En enzymatisk metode til at redde mesenchymale stamceller fra Clotted knoglemarvsprøver
Summary
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.
Abstract
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 – 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.
Introduction
Mesenkymale stamceller (MSC'er) spiller en vigtig rolle i regenerativ medicin og vævsmanipulering. De kan migrere, differentiere i forskellige celletyper 1 og indpode, hvilket gør dem ideelle kandidater til autologe behandlinger 2,3. Sidst, kliniske undersøgelser med MSC'er for knogle og brusk reparation, graft versus host-sygdom eller hjertesygdom blev iværksat 4. Disse MSC'erne kan høstes fra navlestrengen eller fedtvæv, men mest lovende resultater blev opnået fra knoglemarv stamceller 5.
Hoftebenskammen gør det muligt at indsamle en betydelig mængde knoglemarv og tjener derfor som vigtigste sted for aspiration 6. Men kvaliteten af aspirat falder med stigende mængde knoglemarv trukket tilbage. Mens de første 5 ml knoglemarvsaspirat indeholder MSC'er af høj kvalitet, tilbagetrækning af større mængder fører til fortynding af aspirat med perifert blod from den meget vaskulariserede knogle 7. På grund af de nuværende megakaryocytter og blodplader, knoglemarvsaspirater er tilbøjelige til at størkne, medmindre der anvendes antikoagulanter. Men selv med antikoagulantia, kan der forekomme blodpropper.
I knoglemarven, udgør MSC'er kun en lille del af den samlede celle pool 8 og skal udvides i kultur i de fleste væv ingeniør eller terapeutiske anvendelser 4. Kvaliteten af en sådan kultur i høj grad afhænger af den oprindelige celle pool, dvs.., Mangfoldighed og et højt udgangspunkt nummer 9. Lavt antal af MSC'er fra tilbagekøb kan delvis forklares med donor variabilitet. På den anden side, MSC'er fra prøver lav kvalitet kræve længere tid i kultur og udvidet passage for at nå det ønskede antal celler. I begge tilfælde udvides passage er en kilde til celleældning og kan føre til tab af differentiering potentiale 10. Derfor optimerede protokoller, som kan maksimere celle yield og forhindre skadelige virkninger skal udvikles 11,12.
Da vi begyndte at arbejde med hunde MSC, vi var overrasket over at se, at omkring tre ud af fire hunde knoglemarv prøver indeholdt blodpropper, mens heldigvis størknet humane prøver (en i ti) var mindre hyppige. På den anden side var det ikke overraskende, at vi observeret meget lavere udbytter af MSC'er fra koagulerede prøver. For at løse tilbagevendende spørgsmål om koagulerede prøver, vi har udviklet protokollen ved hjælp af trombolytisk lægemiddel urokinase stedet for resampling.
Thrombolytiske terapier kan modvirke livstruende situationer såsom okklusion af blodkar forårsager hjerteanfald, slagtilfælde eller embolier grund af uønsket koagulation. De virker ved nedbrydning af blodpropper ved enzymatisk spaltning af fibrin med plasmin og enzymatisk plasminogenaktivatorer. På trods af den udbredte anvendelse til behandling af patienter, der findes kun ganske få publikationer, udnyttet thrombolytiske aktivitetertil laboratoriebrug at redde koagulerede prøver, for det meste med fokus på lymfocytter. I 1987 Niku et al. beskrev brugen af streptokinase for at opløse blodpropper resulterer i funktionelle lymfocytter 13 og fire år senere, de Vis et al. udvidet anvendelse af streptokinase at isolere leukæmiceller fra blod og knoglemarv til flowcytometrisk applikationer 14. En nyere publikation foreslår anvendelsen af Alteplase for kræftdiagnostik 15. Mens du bruger den samme enzymatiske metode, vores protokol fokuserer på isoleringen af multipotent MSC'er formular knoglemarv til at give et værktøj til forskere på området stamcelle.
