Isolering og Kryopræservering af neonatal rottecardiomyocytter
Summary
The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time.
Abstract
Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is difficult and time consuming. The most commonly used cells are neonatal rat cardiomyocytes (NRCMs), which require isolation every time cells are needed. The birth of the rats can be unpredictable. Cryopreservation is proposed to allow for cells to be stored until needed, yet freezing/thawing methods for primary cardiomyocytes are challenging due to the sensitivity of the cells. Using the proper cryoprotectant, dimethyl sulfoxide (DMSO), cryopreservation was achieved. By slowly extracting the DMSO while thawing the cells, cultures were obtained with viable NRCMs. NRCM phenotype was verified using immunocytochemistry staining for α-sarcomeric actinin. In addition, cells also showed spontaneous contraction after several days in culture. Cell viability after thawing was acceptable at 40-60%. In spite of this, the methods outlined allow one to easily cryopreserve and thaw NRCMs. This gives researchers a greater amount of flexibility in planning experiments as well as reducing the use of animals.
Introduction
Cellekultur af cardiomyocytter er et kritisk værktøj i moderne hjerte- forskning. Nyfødte rottecardiomyocytter (NRCMs) er almindeligt brugt siden isolation og kultur er lettere end voksne rottecardiomyocytter 1. Den NRCM metode stadig har flere begrænsninger, herunder en lang isolation procedure og begrænset celleproliferation i skålen. Der er mange protokoller til isolering af NRCMs med mest almindeligt kræver 4-48 timer af arbejde 2-6. Derudover er cellerne ofte isoleret fra 1 til 2 dage gamle rotteunger 2,4-7; timingen af fødslen kan være uforudsigelig og konflikt med andet arbejde i laboratoriet. Isoleringen kan være ineffektiv og uøkonomisk, hvis kun en lille mængde af celler er nødvendige for eksperimenter. Bestræbelserne på at forbedre arbejdsgangen fokus på at reducere isolation tid, men dette løser ikke problemerne med timing fødslen af hvalpene.
Som alternativer mange laboratorier anvendercardiomyocytter afledt af embryonale stamceller (EKSF) eller inducerede pluripotente stamceller (iPSCs). Imidlertid kan omprogrammering og / eller differentiering proces være meget tidskrævende og dyre samt. Der kan være andre problemer, når du bruger disse celler som in vitro myocyt modeller. Både ESC og iPSC- afledte cardiomyocytter har vist sig at udvise forskelle i elektrofysiologi fra primære cardiomyocytter 8-10.
Dissocierede NRCMs er i stand til at blive lagret i flere dage ved hjælp af køling 11, men dette giver mulighed for langtidsopbevaring. Flydende nitrogen anvendes typisk til at bevare celler for en større tidsperiode, men kræver et kryobeskyttelsesmiddel såsom dimethylsulfoxid (DMSO). Tidligere forskning har vist, at den ideelle koncentration mellem 5-10% DMSO i indefrysning medier giver mulighed for nedfrysning af NRCMs, men selv da levedygtigheden fortsat lav 12. Selvom DMSO hjælper med at beskytte cellerne ved frizing, kan det være toksisk for celler ved koncentrationer over 1,5% 13. Tidligere undersøgelser har vist, at langsomt at fjerne DMSO fra cellerne, kan forbedre cellelevedygtighed 14.
Vi forsøgte at forbedre effektiviteten af NRCM cellebaserede assays ved kryopræservering cellerne efter isolation. Dette gør det muligt for cellerne, der skal optøs og anvendes efter behov, hvilket reducerer hyppigheden af isolationer og forbrug af dyr. Ved hjælp af denne metode, viser vi, at det er muligt at kryopræservere NRCMs og tø dem til brug på et senere tidspunkt. Efter optøning af cellerne opretholde en acceptabel rentabilitet og producere NRCM kulturer, der er positive for α-sarcomerisk actinin (α-SA) og kontrakt spontant.
Protocol
Representative Results
Discussion
Denne protokol giver mulighed for NRCMs skal isoleres, kryopræserveret og optøet. Optøning af celler er en afgørende del af proceduren. En række DMSO fortyndinger anvendes til langsomt at fjerne DMSO fra cellerne 14. Det er vigtigt, at udvindingen af DMSO udføres hurtigt som cellerne er særligt følsomme over for at dø umiddelbart efter optøning. Hvis der kræves mere eller mindre celler til optøning, kan mængden af DMSO løsninger skaleres efter behov.
En u…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by funding from American Heart Association 12BGIA12040477, NC State University Chancellor’s Faculty Excellence Program, and National Natural Science Foundation of China H020381370216.
