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 The samples were transferred to the laboratory environment for processing the same day as withdrawn.
1. Preparation of Urokinase (Prior to 1st Use)
<ol>
	<li>Reconstitute urokinase using sterile phosphate buffered saline (PBS) according to the manufacturer&#8217;s instructions: Add 10 ml PBS to the septum-inlet flask using a 10 ml syringe. Dissolve the dry substance by swirling to obtain 50,000 injection units (U) per ml.</li>
	<li>Prepare 500 &#181;l aliquots (25,000 U each) into sterile tubes. Store aliquots -20 &#176;C and use it when required for at least 6 months.</li>
</ol>
2. Preparative Steps (Prior to Every Bone Marrow Treatment)
<ol>
	<li>Thaw one aliquot of urokinase (25,000 U) per clotted bone marrow aspirate (aspirate volumes up to 25 ml have been successfully treated using a single aliquot of 25,000 U).</li>
	<li>Preheat a water bath or shaking incubator to 37 &#176;C.</li>
</ol>
3. Enzymatic Digest of the Clot
<ol>
	<li>Perform all work in a laminar flow biosafety cabinet to avoid microbial contamination of the bone marrow samples during processing. When working with potentially infectious human material, the use of protective gloves as well as careful handling is advised.</li>
	<li>Place a 100 &#181;m cell strainer on top of a sterile 50 ml tube. Carefully pour the bone marrow aspirate through the cell strainer, tilting the strainer or moving around the clot material. Use a sterile pipet tip for better flow through the filter mesh. 
	NOTE: Avoid any dilution of the clot, e.g., by washing the clot with PBS. Urokinase acts indirectly and requires components of the serum (i.e., plasminogen) from the biopsy for effective digest. While continuing the procedure with the clot material, the filtrate can be kept at RT until further use in step 3.6.</li>
	<li>Transfer the bone marrow clot material into an empty cell culture dish using sterile forceps. Cut the debris into small pieces of approximately 2 mm3 using a sterile scalpel.</li>
	<li>Transfer the small pieces of the clot into a 50 ml reaction tube using the sterile forceps. Ensure that the minced clot has a wet appearance. If it appears to be dry, use some of the filtrate from step 3.1 to moisten.</li>
	<li>Triturate the clot by pipetting up and down for 5 times using a 5 ml microtiter pipet. Add 1 aliquot of urokinase to the sample. Incubate for 30 min at 37 &#176;C either in a water bath or in a shaking (gently) incubator.</li>
	<li>Triturate by pipetting up and down for 5 times using a 5 ml pipet. Perform this step in a biosafety cabinet. Incubate for another 30 min at 37 &#176;C. After the incubation, triturate again 5 times using a 5 ml pipet. Pass over a fresh 100 &#181;m cell strainer to pool with the filtrate from step 3.2.</li>
	<li>Centrifuge the cell suspension at ambient temperature for 10 min at 500 x g. Discard the supernatant. Re-suspend the cell pellet in media as described below or according to the standard procedure of your laboratory for MSC expansion culture.</li>
</ol>
4. Seeding for Expansion Cell Culture and CFU Plates
<ol>
	<li>Resuspend the cells (consisting of erythrocytes and mononuclear cells) in 50 ml of basal medium: Alpha-MEM supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 mg/ml streptomycin and 2.5 mg/ml amphotericin B. Count the cells diluted 1:10 in Trypan Blue solution using a Neubauer chamber.</li>
	<li>Plate the cells in cell culture flasks at the density of 5 x 107 cells/cm2 and incubate in a humidified incubator at 37 &#176;C. If available, use hypoxic (5% oxygen) conditions. For the CFU assay control, plate 109 cells in a cell culture dish of 10 cm diameter.</li>
