Name: preparation of pooled human platelet lysate (phpl) as an efficient supplement for animal serum-free human stem cell cultures
Uploaded: 2009-10-30T07:00:00
Description: Watch this Scientific Journal Video about Preparation of Pooled Human Platelet Lysate pHPL as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures at JoVE.com

Detta arbete har fått stöd av den österrikiska Research Foundation (FWF, bevilja N211-NAN till DS) och den österrikiska Research Promotion Agency (FFG bevilja N200 till DS). Författarna tackar Claudia Url för utmärkt tekniskt bistånd och Monica Farrell för språklig redigering.

1. Utgångsmaterial Börja med trombocyter rich plasma (PRP) enheter som utarbetats av cytapheresis eller härrör från buffy rockar. 2. Sterilitet kontrollera För sterilitet kontrollera att ta ett prov på 20 ml från varje PRP enhet genom att överföra volymen till en ansluten liten väska (Baxter). Koppla denna väska genom svetsning. 3. Frysning av PRP enheter Omedelbart efter...

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I vissa regioner trombocyter rich plasma (PRP) kan erhållas från buffy-rockar annars vara en restprodukt av packade röda blodkroppar från undersökta blod (Figur 1). Optimalt är PRP användas omedelbart för vidare beredning av pHPL, som i föråldrade blodplättar koncentrerar tillgängligheten av tillväxtfaktorer kan minskas på grund av trombocyter lagring skador och nedbrytning 20. Det rekommenderas vidare att producera PRP genom att matcha trombocyter av blodgrupp O med plasma av blodgrupp AB för ...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>Double bag (2 x 3.5L)</td>
<td>&nbsp;</td>
<td>MacoPharma</td>
<td>VDL 8000XQ</td>
<td>originally for ascites puncture</td>
</tr>
<tr>
<td>50 mL Falcon tube</td>
<td>&nbsp;</td>
<td>Falcon BD</td>
<td>2098</td>
<td>&nbsp;</td>
<tr>
<td>50 mL Stripettes</td>
<td>&nbsp;</td>
<td>Costar</td>
<td>4501</td>
<td>&nbsp;</td>
<tr>
<td>Plasma bags (600mL)</td>
<td>&nbsp;</td>
<td>Baxter</td>
<td>R4R2021</td>
<td>&nbsp;</td>
<tr>
<td>Sterile tubing welder</td>
<td>&nbsp;</td>
<td>Terumo</td>
<td>TSCD-II</td>
<td>&nbsp;</td>
<tr>
<td>Welding equipment </td>
<td>&nbsp;</td>
<td>Fresenius Kabi</td>
<td>Compo Seal</td>
<td>&nbsp;</td>
<tr>
<td>Water bath</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sterile scissors</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Clamps</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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preparation of pooled human platelet lysate (phpl) as an efficient supplement for animal serum-free human stem cell cultures

Trombocyter derived growth faktorer har visat att stimulera celltillväxt effektivt<em&gt; In vivo</em<sup&gt; 1,2</sup&gt; Och<em&gt; In vitro</em&gt;. Denna effekt har rapporterats för mesenkymala stromaceller (MSC), fibroblaster och endotel kolonibildande celler med trombocyter aktiveras av trombin<sup&gt; 3-5</sup&gt; Eller lyseras av frys / tö cykler<sup&gt; 6-14</sup&gt; Innan trombocyter releasate läggs till cellkulturmedium. Den trofiska effekten av trombocyter härrör tillväxtfaktorer har redan testats i flera studier för tissue engineering och regenerativ terapi.<sup&gt; 1,15-17</sup&gt; Varierande effektivitet anses vara åtminstone delvis beror på individuellt olika halter av tillväxtfaktorer<sup&gt; 18,19</sup&gt; Och en nuvarande bristen på standardiserade protokoll för trombocyter beredning.<sup&gt; 15,16</sup&gt; Detta protokoll utgör ett praktiskt förfarande för att skapa en pool av mänskliga blodplättar lysat (pHPL) från rutinmässigt producerade trombocyter rich plasma (PRP) på 4-50 enda blodgivning. Av flera frys / tö cykler trombocytens membran är skadade och tillväxtfaktorer effektivt utsöndras i plasma. Slutligen är trombocyter fragment avlägsnas genom centrifugering för att undvika omfattande samlade bildning och bryter potentiella antigener. Genomförandet av pHPL i standard kulturen protokoll är ett lovande verktyg för vidare utveckling av Cell Therapeutics odlat i ett animaliskt protein-free-system.

Watch this Scientific Journal Video about Preparation of Pooled Human Platelet Lysate pHPL as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures at JoVE.com

Preparation of Pooled Human Platelet Lysate pHPL as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Mänskliga trombocyter lysat är en rik källa av tillväxtfaktorer och ett potent tillskott i cellkultur. Detta protokoll visar att förbereda en stor pool av mänskliga trombocyter lysat genom att utgå från trombocyter rich plasma, utför flera frys-tö cykler och en uttunning av trombocyter fragment.

Beredning av poolade humana trombocyter lysat (pHPL) som en effektiv Tillägg för Animal serumfritt mänskliga kulturer Stem Cell

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for ...

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Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

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