酶法从凝块骨髓样本拯救间充质干细胞
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
间充质干细胞（MSCs）起到在再生医学和组织工程的主要作用。他们可以迁移，分化成各种细胞类型1和嫁接，这使得他们的理想人选自体疗法2,3。最近，利用干细胞的骨和软骨修复的临床试验，移植物抗宿主病或心脏疾病患者推出了4款。这些干细胞可以从脐带或脂肪组织收获，但最有希望的结果，从骨髓来源的干细胞5中得到。
髂嵴允许收集了相当数量的骨髓，因此作为吸6主站点。然而，抽吸质量而增加骨髓抽出的体积减小。而第一5毫升骨髓抽吸的含有高品质的MSCs，撤回较大体积导致稀释外周血f显示抽吸的ROM的高度血管骨7。因为本巨核细胞和血小板，骨髓抽吸容易凝结，除非抗凝剂被使用。但即使有抗凝血剂，可能发生血栓。
在骨髓，干细胞代表总细胞池8的只有一小部分，并且必须在培养对于大多数组织工程或治疗应用4展开。这种文化的质量在很大程度上取决于初始细胞库， 即 ，多样性和高起点9号。从取款低数量的干细胞可以通过捐助变异的部分原因。另一方面，从低质量样品的MSC需要更长的时间，培养和传代扩大到达细胞的期望数量。在这两种情况下，扩展的传代是细胞衰老的来源，并可能导致分化潜能10的损失。因此，优化的协议，可以最大限度地提高小区Y屈服和防止有害作用必须开发11,12。
当我们开始与犬间充质干工作，我们惊讶地看到，约四分之三的犬骨髓样本中含有血块，而幸运的是凝结人体样本（十分之一）不太频繁。在另一方面这是毫不奇怪，我们观察到凝血样本的MSCs低得多的产量。为了解决凝血样本的反复出现的问题，我们使用重采样的溶栓药物尿激酶，而不是开发的协议。
溶栓疗法可以抵消危及生命的情况下，如血管造成心脏发作，中风或因不必要的凝血栓塞闭塞。它们通过纤维蛋白溶酶由酶和纤维蛋白溶酶原活化剂的酶裂解的工作由凝块的降解。尽管广泛使用的治疗患者中，只有极少数出版物存在使用溶栓活动实验室应用抢救凝结样品，主要是着眼于淋巴细胞。 1987年，尼库等 。描述了使用链激酶溶解导致功能性淋巴细胞13和四年后血块，可见德等人 。延长的使用链激酶从血液和骨髓供流式细胞应用14分离白血病细胞。最近的一个出版物建议使用阿替普酶的癌症诊断15。而使用相同的酶的方法，我们的协议的重点是多能间充质干形式的骨髓中分离，以提供对研究人员在干细胞领域的工具。
Protocol
Representative Results
Discussion
常规我们采样骨髓当患者接受手术（在我们的情况下，主要的脊柱外科手术），条件是只有很少的额外的工作，必须进行的人员在手术室的优点。即使将样品撤出后立即用柠檬酸钠混合，许多样品时，他们在实验室中进行处理到达被部分凝结。在此阶段，重采样，以取代凝块标本将是一个独立的额外介入再次迫使局部或全身麻醉6。这需要双方的医护人员和捐助的意愿作出贡献，并消?…
Disclosures
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 |
References
- Jiang, Y., et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature. 418 (6893), 41-49 (2002).
- Uccelli, A., Moretta, L., Pistoia, V. Mesenchymal stem cells in health and disease. Nat Rev Immunol. 8 (9), 726-736 (2008).
- Bartholomew, A., et al. Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp Hematol. 30 (1), 42-48 (2002).
- Wang, S., Qu, X., Zhao, R. C. Clinical applications of mesenchymal stem cells. J Hematol Oncol. 5, 19 (2012).
- Baksh, D., Song, L., Tuan, R. S. Adult mesenchymal stem cells: characterization, differentiation, and application in cell and gene therapy. J Cell Mol Med. 8 (3), 301-316 (2004).
- Malempati, S., Joshi, S., Lai, S., Braner, D. A., Tegtmeyer, K. Videos in clinical medicine. Bone marrow aspiration and biopsy. N Engl J Med. 361 (15), e28 (2009).
- Cuthbert, R., et al. Single-platform quality control assay to quantify multipotential stromal cells in bone marrow aspirates prior to bulk manufacture or direct therapeutic use. Cytotherapy. 14 (4), 431-440 (2012).
- Veyrat-Masson, R., et al. Mesenchymal content of fresh bone marrow: a proposed quality control method for cell therapy. Br J Haematol. 139 (2), 312-320 (2007).
- Lazarus, H. M., Haynesworth, S. E., Gerson, S. L., Rosenthal, N. S., Caplan, A. I. Ex vivo expansion and subsequent infusion of human bone marrow-derived stromal progenitor cells (mesenchymal progenitor cells): implications for therapeutic use. Bone Marrow Transplant. 16 (4), 557-564 (1995).
- Bertolo, A., et al. An in vitro expansion score for tissue-engineering applications with human bone marrow-derived mesenchymal stem cells. J Tissue Eng Regen Med. , (2013).
- Casiraghi, F., Remuzzi, G., Abbate, M., Perico, N. Multipotent mesenchymal stromal cell therapy and risk of malignancies. Stem Cell Rev. 9 (1), 65-79 (2013).
- Bonab, M. M., et al. Aging of mesenchymal stem cell in vitro. BMC Cell Biol. 7, 14 (2006).
- Niku, S. D., Hoon, D. S., Cochran, A. J., Morton, D. L. Isolation of lymphocytes from clotted blood. J Immunol Methods. 105 (1), 9-14 (1987).
- De Vis, J., Renmans, W., Segers, E., Jochmans, K., De Waele, M. Flow cytometric immunophenotyping of leukemia cells in clotted blood and bone marrow. J Immunol Methods. 137 (2), 193-197 (1991).
- St Antoine, A., et al. Application of thrombolytic drugs on clotted blood and bone marrow specimens to generate usable cells for cytogenetic analyses. Arch Pathol Lab Med. 135 (7), 915-919 (2011).
- Kisiel, A. H., et al. Isolation, characterization, and in vitro proliferation of canine mesenchymal stem cells derived from bone marrow, adipose tissue, muscle, and periosteum. Am J Vet Res. 73 (8), 1305-1317 (2012).
- Bertolo, A., et al. Influence of different commercial scaffolds on the in vitro differentiation of human mesenchymal stem cells to nucleus pulposus-like cells. Eur Spine J. 21, S826-S838 (2012).
- Makridakis, M., Roubelakis, M. G., Vlahou, A. Stem cells: insights into the secretome. Biochim Biophys Acta. 1834 (11), 2380-2384 (2013).
- Benvenuti, S., et al. Rosiglitazone stimulates adipogenesis and decreases osteoblastogenesis in human mesenchymal stem cells. J Endocrinol Invest. 30 (9), RC26-RC30 (2007).
- Jaiswal, N., Haynesworth, S. E., Caplan, A. I., Bruder, S. P. Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro. J Cell Biochem. 64 (2), 295-312 (1997).
- Patel, A. Tissue banking for research–bench to bedside and back–myth, reality or fast fading reality at the dawn of a personalised healthcare era. Cell Tissue Bank. 12 (1), 19-21 (2011).
- Smith, H. W., Marshall, C. J. Regulation of cell signalling by uPAR. Nat Rev Mol Cell Biol. 11 (1), 23-36 (2010).