	<li>After 3 days of culture, mesenchymal stem cells are attached to the cell culture dish while other cells remain in suspension. Change the media with basal medium supplemented with 5 ng/ml basic fibroblast growth factor (bFGF). For the CFU assay control, treat the cell culture dish likewise.</li>
	<li>Continue cell culture for a total of 2 weeks, exchanging media three times a week. If cells exceed 80% confluency, split by trypsinization. For the CFU assay control, exchange the media likewise, but do not split the cells for the course of the two weeks.</li>
</ol>
5. Giemsa Stain for CFU Assay
<ol>
	<li>Prepare 10 ml of Giemsa-solution per dish: dilute the stock solution (7.6 g/l Giemsa in glycerol:methanol) 1:10 in sterile water (always prepare a fresh working dilution).</li>
	<li>Remove the media from the cell culture dish and wash the cells with PBS. Be very precautious when applying liquid to the petri dish and avoid washing off the cells.</li>
	<li>Fix cells in pure Methanol for 5 min at RT. Discard the Methanol. Add the Giemsa-solution and incubate for 60 min in a humidified incubator at 37 &#176;C.</li>
	<li>Wash two times with PBS. Air dry the plate head first on a paper towel. Count the colonies manually. This is best achieved using a marker pen on the back of the plate.</li>
</ol>
6. MSC Differentiation
<ol>
	<li>Canine MSCs differentiation 
	NOTE: Canine MSCs were differentiated into chondrogenic, osteogenic and adipogenic lineages by stimulation with the appropriate media for four weeks.</li>
	<li>For Adipogenic differentiation, culture MSCs in monolayer at 4 x 105 cells/cm2 alternating two different culture conditions as follows;
	<ol>
		<li>Culture in adipogenesis maintenance media containing DMEM + GlutaMAX, 3% FBS, 100 units/ml penicillin, 100 mg/ml streptomycin, 2.5 &#181;g/ml amphotericin B and 170 mM insulin.</li>
		<li>Culture in adipogenesis inducing media with maintenance medium supplemented with 3% FBS, 5% rabbit serum, 1 &#181;M dexamethasone, 500 &#181;M 3-Isobutyl-1-methylxanthine, 33 &#181;M biotin, 5 &#181;M rosiglitazone and 17 &#181;M pantothenate 16.</li>
		<li>Revel the lipid droplets by staining with Oil Red-O. Briefly, fix cells with 10% formaldehyde, wash with PBS and stain with 0.35% Oil red O in isopropanol.</li>
	</ol>
	</li>
	<li>For Osteogenic differentiation culture MSCs in monolayer at 7 x 103 cells/cm2 and stimulate in the following:
	<ol>
		<li>Advanced DMEM (GIBCO) + GlutaMAX, 5 % FBS, 100 units/ml penicillin, 100 mg/ml streptomycin, 2.5 &#181;g/ml amphotericin B, 50 &#181;M L-ascorbic acid 2-phosphate, 10 mM &#223;-glycerophosphate and 100 nM dexamethasone.</li>
		<li>Identify the mineralization deposits by Von Kossa stain (5 % AgNO3). Briefly, fix the cells with 10% formaldehyde, wash with PBS and stain with 5 % AgNO3in distilled water.</li>
	</ol>
	</li>
	<li>Chondrogenic differentiation
	<ol>
		<li>Cut cubes (3 mm per side) from a sponge shaped medical device, constituted from lyophilized collagen type I, and used as scaffold material to support cells 17.</li>
		<li>Seed MSCs on the top of the cubes at the concentration of 4 x 106 cells/ml (~70,000 cells/cube).</li>
		<li>Prior to the addition of media, allow the cells to adhere to the cubes for 30 min.</li>
		<li>Maintain MSC-collagen constructs in chondrogenic media consisting of DMEM/F12 + GlutaMAX, 2.5 % FBS, 100 units/ml penicillin, 100 mg/ml streptomycin, 2.5 &#181;g/ml amphotericin B, 40 ng/ml dexamethasone, 50 &#181;g/ml ascorbic acid 2-phosphate, 50 &#181;g/ml L-proline, 1x Insulin-Transferrin-Selenium X, and 10 ng/ml transforming growth factor-&#946;1.</li>
		<li>Use alcian blue staining to visualize accumulation of proteoglycans in constructs sections. Briefly, stain the sections O/N with 0.4% alcian blue dissolved in 0.01% H2SO4 and 0.5M guanidine hydrochloride. Next, wash the sections were washed for 30 min in 40% DMSO and 0.05M MgCl2. Cells were counterstained with nuclear fast red.</li>
	</ol>
	</li>
</ol>