Protocol
Representative Results
Discussion
Rutinemæssigt vi prøve knoglemarv, mens patienten er i kirurgi (i vores tilfælde hovedsagelig rygkirurgi), med den fordel, at kun lidt ekstra arbejde der skal udføres af personalet i driften teater. Selvom prøverne blandes med natriumcitrat umiddelbart efter tilbagetrækningen, mange prøver var delvis størknet, da de ankom i laboratoriet til forarbejdning. På dette stadium, vil resampling at erstatte koagulerede prøver være en separat ekstra indgreb nødvendiggør igen lokale eller generelle anæstesi 6.<…
Divulgations
The authors have nothing to disclose.
Acknowledgements
This work was supported by the Swiss National Foundation Grant CR3I3_140717/1 and the Swiss Paraplegic Foundation.
Materials
| Basal Medium Components | ||
| PenStrep 100X | Gibco | 15140122 |
| Human FGF-basic | Peprotech | 100-18B |
| MEM Alpha w/ Nucleoside, w/ stable Glutamine | Amimed | 1-23S50-I |
| FBS Heat Inactivated | Amimed | 2-01F36-I |
| Amphotericin B | Applichem | A1907 |
| Adipogenic Medium Components | ||
| DMEM-HAM F12 + GlutaMAX | Amimed | 1-26F09-I |
| Insulin | Sigma | I5500 |
| Rabbit serum | Gibco | 16120099 |
| Dexamethasone | Applichem | D4902 |
| 3-Isobutyl-1-methylxanthine | Sigma | I5879 |
| Biotin | Sigma | B4639 |
| Rosiglitazone | Sigma | R2408 |
| Pantothenate | Sigma | P5155 |
| Oil Red-O | Sigma | O0625 |
| Osteogenic Medium Components | ||
| L-ascorbic acid 2-phosphate | Sigma | A8960 |
| ß-glycerophosphate | Sigma | G9422 |
| Silver nitrate (AgNO3) | Sigma | S6506 |
| Chondrogenic Medium Components | ||
| Biopad – sponge shaped medical device | Euroresearch | |
| L-proline | Sigma | P5607 |
| Insulin-Transferrin-Selenium X | Gibco | 51500056 |
| Human transforming growth factor-β1 | Peprotech | 100-21 |
| Alcian Blue 8GX | Sigma | A3157 |
| Nuclear fast red | Sigma | N8002 |
| Generic | ||
| Tri-Sodium citrate dihydrate | Applichem | A3901 |
| PBS | Applichem | 964.9100 |
| Urokinase | Medac | 1976826 |
| 0.5% Trypsin-EDTA | Gibco | 15400054 |
| Giemsa stain | Applichem | A0885 |
| Formaldehyde | Applichem | A0877 |
| Sulfuric acid (H2SO4) | Applichem | A0655 |
| Dimethyl sulfoxide (DMSO) | Applichem | A1584 |
| Magnesium chloride (MgCl2) | Applichem | A3618 |
| Guanidine hydrochloride | Applichem | A1499 |
| Consumables | ||
| 50 mL reaction tube | Axygen | SCT-50ML-25-S |
| 10 mL syringe | Braun | 4606108V |
| Sterican needle (22G) | Braun | 4657624 |
| 1.7 mL Microtubes | Brunschwig | MCT-175-C |
| 100 μm cell strainer | Falcon | 6.05935 |
| sterile forceps | Bastos Viegas, SA | 489-001 |
| sterile scalpel | Braun | 5518059 |
| Primaria cell cuture dish | Falcon | 353803 |
| C-Chip Neubauer Improved | Bioswisstech | 505050 |
| cell culture flask – Flask T300 | TPP | 90301 |
| Equipment | ||
| Microbiological biosafety cabinet class II | Skan | 82011500 |
| water bath | Memmert | 1305.0377 |
| Stripettes Serological Pipette 5ml | Corning | 4487-200ea |
| microscope | Olympus | CKX41 |
| humidified incubator Heracells 240 | Thermo scientific | 51026331 |
| Heraeus Multifuge 1S-R | Thermo scientific | 75004331 |
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