Name: an enzymatic method to rescue mesenchymal stem cells from clotted bone marrow samples
Uploaded: 2015-04-12T15:00:00
Description: Watch this Scientific Journal Video about An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples at JoVE.com

This work was supported by the Swiss National Foundation Grant CR3I3_140717/1 and the Swiss Paraplegic Foundation.

NOTE: Human bone marrow aspirates from the iliac crest were collected from consenting donors with the approval of the ethics committee of the canton of Lucerne. Canine bone marrow aspirates from the iliac crest were collected with dog owner&#8217;s consent.Human (approx. 20 ml) and canine (approx. 10 ml) bone marrow aspirates were anti-coagulated by addition of 15 ml of 3.8% sodium citrate immediately after withdrawal in the operation theatre. The samples were transferred to the laboratory environment for processing the ...

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Routinely we sample bone marrow while the patient is undergoing surgery (in our case mainly spine surgery), with the advantage that only little additional work has to be carried out by the personnel in the operation theatre. Even though the samples are mixed with sodium citrate immediately after withdrawal, many samples were partially clotted when they arrived in the laboratory for processing. At this stage, resampling to replace clotted specimens would be a separate additional intervention necessitating again local or g...

Mesenchymal stem cells (MSCs) play a major role in regenerative medicine and tissue engineering. They can migrate, differentiate into various cell types 1 and engraft, which renders them the ideal candidates for autologous therapies 2,3. Lately, clinical trials using MSCs for bone and cartilage repair, graft versus host disease or heart disease were launched 4. These MSCs can be harvested from the umbilical cord or adipose tissue but most promising results were obtained from bone marrow derived stem cells 5.
The iliac crest allows to collect a considerable amount of bone marrow and therefore serves as main site of aspiration 6. However, the quality of the aspirate decreases with increasing volume of bone marrow withdrawn. While the first 5 ml of bone marrow aspirate contain MSCs of high quality, withdrawal of larger volumes leads to dilution of the aspirate with peripheral blood from the highly vascularized bone 7. Because of the present megakaryocytes and platelets, bone marrow aspirates are prone to clotting, unless anticoagulants are used. But even with anticoagulants, clots may occur.
In bone marrow, MSCs represent only a small proportion of the total cell pool 8 and have to be expanded in culture for most tissue engineering or therapeutic applications 4. The quality of such a culture largely depends on the initial cell pool, i.e., diversity and a high starting number 9. Low numbers of MSCs from withdrawals may be partly explained by donor variability. On the other hand, MSCs from low quality samples require longer time in culture and extended passaging to reach the desired number of cells. In either case, extended passaging is a source of cell senescence and can lead to the loss of differentiation potential 10. Therefore, optimized protocols that can maximize cell yield and prevent from detrimental effects have to be developed 11,12.
When we began to work with canine MSCs, we were astonished to see that about three in four canine bone marrow samples contained clots, while fortunately clotted human samples (one in ten) were less frequent. On the other hand it was no surprise, that we observed much lower yields of MSCs from clotted samples. To solve the recurring issue of clotted samples, we developed the protocol using the thrombolytic drug urokinase instead of resampling.
Thrombolytic therapies can counteract life threatening situations such as occlusion of blood vessels causing heart attack, stroke or embolisms because of unwanted clotting. They work by degradation of the clots through enzymatic cleavage of fibrin by plasmin and enzymatic plasminogen activators. Despite the wide use for treatment of patients, only very few publications exist that utilized thrombolytic activities for laboratory applications to rescue clotted samples, mostly focusing on lymphocytes. In 1987, Niku et al. described the use of streptokinase for dissolving blood clots resulting in functional lymphocytes 13 and four years later, De Vis et al. extended the use of streptokinase to isolate leukemia cells from blood and bone marrow for flow cytometric applications 14. A more recent publication suggests the use of Alteplase for cancer diagnostics 15. While using the same enzymatic approach, our protocol focuses on the isolation of multipotent MSCs form bone marrow to provide a tool for researchers in the stem cell field.