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</ol>

The authors have nothing to disclose.

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 general anesthesia 6. This requires the willingness of both the clinical staff and the donor to contribute and consumes a lot of resources 21.
Here we provide a protocol that uses recombinant human urokinase on clotted bone marrow samples to isolate multipotent MSCs. We could demonstrate that the urokinase treatment does not harm the cells and the differentiation potential is retained. The protocol is short since it prolongs sample processing only by approximately 1 &#189; hr as compared to unclotted samples while yielding comparable cell pools. To date, this protocol has been successfully applied on bone marrow clots from different individuals and dogs using urokinase from several lots and has shown robust and reproducible success. Apart from this, urokinase acts indirectly via activation of sample-intrinsic plasminogen. This implies that the urokinase reaction requires the plasma-environment. It is therefore unadvisable to introduce steps of clot rinsing or alike with e.g., PBS. This would dilute the serum environment and lead to a significant slow-down of the enzymatic reaction.
The decision to use urokinase for our method and not another thrombolytic drug was trivial: we developed the protocol based on the thrombolytic drug readily available in our hospital pharmacy. For other research groups it may therefore be easier to use a tissue-type plasminogen activator (i.e., a different drug product like alteplase, reteplase or tenecteplase) if urokinase is not available. However, potentially important differences between the drugs exist: unlike tissue-type plasminogen activators, urokinase can trigger cell activation and proliferation when bound to the urokinase-type plasminogen activator receptor (uPAR) 22. Upon binding, intracellular signaling cascades are activated that lead to cell migration and proliferation. In a physiological situation this is further supported by the secretion of matrix metalloproteinases by activated cells. However, in our system, urokinase is diluted and gradually removed upon expansion culture without showing detrimental effects. Still, with respect to the intended use of isolated MSCs, the suitability of any of the thrombolytic drugs needs to be determined individually.
Generally, high dosages for rapid and complete clot digest are recommended to minimize unwanted selection during clot processing. Noteworthy, many previous studies used bacterial streptokinase for digesting blood and bone marrow clots 13,14. However, this drug may provoke unwanted activation and response of the immune system, which may be a major drawback especially when application of cells to patients is intended.
We therefore believe that our protocol does not only help researchers and medical professionals to save time and reduce costs. Using urokinase &#8211; an approved enzymatic drug without known antigenicity &#8211; may even provide a valuable tool for many translational research laboratories aiming at therapeutic use of MSC preparations.

