler fra forskellige gruppe...

Primære murine BMSCs er almindeligt anvendt som en in vitro- model af mesenkymale stamceller siden deres opdagelse i den tidlige 1980s1. Faktisk, kulturer af plast-tilhænger celler skyllet fra knoglemarven hulrummet af lange knogler opretholde kapacitet skal differentieres i osteoblaster, osteoklaster, chondrocytter eller adipocytter i mange studier2,3, 4 , 5. dog knoglemarven er en unik væv består af mange forskellige cellepopulationer, herunder, men ikke begrænset til, BMSCs, HSCs, endotel og immun celler. Således, isolation og kultur teknikker kan give cellepopulationer med forskellige homogenitet. Ved hjælp af disse teknikker til at teste den potentielle differentiering fra celler kan være udfordrende. For eksempel, når man sammenligner celler fra mus med forskellige genotyper, begyndende med en blandet celle population begrænser fortolkning af data. Omvendt, at opnå homogen populationer af BMSCs og HSCs kan være teknisk vanskeligt og måske ikke som repræsentant for en ex vivo -model.
I vores laboratorium er vi primært interesseret i udnyttelsen af BMSCs på grund af deres potentiale skal differentieres i osteoblaster og osteoklaster adipocytter. Vi præsenterer her, teknikker til BMSCs og HSCs isolation og kultur bruges til at vurdere osteoblastogenesis eller adipogenesis i vitroog kulturer af HSCs til at differentiere i osteoklaster. Én metode bruger en blandet population af knoglemarv celler (BMCs) der indeholder BMSCs og HSCs direkte egnet til adipogenesis, osteoblastogenesis og osteoclastogenesis (kaldet samlede BMCs). Denne metode er en tættere ex vivo repræsentation af heterogenitet fundet blandt cellerne i knoglemarven mikromiljø. En anden metode adskiller tilhænger fra ikke-tilhænger celler i et forsøg på at kultur "renere" populationer af BMSCs og HSCs (kaldet tilhænger BMSCs). Den senere metode tillader cellekultur eksperimenter for at starte med en mere præcis antal BMSCs eller HSCs og reducerer potentiale af komplekse indirekte virkninger af andre cellepopulationer, der forbliver i kulturen. Begge metoder har været tidligere offentliggjort og bruges til at løse forskellige forskning spørgsmål6,7,8,9.

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			<td>190764</td>
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			<td height="21" style="height:21px;">10% Neutral Buffered Formalin</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td>F5554-4L</td>
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			<td height="21" style="height:21px;width:216px;">IBMX</td>
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			<td height="21" style="height:21px;width:216px;">Dexamethasone</td>
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			<td style="width:119px;">D4902</td>
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			<td height="21" style="height:21px;width:216px;">RANKL</td>
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			<td height="42" style="height:42px;width:216px;">Axio Observer inverter microscope</td>
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isolation, culture, and differentiation of bone marrow stromal cells and osteoclast progenitors from mice

Knoglemarven stromale celler (BMSCs) udgør en celle population rutinemæssigt anvendes som en repræsentation af mesenkymale stamceller in vitro-. De bor i knoglemarven hulrum sammen med hæmatopoietisk stamceller (HSCs), som kan give anledning til røde blodlegemer, immun ophav og osteoklaster. Således ekstraktioner af cellepopulationer fra knoglemarven resultater i en meget heterogen blanding af forskellige cellepopulationer, der kan præsentere udfordringer i eksperimentelle design og forvirre data fortolkning. Flere isolation og kultur teknikker er blevet udviklet i laboratorier for at opnå mere eller mindre homogene befolkninger af BMSCs og HSCs invitro. Her præsenterer vi to metoder til isolering af BMSCs og HSCs fra mus lange knogler: en metode, der giver en blandet population af BMSCs og HSCs og en metode, der forsøger at adskille to cellepopulationer baseret på overholdelse. Begge metoder giver celler velegnet til osteogenic og adipogenic differentiering eksperimenter såvel som funktionelle assays.

Oversigt over cellekulturer To kultur teknikkerne giver mulighed for vurdering af differentiering på en blandet population af BMCs består af BMSCs og HSCs (Total BMCs, figur 1A) eller en befolkning på BMSCs skilt fra HSCs (tilhænger BMSCs, figur 1B). 48 timer efter celler fra marven isoleres og forgyldt (figur 1 c), de hurtigt tillægger undersiden af plast kultur plade og har...

Watch this Scientific Journal Video about Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice at JoVE.com

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

I denne artikel præsenterer vi metoder for at isolere og differentiere knoglemarv stromale celler og hæmatopoietisk stamceller fra mus lange knogler. To forskellige protokoller præsenteres højtydende forskellige cellepopulationer egnet til udvidelse og differentiering af osteoblaster, adipocytter og osteoklaster.

Isolation, kultur og differentiering af knoglemarven stromale celler og osteoklast ophav fra mus

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

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Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the ...

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