Materials
| IMDM (+25mM HEPES +L-glutamine) | Gibco | 12440-046 | With added 25mM HEPES and L-glutamine |
| L-glutamine | Gibco | 25030-081 | |
| FBS | Hyclone | SH30070.03 | |
| Gentamicin | Gibco | 15710-064 | |
| 2-Mercaptoethanol | Gibco | 21985-023 | |
| HBSS (+Ca +Mg) | Corning | 21-020-CV | With added calcium and magnesium, pH 7.1-7.4 |
| Trypsin 0.25% | Gibco | 25300-056 | |
| Trypsin 0.05% | Gibco | 25300-054 | |
| Cryostor CS5 | BioLife Solutions | 205102 | Freezing media |
| Cryogenic Vial | Corning | 430659 | |
| Collagenase | Sigma | C1889-50MG | |
| 40μm Cell Strainer | Greiner Bio-One | 542040 | |
| Sterilizing Vacuum Filter (0.22μm) | Corning | 431118 | |
| 50mL Conical | Corning | 430828 | |
| 15mL Conical | Corning | 430790 | |
| trypan blue | Cellgro | 25-900-CI | |
| Mr Frosty Freezing Container | ThermoScientific | 5100-0001 | |
| Millicell EZ SLIDES | Millipore | PEZGS0416 | |
| α-sarcomeric actinin antibody | Sigma | A7811 | |
| Fibronectin | Corning | 356008 | |
| Bromodeoxyuridine | BD Biosciences | 51-7581KZ |
References
- Louch, W. E., Sheehan, K. Methods in cardiomyocyte isolation, culture, and gene transfer. Journal of Molecular and Cellular Cardiology. 51 (3), 288-298 (2011).
- Miragoli, M., Salvarani, N., Rohr, S. Myofibroblasts Induce Ectopic Activity in Cardiac Tissue. Circulation Research. 101, 755-758 (2007).
- Simpson, P., Savion, S. Differentiation of rat myocytes in single cell cultures with and without proliferating nonmyocardial cells. Cross-striations, ultrastructure, and chronotropic response to isoproterenol. Circulation Research. 50 (1), 101-116 (1982).
- Simpson, P., McGrath, a., Savion, S. Myocyte hypertrophy in neonatal rat heart cultures and its regulation by serum and by catecholamines. Circulation Research. 51 (6), 787-801 (1982).
- Rohr, S., Flückiger-Labrada, R., Kucera, J. P. Photolithographically defined deposition of attachment factors as a versatile method for patterning the growth of different cell types in culture. Pflügers Archive : European Journal of Physiology. 446 (1), 125-132 (2003).
- Zhang, B., Green, J. V., Murthy, S. K., Radisic, M. Label-free enrichment of functional cardiomyocytes using microfluidic deterministic lateral flow displacement. PloS One. 7 (5), e37619 (2012).
- Boateng, S. Y., Hartman, T. J., Ahluwalia, N., Vidula, H., Desai, T. Inhibition of fibroblast proliferation in cardiac myocyte cultures by surface microtopography. American Journal of Physiology. Cell Physiology. 285 (1), C171-C182 (2003).
- Fu, J. -. D., Li, J., et al. Crucial role of the sarcoplasmic reticulum in the developmental regulation of Ca2+ transients and contraction in cardiomyocytes derived from embryonic stem cells. FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology. 20 (1), 181-183 (2006).
- Liu, J., Fu, J. D., Siu, C. W., Li, R. a Functional sarcoplasmic reticulum for calcium handling of human embryonic stem cell-derived cardiomyocytes: insights for driven maturation. Stem Cells. 25 (12), 3038-3044 (2007).
- Knollmann, B. C. Induced pluripotent stem cell-derived cardiomyocytes: boutique science or valuable arrhythmia model. Circulation Research. 112 (6), 969-976 (2013).
- Uchida, T., Nagayama, M., Taira, T., Shimizu, K., Sakai, M., Gohara, K. Optimal temperature range for low-temperature preservation of dissociated neonatal rat cardiomyocytes. Cryobiology. 63 (3), 279-284 (2011).
- Miyamura, K., Nagayama, M., et al. Evaluation of viability of cryopreserved rat cardiac myocytes and effects of dimethyl sulfoxide concentration on cryopreservation. Cryobiology. 59 (3), 375 (2010).
- Rana, P., Anson, B., Engle, S., Will, Y. Characterization of human-induced pluripotent stem cell-derived cardiomyocytes: bioenergetics and utilization in safety screening. Toxicological Sciences: An Official Journal of the Society of Toxicology. 130 (1), 117-131 (2012).
- Yokomuro, H., Mickle, D. a. G., Weisel, R. D., Li, R. -. K. Optimal conditions for heart cell cryopreservation for transplantation. Molecular and Cellular Biochemistry. 242 (1-2), 109-114 (2003).
- Simpson, P., McGrath, a., Savion, S. Myocyte hypertrophy in neonatal rat heart cultures and its regulation by serum and by catecholamines. Circulation Research. 51 (6), 787-801 (1982).
- Evans, H. J., Western Goodwin, R. L. array analysis of cell cycle protein changes during the hyperplastic to hypertrophic transition in heart development. Molecular and Cellular Biochemistry. 303 (1-2), 189-199 (2007).
- Golden, H. B., Gollapudi, D., et al. Methods in Molecular Biology. , 205-214 (2012).
- Zlochiver, S., Muñoz, V., Vikstrom, K. L., Taffet, S. M., Berenfeld, O., Jalife, J. Electrotonic myofibroblast-to-myocyte coupling increases propensity to reentrant arrhythmias in two-dimensional cardiac monolayers. Biophysical Journal. 95 (9), 4469-4480 (2008).
- Chang, M. G., Zhang, Y., et al. Spiral waves and reentry dynamics in an in vitro model of the healed infarct border zone. Circulation Research. 105 (11), 1062-1071 (2009).