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><tbody><tr><td>Basal Medium Components</td><td></td><td></td><td></td></tr><tr><td>PenStrep 100X</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Human FGF-basic</td><td>Peprotech</td><td>100-18B</td><td></td></tr><tr><td>MEM Alpha w/ Nucleoside, w/ stable Glutamine</td><td>Amimed</td><td>1-23S50-I</td><td></td></tr><tr><td>FBS Heat Inactivated</td><td>Amimed</td><td>2-01F36-I</td><td></td></tr><tr><td>Amphotericin B</td><td>Applichem</td><td>A1907</td><td></td></tr><tr><td>Adipogenic Medium Components</td><td></td><td></td><td></td></tr><tr><td>DMEM-HAM F12 + GlutaMAX</td><td>Amimed</td><td>1-26F09-I</td><td></td></tr><tr><td>Insulin&amp;nbsp;</td><td>Sigma</td><td>I5500</td><td></td></tr><tr><td>Rabbit serum&amp;nbsp;</td><td>Gibco</td><td>16120099</td><td></td></tr><tr><td>Dexamethasone</td><td>Applichem</td><td>D4902</td><td></td></tr><tr><td>3-Isobutyl-1-methylxanthine</td><td>Sigma</td><td>I5879</td><td></td></tr><tr><td>Biotin&amp;nbsp;</td><td>Sigma</td><td>B4639</td><td></td></tr><tr><td>Rosiglitazone&amp;nbsp;</td><td>Sigma</td><td>R2408</td><td></td></tr><tr><td>Pantothenate&amp;nbsp;</td><td>Sigma</td><td>P5155</td><td></td></tr><tr><td>Oil Red-O&amp;nbsp;</td><td>Sigma</td><td>O0625</td><td></td></tr><tr><td>Osteogenic Medium Components</td><td></td><td></td><td></td></tr><tr><td>L-ascorbic acid 2-phosphate</td><td>Sigma</td><td>A8960</td><td></td></tr><tr><td>&amp;szlig;-glycerophosphate</td><td>Sigma</td><td>G9422</td><td></td></tr><tr><td>Silver nitrate (AgNO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma</td><td>S6506</td><td></td></tr><tr><td>Chondrogenic Medium Components</td><td></td><td></td><td></td></tr><tr><td>Biopad - sponge shaped medical device&amp;nbsp;</td><td>Euroresearch</td><td></td><td></td></tr><tr><td>L-proline&amp;nbsp;</td><td>Sigma</td><td>P5607</td><td></td></tr><tr><td>Insulin-Transferrin-Selenium X</td><td>Gibco</td><td>51500056</td><td></td></tr><tr><td>Human transforming growth factor-&amp;beta;1&amp;nbsp;</td><td>Peprotech</td><td>100-21</td><td></td></tr><tr><td>Alcian Blue 8GX</td><td>Sigma</td><td>A3157</td><td></td></tr><tr><td>Nuclear fast red</td><td>Sigma</td><td>N8002</td><td></td></tr><tr><td>Generic</td><td></td><td></td><td></td></tr><tr><td>Tri-Sodium citrate dihydrate</td><td>Applichem</td><td>A3901</td><td></td></tr><tr><td>PBS</td><td>Applichem</td><td>964.9100</td><td></td></tr><tr><td>Urokinase</td><td>Medac</td><td>1976826</td><td></td></tr><tr><td>0.5% Trypsin-EDTA</td><td>Gibco</td><td>15400054</td><td></td></tr><tr><td>Giemsa stain</td><td>Applichem</td><td>A0885</td><td></td></tr><tr><td>Formaldehyde</td><td>Applichem</td><td>A0877</td><td></td></tr><tr><td>Sulfuric acid (H2SO4)</td><td>Applichem</td><td>A0655</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Applichem</td><td>A1584</td><td></td></tr><tr><td>Magnesium chloride (MgCl2)</td><td>Applichem</td><td>A3618</td><td></td></tr><tr><td>Guanidine hydrochloride</td><td>Applichem</td><td>A1499</td><td></td></tr><tr><td>Consumables</td><td></td><td></td><td></td></tr><tr><td>50 ml reaction tube</td><td>Axygen</td><td>SCT-50ML-25-S</td><td></td></tr><tr><td>10 ml syringe</td><td>Braun</td><td>4606108V</td><td></td></tr><tr><td>Sterican needle (22G)</td><td>Braun</td><td>4657624</td><td></td></tr><tr><td>1.7 ml Microtubes</td><td>Brunschwig</td><td>MCT-175-C</td><td></td></tr><tr><td>100 &amp;mu;m cell strainer</td><td>Falcon</td><td>6.05935</td><td></td></tr><tr><td>sterile forceps</td><td>Bastos Viegas, SA</td><td>489-001</td><td></td></tr><tr><td>sterile scalpel</td><td>Braun</td><td>5518059</td><td></td></tr><tr><td>Primaria cell cuture dish</td><td>Falcon</td><td>353803</td><td></td></tr><tr><td>C-Chip Neubauer Improved</td><td>Bioswisstech</td><td>505050</td><td></td></tr><tr><td>cell culture flask - Flask T300</td><td>TPP</td><td>90301</td><td></td></tr><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>Microbiological biosafety cabinet class II</td><td>Skan</td><td>82011500</td><td></td></tr><tr><td>water bath</td><td>Memmert</td><td>1305.0377</td><td></td></tr><tr><td>Stripettes Serological Pipette 5ml</td><td>Corning</td><td>4487-200ea</td><td></td></tr><tr><td>microscope</td><td>Olympus</td><td>CKX41</td><td></td></tr><tr><td>humidified incubator Heracells 240</td><td>Thermo scientific</td><td>51026331</td><td></td></tr><tr><td>Heraeus Multifuge 1S-R</td><td>Thermo scientific</td><td>75004331</td><td></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 protocol using urokinase. Typically clots disappear almost completely when applying our protocol. However, during method development we addressed the question of clot digest systematically by weighing before and after digest. These experiments revealed that the undigested remainders were 15% (dog) or 9% (human) of the initial clot weight (Figure 2A).