<table border="0" cellpadding="0" cellspacing="0" width="674">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Basal Medium Components</td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">PenStrep 100X</td>
			<td style="width:227px;">Gibco</td>
			<td style="width:119px;">15140122</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Human FGF-basic</td>
			<td style="width:227px;">Peprotech</td>
			<td style="width:119px;">100-18B</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">MEM Alpha w/ Nucleoside, w/ stable Glutamine</td>
			<td style="width:227px;">Amimed</td>
			<td style="width:119px;">1-23S50-I</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:328px;">FBS Heat Inactivated</td>
			<td style="width:227px;">Amimed</td>
			<td style="width:119px;">2-01F36-I</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Amphotericin B</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">A1907</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;"></td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Adipogenic Medium Components</td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">DMEM-HAM F12 + GlutaMAX</td>
			<td style="width:227px;">Amimed</td>
			<td style="width:119px;">1-26F09-I</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Insulin&#160;</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">I5500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Rabbit serum&#160;</td>
			<td style="width:227px;">Gibco</td>
			<td style="width:119px;">16120099</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Dexamethasone</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">D4902</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">3-Isobutyl-1-methylxanthine</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">I5879</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Biotin&#160;</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">B4639</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Rosiglitazone&#160;</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">R2408</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Pantothenate&#160;</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">P5155</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Oil Red-O&#160;</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">O0625</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;"></td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Osteogenic Medium Components</td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">L-ascorbic acid 2-phosphate</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">A8960</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">&#223;-glycerophosphate</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">G9422</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:328px;">Silver nitrate (AgNO3)</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">S6506</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;"></td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Chondrogenic Medium Components</td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Biopad - sponge shaped medical device&#160;</td>
			<td style="width:227px;">Euroresearch</td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">L-proline&#160;</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">P5607</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Insulin-Transferrin-Selenium X</td>
			<td style="width:227px;">Gibco</td>
			<td style="width:119px;">51500056</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Human transforming growth factor-&#946;1&#160;</td>
			<td style="width:227px;">Peprotech</td>
			<td style="width:119px;">100-21</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Alcian Blue 8GX</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">A3157</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Nuclear fast red</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">N8002</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;"></td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Generic</td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Tri-Sodium citrate dihydrate</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">A3901</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">PBS</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">964.9100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Urokinase</td>
			<td style="width:227px;">Medac</td>
			<td style="width:119px;">1976826</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">0.5% Trypsin-EDTA</td>
			<td style="width:227px;">Gibco</td>
			<td style="width:119px;">15400054</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Giemsa stain</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">A0885</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formaldehyde</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">A0877</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sulfuric acid (H2SO4)</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">A0655</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">A1584</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium chloride (MgCl2)</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">A3618</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Guanidine hydrochloride</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">A1499</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;"></td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Consumables</td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">50 mL reaction tube</td>
			<td style="width:227px;">Axygen</td>
			<td style="width:119px;">SCT-50ML-25-S</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">10 mL syringe</td>
			<td style="width:227px;">Braun</td>
			<td style="width:119px;">4606108V</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Sterican needle (22G)</td>
			<td style="width:227px;">Braun</td>
			<td style="width:119px;">4657624</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">1.7 mL Microtubes</td>
			<td style="width:227px;">Brunschwig</td>
			<td style="width:119px;">MCT-175-C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">100 &#956;m cell strainer</td>
			<td style="width:227px;">Falcon</td>
			<td style="width:119px;">6.05935</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">sterile forceps</td>
			<td style="width:227px;">Bastos Viegas, SA</td>
			<td style="width:119px;">489-001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">sterile scalpel</td>
			<td style="width:227px;">Braun</td>
			<td style="width:119px;">5518059</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Primaria cell cuture dish</td>
			<td style="width:227px;">Falcon</td>
			<td style="width:119px;">353803</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">C-Chip Neubauer Improved</td>
			<td style="width:227px;">Bioswisstech</td>
			<td style="width:119px;">505050</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">cell culture flask - Flask T300</td>
			<td style="width:227px;">TPP</td>
			<td style="width:119px;">90301</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;"></td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Equipment</td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Microbiological biosafety cabinet class II</td>
			<td style="width:227px;">Skan</td>
			<td style="width:119px;">82011500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">water bath</td>
			<td style="width:227px;">Memmert</td>
			<td style="width:119px;">1305.0377</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Stripettes Serological Pipette 5ml</td>
			<td style="width:227px;">Corning</td>
			<td>4487-200ea</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">microscope</td>
			<td style="width:227px;">Olympus</td>
			<td style="width:119px;">CKX41</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">humidified incubator Heracells 240</td>
			<td style="width:227px;">Thermo scientific</td>
			<td>51026331</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Heraeus Multifuge 1S-R</td>
			<td style="width:227px;">Thermo scientific</td>
			<td style="width:119px;">75004331</td>
		</tr>
	</tbody>
</table>

an enzymatic method to rescue mesenchymal stem cells from clotted bone marrow samples

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade clots in the bone marrow and release MSCs for further use. This protocol provides a rapid and inexpensive alternative to bone marrow resampling. Bone marrow is a major source of MSCs, which are interesting for tissue engineering and autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system. The resulting clots contain also MSCs that are lost for expansion culture or direct stem cell therapy. We experienced that 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Thus, we developed a protocol for enzymatic digestion of those clots to avoid labor-intense and costly bone marrow resampling. Urokinase - a clinically approved and readily available thrombolytic drug &#8211; clears away the bone marrow clots almost completely. As a consequence, treated bone marrow aspirates yield similar numbers of MSCs as unclotted samples. Also, after urokinase treatment the cells kept their metabolic activity and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. Our protocol salvages clotted blood and bone marrow samples without affecting the quality of the cells. This obsoletes resampling, considerably reduces sampling costs and enables the use of clotted samples for research or therapy.

The facts that 74% of canine bone marrow samples (n=54) contained clots when they arrived in our laboratory (Figure 1A) along with decreased MSC yields from these samples, made us believe that a considerable number of MSCs was trapped within the clots. Indeed, a simple DAPI-stain of sectioned clot material confirms the presence of nucleated cells in high density (Figure 1B). This ultimately leads to low numbers of MSCs available for expansion culture, which triggered us to develop the pr...

Watch this Scientific Journal Video about An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples at JoVE.com

An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Mesenchymal stem cells are usually obtained from bone marrow and require expansion culture. When samples clot before processing, a protocol using the (enzymatic) thrombolytic drug urokinase can be applied to degrade the clot. Thus, cells are released and available for expansion culture. This protocol provides a rapid and inexpensive alternative to resampling.

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade ...

Research

JoVE Journal

Developmental Biology

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