REJUVENATION RESEARCH Volume 17, Number 2, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/rej.2013.1488

A Fast and Mild Decellularization Protocol for Obtaining Extracellular Matrix Ariana Mirzarafie, Rhian K. Grainger, Ben Thomas, William Bains, Fatma I. Ustok, and Chris R. Lowe

Abstract

Degradation of extracellular matrix (ECM) function with age is a major cause of loss of tissue function with age that we would wish to reverse. Tissue engineering to provide replacement tissue requires an ECM-mimicking scaffold for cell organization. The standard protocols for achieving this take 10 days and include steps that may change the protein structure of the ECM. Here we describe a much shorter protocol for decellularizing chicken muscle, skin, and tendon samples that achieves the same efficiency as the original protocol without protein cross-link interference. Our protocol can be completed in 72 hr.

DNA, so it can be expected that 100% decellularization is not achieved by present methods.6,9

Introduction

A

ging of the extracellular matrix (ECM), the protein matrix that surrounds and penetrates the tissues and binds the body together, contributes significantly to functional aging of tissues. Aging of the ECM affects the mechanical properties of tissues as well as the biology of the cells within the matrix (for review, see ref. 1) Enzymic cross-linking2–4 and non-enzymic glycation5 all contribute to changes in ECM function and are targets for therapies for the diseases and disabilities of age. To identify the contribution of ECM aging to overall tissue aging, and to test cross-link breaker therapeutics, a quick, simple method of preparing ECM is needed. Specifically, the method needs to preserve all the major extracellular proteins in the ECM, remove the large majority of cellular proteins, not involve low pH conditions or oxidizing chemicals (such as those used in the rapid methods of (Gilbert et al. 20096) that might change the cross-link densities in the ECM, and be applicable to a wide range of tissues, including tissues such as cartilage and bone, which are difficult to manipulate into thin sheets. This article describes the development of such a method. We started with the ECM preparation method of Lang et al. 2011.7 This protocol was reported to remove up to 93% of cellular components, while preserving collagens I, III, and IV, proteoglycans, glycosaminoglycans, fibronectin, elastin, and laminin of the ECM. The purpose of the protocol was to have a facile method of preparing ECM for chemical analysis, and not to remove all traces of cellular immunogens for transplant applications.8 Most commercial ECM preparations retain some

Methods

Sainsbury’s chicken breast muscle tissue samples of up to 1 cm3 were prepared, weighed, and decellularized according to an initial optimized protocol based on an older protocol,7 which involved three buffer changes over 10 days (2% vol/vol Triton-X-100 for 3 days, 0.1% wt/vol sodium dodecyl sulfate [SDS]/0.1 M NaCl for 5 days, and deionized (DI) H2O for 3 days). Samples were collected each day, and their DNA concentration and residual enzyme activity were measured (QIAGEN DNA Blood & Tissue kit straight away, Invitrogen Quant-iT DNA kit, and the MTT assay once all samples were taken after 10 days) to determine the extent of decellularization. On the basis of those results, a shorter protocol was designed by omitting the apparently insignificant steps. Skin and tendon tissue samples of up to 1 cm3 were prepared, weighed, and shaken at 4C in 2% (vol/vol) Triton-X-100 buffer (roughly 10 mL of buffer for every gram of tissue for all buffers) for 24 hr, changing the buffer after the first 4 hr, then again after 4 hr. After 24 hr, a part of the samples was retrieved and stored at 0C for subsequent analysis, the buffer was changed to 0.1% (wt/vol) SDS, 0.1 M NaCl, and the rest of the samples returned to shaking at 4C for another 24 hr, after which another part of the samples was taken. Finally, the SDS/NaCl buffer was replaced by deionized water, and the samples were returned to 4C shaking for 24 hr. Then all of the samples were retrieved and underwent a DNA assay to assess remaining DNA concentration and the MTT assay to measure residual enzyme activity. Finally,

Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, United Kingdom.

159

160

MIRZARAFIE ET AL.

FIG. 1. Extent of decellularization of tendon samples over 3 days using the new protocol. (A) DNA concentration over 3 days. (B) Residual dehydrogenase activity over 3 days. DNA concentration and enzyme activity were measured and used to determine the extent of decellularization in chicken tendon samples before decellularization and every day thereafter using the new protocol. Both measures support the *90% cell loss achieved with previous protocols. a control and third-day muscle samples were examined by microscopy after Methylene Blue staining.