A typical feature of bone marrow derived MSCs is the ability to adhere to cell culture dishes. Researchers make use of this characteristic to select for MSCs. As a consequence, a colony forming assay allows to evaluate the quality of the cell pool in a simple, quantitative and reliable way. In our laboratory, the colony forming assay described herein has been applied routinely to all bone marrow samples processed (also non-clotted ones). This allowed us to use the assay as main criterion for determining the efficacy of the urokinase digest. To allow direct comparison, cells from clotted sample filtrates (i.e. as if clotted samples were just filtered but not enzymatically treated) and digested clots were seeded on separate 10 cm dishes followed by incubation for two weeks. When visualizing colony forming units (CFU) with Giemsa stain (as shown in Figure 2B), we observed 3.8 times more CFU from the urokinase reactions of canine samples than from the corresponding initial filtrates (Figure 2C). Although less prominent, human samples followed a similar trend with 1.6 fold more CFU from treated samples, confirming the suitability of the protocol. Indeed, the plates of digested clot illustrated in the right row in Figure 2B correspond to the typical result seen for a pooled sample. However, donor variation may easily result in CFU numbers from half to double of what is depicted.
Taking the colony-size as indicator of cell pool quality, more medium (2-5 mm) and large (&#62;5 mm) colonies appeared on the plates seeded from clots compared to the CFU from filtrates (Figure 2C). This generally indicates that MSCs grow normally after urokinase treatment. Also in the aggregate, the comparison of digested canine specimens (n = 21) to samples without clot (n = 7) yielded comparable numbers of total MSCs for treated samples, although a big inter-sample variation was observed due to the heterogeneous donor population (Figure 2D).
As a last functional test of suitability, we induced differentiation of MSCs derived from digested bone marrow samples. Applications in autologous stem cell therapies are based on MSC differentiation into cartilage, bone or adipose tissue or MSC paracrine signaling 18. Cells can be guided towards the desired path of differentiation by choosing the appropriate culture conditions for adipogenic differentiation 19, osteogenic differentiation 20 or chondrogenic lineage 17. Hence, we compared MSCs from bone marrow clot to MSCs derived from unclotted bone marrow sample. After four weeks in culture, it was possible to differentiate MSCs into all three above mentioned lineages. This was histologically tested with Von Kossa (for osteogenic lineage), Oil Red O (adipogenic) and Alcian Blue (chondrogenic) stainings, showing no differences in the grade of differentiation between the groups (Figure 3).
<img alt="Figure 1" src="/files/ftp_upload/52694/52694fig1.jpg" /> 
Figure 1.&#160;Bone marrow clots. Percentage of canine and human bone marrow samples in partially clotted state upon arrival (A). Furthermore, we found that MSC yields from clotted samples were strongly reduced due to a high number of cells trapped within the clots. A high number of nucleated cells are present within the clots as demonstrated by DAPI-staining of a cryosection from a canine bone marrow clot (B). Scale bar represents 200 &#181;m.
<img alt="Figure 2" src="/files/ftp_upload/52694/52694fig2.jpg" /> 
Figure 2.&#160;Isolation of MSCs from bone marrow clots digested with urokinase. (A) Typically, bone marrow clots disappear almost completely upon urokinase digest. Upon method development, we assessed clot weights for canine (n = 5, mean &#177; SD, **p &#8804;0.01) and human samples (n = 3, mean &#177; SD, n.s. = not significant p = 0.10) that were strongly reduced upon urokinase digest. (B) A simple CFU assay can serve as an informative tool for assessing the quality of an MSC preparation. To assess the efficacy of the digest, the CFU assay was performed for filtrates and digested clots from bone marrow aspirates separately. After two weeks in culture, 10 cm control plates were stained with GIEMSA and CFU were counted. The assay confirmed that high number of CFU can be released from the clot by urokinase digest and remain functional. This is also confirmed by the high frequency of large colonies (class-division: &#62;5 mm in black, 2-5 mm in dark grey and &#60;2 mm in light grey. Error bars represent SD of total CFU (n = 5 for dog, n = 3 for human, **p &#8804;0.01, n.s. = not significant, p = 0.17). (C) Pictures from Giemsa-stained plates confirm the results shown. The plates shown for digested clot correspond to a typical result for a digested bone marrow sample &#8211; however, numbers can vary largely due to donor variability. (D) In the sum, applying the urokinase digest protocol (n = 21) results in comparable total MSC yields after expansion culture to naturally clot free samples (n = 7, mean &#177; SD). Statistical analysis for the entire Figure 2 was performed using Student&#8217;s t-test.
<img alt="Figure 3" src="/files/ftp_upload/52694/52694fig3.jpg" /> 
Figure 3.&#160;Comparison of differentiation potential of digested versus natively unclotted samples. Canine MSCs isolated from clotted bone marrow treated with Urokinase (top row) and unclotted bone marrow (bottom row) were differentiated in osteogenic phenotype and stained by Von Kossa (bar = 200 &#181;m), adipogenic phenotype was stained by Oil Red O (bar =100 &#181;m; in the insets larger magnification of fat vacuoles), while chondrogenesis was revealed by Alcian blue staining (bar =100 &#181;m; counterstaining nuclear fast red). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52694/52694fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

an-enzymatic-method-to-rescue-mesenchymal-stem-cells-from-clotted

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

9.2K Views.  Swiss Paraplegic Research. 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.

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

Isolation of Human Mesenchymal Stem Cells and their Cultivation on the Porous Bone Matrix

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs bovine bone matrix Nukbone (NKB) as a scaffold. Thus, the application of MSCs in repair and tissue regeneration processes depends principally on the efficient implementation of the techniques for placing these cells in a host tissue. For this reason, the design of biomaterials and cellular scaffolds has gained importance in recent years because the topographical characteristics of the selected scaffold must ensure adhesion, proliferation and differentiation into the desired cell lineage in the microenvironment of the injured tissue. This option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) employs bovine bone matrix as a cellular scaffold and is an efficient culture technique because the cells respond to the topographic characteristics of the bovine bone matrix Nukbone (NKB), i.e., spreading on the surface, macroporous covering and colonizing the depth of the biomaterial, after the cell isolation process. We present the procedure for isolating and culturing MSCs on a bovine matrix.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs the bovine bone matrix Nukbone (NKB) as a scaffold.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific ...

Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, milk and urine. One subset of these RNAs are the posttranscriptional regulators &#8211; microRNAs (miRNAs). To delineate the miRNAs produced by specific cell types, in vitro culture systems can be used to harvest and profile exRNAs derived from one subset of cells. The secreted factors of mesenchymal stem cells are implicated in alleviating numerous diseases and is used as the in vitro model system here. This paper describes the process of collection, purification of small RNA and library generation to sequence extracellular miRNAs. ExRNAs from culture media differ from cellular RNA by being low RNA input samples, which calls for optimized procedures. This protocol provides a comprehensive guide to small exRNA sequencing from culture media, showing quality control checkpoints at each step during exRNA purification and sequencing.

This protocol demonstrates how to purify extracellular microRNAs from cell culture media for small RNA library construction and next generation sequencing. Various quality control checkpoints are described to allow readers to understand what to expect when working with low input samples like exRNAs.

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, ...