Author Disclosure Statement

Results

References

In tendon samples, a 94% ( – 2.9) decrease in DNA concentration was observed by the third day using the new protocol, which was virtually identical to the decrease observed by the tenth day using the old protocol on the much less tough muscle samples. Using the new protocol, skin and muscle samples lost a comparable concentration of DNA (Fig. 1). The MTT assay showed a similar trend for tendon samples using the new protocol, as 89% of residual dehydrogenase activity was lost by the third day, and 78% was lost as early as after 24 hr. Visual analysis of the nuclear staining with Methylene Blue of muscle samples confirmed the extensive decellularization, because virtually no nuclei were present in the third-day decellularized sample, in stark contrast with the control sample. Discussion

All measures of decellularization (DNA concentration, residual dehydrogenase activity, and nuclear staining) confirm the efficiency of the new protocol at removing cellular content of muscle, skin, and tendon tissue samples while maintaining the ECM scaffold. The result is practically cell-free tissue that can be used in chemical analysis and facilitate enzyme-linked immunosorbent assay (ELISA) for extracellular-advanced glycation endproducts (AGE) antibodies. All harsh conditions were avoided in this protocol, including extreme temperature and pH, which are common in alternative decellularization methods, such as acid hydrolysis and mechanical pressure. We have not tested the ability of the ECM prepared by this method to support cell growth. Decellularized ECM is a widely used scaffold for regenerative medicine applications: Badylak et al. 20099 report 28 commercially available ECM scaffolds derived from animal tissues. It remains to be seen if our protocol can be adapted for this application.

No competing financial interests exist.

1. Karsdal MA, Nielsen MJ, Sand JM, Henriksen K, Genovese F, Bay-Jensen A-C, Smith V, Adamkewicz JI, Christiansen C, Leeming DJ. Possibilities for evaluation and current understanding of the matrix as more than a passive architecture, but a key player in tissue failure. Assay Drug Dev Technol 2013;11:70–92. 2. Bailey AJ, Paul RG, Knott L. Mechanisms of maturation and ageing of collagen. Mech Ageing Dev 1998;106:1–56. 3. Fritze O, Romero B, Schleicher M, Jacob MP, Oh DY, Starcher B, Schenke-Layland K, Bujan J, Stock UA. Agerelated changes in the elastic tissue of the human aorta. J Vascular Res 2012;49:77–86. 4. Bains W. Transglutaminse 2 and EGGL, the transglutaminse 2 catalyzed cross-link, as therapeutic targets for disabilities of old age. Rejuvenation Res 2013; in press. 5. Sjoberg JS, Bulterijs S. Characteristics, formation, and pathophysiology of glucosepane: A major protein cross-link. Rejuvenation Res 2009;12:137–148. 6. Gilbert TW, Freund JM, Badylak SF. Quantification of DNA in biologic scaffold materials. J Surgical Res 2009;152:135–139. 7. Lang R, Stern MM, Smith L, Liu Y, Bharadwaj S, Liu G, Baptista PM, Bergman CR, Soker S, Yoo JJ, Atala A, Zhang Y. Three-dimensional culture of hepatocytes on porcine liver tissue-derived extracellular matrix. Biomaterials 2011;32: 7042–7052. 8. Keane TJ, Londono R, Turner NJ, Badylak SF. Consequences of ineffective decellularization of biologic scaffolds on the host response. Biomaterials 2012;33:1771–1781. 9. Badylak SF, Freytes DO, Gilbert TW. Extracellular matrix as a biological scaffold material: Structure and function. Acta Biomater 2009;5:1–13.

Address for correspondence and reprints: Ariana Mirzarafie 463 Green Lanes London N13 4BS United Kingdom

Acknowledgments

We are grateful to SENS Research Foundation for funding to support this work, as well as to Prof. Laura Nicklason, Yale University, for help with protocol design.

E-mail: [email protected] Received: September 3, 2013 Accepted: September 8, 2013

A fast and mild decellularization protocol for obtaining extracellular matrix.

Degradation of extracellular matrix (ECM) function with age is a major cause of loss of tissue function with age that we would wish to reverse. Tissue...
70KB Sizes 0 Downloads 0 Views