Genetics

Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular cartilage and subchondral bone in recent years led to increased attention to restoration of the entire osteochondral unit. In comparison to chondral lesions the regeneration of osteochondral defects is much more complex and a far greater surgical and therapeutic challenge. The damaged tissue does not only include the superficial cartilage layer but also the subchondral bone. For deep, osteochondral damage, as it occurs for example with osteochondrosis dissecans, the full thickness of the defect needs to be replaced to restore the joint surface 1. Eligible therapeutic procedures have to consider these two different tissues with their different intrinsic healing potential 2. In the last decades, several surgical treatment options have emerged and have already been clinically established 3-6.
Autologous or allogeneic osteochondral transplants consist of articular cartilage and subchondral bone and allow the replacement of the entire osteochondral unit. The defects are filled with cylindrical osteochondral grafts that aim to provide a congruent hyaline cartilage covered surface 3,7,8. Disadvantages are the limited amount of available grafts, donor site morbidity (for autologous transplants) and the incongruence of the surface; thereby the application of this method is especially limited for large defects.
New approaches in the field of tissue engineering opened up promising possibilities for regenerative osteochondral therapy. The implantation of autologous chondrocytes marked the first cell based biological approach for the treatment of full-thickness cartilage lesions and is now worldwide established with good clinical results even 10 to 20 years after implantation 9,10. However, to date, this technique is not suitable for the treatment of all types of lesions such as deep defects involving the subchondral bone 11.
The sandwich-technique combines bone grafting with current approaches in Tissue Engineering 5,6. This combination seems to be able to overcome the limitations seen in osteochondral grafts alone. After autologous bone grafting to the subchondral defect area, a membrane seeded with autologous chondrocytes is sutured above and facilitates to match the topology of the graft with the injured site. Of course, the previous bone reconstruction needs additional surgical time and often even an additional surgery. Moreover, to date, long-term data is missing 12.
Tissue Engineering without additional bone grafting aims to restore the complex structure and properties of native articular cartilage by chondrogenic and osteogenic potential of the transplanted cells. However, again, it is usually only the cartilage tissue that is more or less regenerated. Additional osteochondral damage needs a specific further treatment. In order to achieve a regeneration of the multilayered structure of osteochondral defects, three-dimensional tissue engineered products seeded with autologous/allogeneic cells might provide a good regeneration capacity 11.
Beside autologous chondrocytes, mesenchymal stem cells (MSC) seem to be an attractive alternative for the development of a full-thickness cartilage tissue. In numerous preclinical in vitro and in vivo studies, mesenchymal stem cells have displayed excellent tissue regeneration potential 13,14. The important advantage of mesenchymal stem cells especially for the treatment of osteochondral defects is that they have the capacity to differentiate in osteocytes as well as chondrocytes. Therefore, they potentially allow a multilayered regeneration of the defect.
In recent years, several scaffolds with osteochondral regenerative potential have therefore been developed and evaluated with promising preliminary results 1,15-18. Furthermore, fibrin glue as a cell carrier became one of the preferred techniques in experimental cartilage repair and has already successfully been used in several animal studies 19-21 and even first human trials 22.
The following protocol will demonstrate an experimental technique for isolating mesenchymal stem cells from a rabbit's bone marrow, for subsequent proliferation in cell culture and for preparing a standardized in vitro-model for fibrin-cell-clots. Finally, a technique for the implantation of pre-established fibrin-cell-clots into artificial osteochondral defects of the rabbit's knee joint will be described.

An experimental technique for the treatment of osteochondral defects in the rabbit's knee joint is described. The implantation of allogeneic mesenchymal stem cells into osteochondral defects provides a promising development in the field of tissue engineering. The preparation of fibrin-cell-clots in vitro offers a standardized method for implantation.

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular ...

Medicine

Isolation and Enrichment of Rat Mesenchymal Stem Cells (MSCs) and Separation of Single-colony Derived MSCs

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and can be easily separated from the hematopoietic stem cells (HSCs) due to their plastic adherence. This protocol describes how to isolate MSCs from rat femurs and tibias. The isolated cells were further enriched against two MSCs surface markers CD54 and CD90 by magnetic cell sorting. Expression of surface markers CD54 and CD90 were then confirmed by flow cytometry analysis. HSC marker CD45 was also included to check if the sorted MSCs were depleted of HSCs. MSCs are naturally quite heterogeneous. There are subpopulations of cells that have different shapes, proliferation and differentiation abilities. These subpopulations all express the known MSCs markers and no unique marker has yet been identified for the different subpopulations. Therefore, an alternative approach to separate out the different subpopulations is using cloning cylinders to separate out single-colony derived cells. The cells derived from the single-colonies can then be cultured and evaluated separately.

Isolation and Enrichment of Rat Mesenchymal Stem Cells MSCs and Separation of Single-colony Derived MSCs

Rat MSCs were isolated from femurs and tibias and then enriched by magnetic cell sorting. Sorted cells were confirmed for the expression of surface markers by flow cytometry. These cells were also cultured at clonal density to form single colonies and then these colonies were separated by cloning cylinders.

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and ...

Biology

Mesenchymal Stromal Cell Culture and Delivery in Autologous Conditions: A Smart Approach for Orthopedic Applications

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on potential biohazard and inflammation risks exerted by xenogeneic nutrients for their culture. Human derivatives or recombinant materials are the first choice candidates to reduce these reactions. Therefore, culture supplements and materials of autologous origin represent the best nutrients and the safest products.
Here, we describe a new protocol for the isolation and culture of bone marrow hMSCs in autologous conditions &#8212;&#160;namely,&#160;patient-derived serum as a supplement for the culture medium and fibrin as a scaffold for hMSC administration. Indeed, hMSC/fibrin clot constructs could be extremely useful for several clinical applications. In particular, we focus on their use in orthopedic surgery, where the fibrin clot derived from the donor&#39;s own blood allowed effective cell delivery and nutrient/waste exchanges. To ensure optimal safety conditions, it is of the utmost importance to avoid the risks of hMSC transformation and tissue overgrowth. For these reasons, the approach described in this paper also indicates a minimally ex vivo hMSC expansion, to reduce cell senescence and morphologic changes, and short-term osteo-differentiation before implantation, to induce osteogenic lineage specification, thus decreasing the risk of subsequent uncontrolled proliferation.

Culturing human Mesenchymal Stromal Cells (hMSCs) with autologous serum, reduces the risks of rejection by xenogeneic material and other negative effects. It also allows for the recovery of a subset of mesodermal progenitors, which can deliver fresh hMSCs. Embedding hMSCs in an autologous fibrin clot enables easy handling and effective surgical implantation.

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on ...

Bioengineering

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates after injection into injured tissues as well as the observed massive cell death rates lead to very restricted therapeutic effects. To overcome these limitations, we sought to establish a non-viral based protocol for suitable cell engineering prior to their administration. The modification of human CD133+ expressing SCs using microRNA (miR) loaded magnetic polyplexes was addressed with respect to uptake efficiency and safety as well as the targeting potential of the cells. Relying on our protocol, we can achieve high miR uptake rates of 80&#8211;90% while the CD133+ stem cell properties remain unaffected. Moreover, these modified cells offer the option of magnetic targeting. We describe here a safe and highly efficient procedure for the modification of CD133+ SCs. We expect this approach to provide a standard technology for optimization of therapeutic stem cell effects and for monitoring of the administered cell product via magnetic resonance imaging (MRI).

This protocol illustrates a safe and efficient procedure to modify CD133+ hematopoietic stem cells. The presented non-viral, magnetic polyplex-based approach may provide a basis for the optimization of therapeutic stem cell effects as well as for monitoring the administered cell product via magnetic resonance imaging.

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

An Enzyme-free Method for Isolation and Expansion of Human Adipose-derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. As a clinically relevant cell type, optimal methods are needed to isolate and expand these cells in vitro. Most methods to isolate adipose-derived MSCs (ADSCs) rely on harsh enzymes, such as collagenase, to digest the adipose tissue. However, while effective at breaking down the adipose tissue and yielding a high ADSC recovery, these enzymes are expensive and can have detrimental effect on the ADSCs &#8212; including the risks of using xenogeneic components in clinical applications. This protocol details a method to isolate ADSCs from fresh lipoaspirate and abdominoplasty samples without enzymes. Briefly, this method relies on mechanical disassociation of any bulk tissue followed by an explant-type culture system. The ADSCs are permitted to migrate out of tissue and onto the tissue culture plate, after which the ADSCs can be cultured and expanded in vitro for any number of research and/or clinical applications.

This protocol provides an enzyme-free method for isolating mesenchymal stem cells from abdominoplasty and lipoaspirate samples using an explant method. The absence of harsh enzymes or centrifugation steps provides for clinically relevant stem cells that can be used for studies in vitro or transferred back to the clinic.

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Optimizing Stromal Cell Isolation from Thymus and Bone Marrow

Stromal Cell Isolation From Hematopoietic Organs

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens through which to study previously unresolved heterogeneity at steady state, as well as changes in cell type representation induced by extrinsic stresses or during aging. Here, we present step-wise protocols for the isolation of high-quality stromal cell populations from murine and human thymus, as well as murine bone and bone marrow. Cells isolated through these protocols are suitable for generating high-quality single-cell multiomics datasets. The impacts of sample digestion, hematopoietic lineage depletion, FACS analysis/sorting, and how these factors influence compatibility with single-cell sequencing are discussed here. With examples of FACS profiles indicating successful and inefficient dissociation and downstream stromal cell yields in post-sequencing analysis, recognizable pointers for users are provided. Considering the specific requirements of stromal cells is crucial for acquiring high-quality and reproducible results that can advance knowledge in the field.

Author Spotlight: Advancing Hematopoietic Research Using Stromal Cell Isolation for Single Cell Sequencing

Here we present protocols that enable isolation of stromal cells from murine bone, bone marrow, thymus and human thymic tissue compatible with single-cell multiomics.

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens ...

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

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