Significance and Use
4.1 Decellularization is used in the preparation of medical products that make use of the native structure and/or composition of the extracellular matrix derived from a specific tissue source. Upon implantation or placement, the decellullarized product is commonly intended to undergo and/or induce constructive remodeling and incorporation into the native host tissue instead of being recognized as foreign material. Typically, immune system recognition of foreign material leads to encapsulation of the material and an aggressive inflammatory response, causing the ultimate rejection or other failure of the product.
4.2 As described above, decellularization is a recognized technique which allows the use of ECM-derived products in medical treatments with a reduced risk of an adverse host immune response and immune rejection by disrupting and removing cells and/or cell contents while aiming to preserve significant features of the ECM structure and/or composition. More complete decellularization is often associated with a beneficial response (1, 2)6 but can also be associated with the loss of important ECM components and the loss of structural or biomechanical integrity from the tissue during the decellularization process (3, 4, 5, 6). Therefore, given the typical objective of producing a product that does not elicit an adverse immune response while maintaining the integrity of the tissue for its intended surgical application, this guide presents a standard approach to the evaluation of decellularization processes, including assessment of adequate decellularization to achieve this end.
4.3 An ideal decellularization process would completely remove source tissue cells and associated cellular content from a tissue or organ, while minimizing unwanted effects on the remaining ECM. However, a more widely encountered and practical representation of an optimized decellularization process exhibits partial removal and/or disruption of resident cells and cellular material to levels within a set of product-specific ranges (acceptance criteria). This guide is intended to aid in evaluating a decellularization process through the mechanisms and extent of decellularization and any potential impacts on the remaining dECM.
4.4 This standard provides a guide to the following steps in evaluating an extracellular matrix decellularization process:
4.4.1 Selecting attributes and test methods for characterization (Section 5)
4.4.2 Developing decellularization acceptance criteria for selected attributes (Section 6)
4.4.3 Documenting and analyzing the decellularization process flow (Section 7)
4.4.4 Performing a characterization of the decellularization process by testing decellularized ECM materials using the selected attributes, methods, and acceptance criteria (Section 8)
4.5 Decellularization processes vary widely in practice, utilizing a variety of reagents, temperatures, pressures, and/or mechanical forces in parallel and/or in sequence. While any one factor may act through consistent mechanisms, its effect will vary according to the decellularization process in its entirety as well as the particular tissue structure. As such, each part of a decellularization process should be understood and analyzed within the context of the complete process sequence and its action upon the type of tissue. For example, a process developed for dermis will likely not translate directly to a heart valve and the doubling of process time will affect each process differently, so the decellularization process will have to be adjusted to account for the difference in tissue properties and desired attributes at the conclusion of the process. Within the context of this guide, analysis of a processing step should not suggest material testing. Analysis is meant to demonstrate an understanding of the relevant mechanisms of decellularization and the relevant mechanisms of adverse effects on the ECM material.
4.6 Decellularization acceptance criteria and ECM integrity acceptance criteria should be developed based on the intended use of the dECM material. This guide suggests some considerations that should be used to develop and justify acceptance criteria.
4.6.1 Decellularization acceptance criteria already established for a source ECM and decellularization process allow for controlled changes to the decellularization process. Significant changes include changes to the processing mechanisms, reagents/materials, reagent concentrations, and controls as well as changes in source ECM materials. Prior to any significant change to a decellularization process, a decellularization process analysis should be conducted on the process steps which are subject to change. In addition, testing against the established decellularization acceptance criteria should be conducted on dECM material produced with the proposed process changes. A risk management process may then be utilized to ensure that any risks associated with the proposed changes are acceptable.
4.7 Measurements of decellularization attributes using the source extracellular matrix material as a reference can provide a valuable frame of reference and determination of percent change for exploratory and informational purposes. However, acceptance criteria based on percent change from the source material are more prone to variability in the final product due to variability in the source material. Acceptance criteria based on measurements of the dECM alone are more stable and simpler to implement.
4.7.1 The preparation of decellularized medical products involves variability originating in the source material as well as the processing; both types of variability can affect the consistency of the end product (dECM) and its performance in meeting predetermined acceptance criteria. A complete characterization of a decellularization process will include statistical ranges for each measured attribute. Statistical correlations may be explored to connect variation in source material and processing to end product attributes. These correlations can help prioritize source material and process controls to address uncontrolled variability.
Scope
1.1 This document provides guidance on the characterization and evaluation of the decellularization processes used to produce decellularized extracellular matrix (dECM) materials which will be used as medical products in direct or indirect contact with the body. The decellularization process may be performed on tissue from human or other mammalian sources or produced in vitro from human or other mammalian cells. The dECM may or may not be recellularized prior to use. Decellularized ECM material derived from non-mammalian tissue or cells and decellularized ECM material used for non-medical purposes may follow the framework provided but may require additional considerations outside the scope of this document.
1.2 Biological tissues are composed of a structural extracellular matrix (ECM) and embedded cells. The intent of a decellularization process is to disrupt and/or remove cells and cellular components from an ECM material while maintaining key structural and/or compositional properties of the material. Decellularization comprises process steps intended or expected to result or aid in the disruption of source tissue cells and/or removal of cellular content from the material undergoing decellularization. Actions that are intended to rinse or otherwise remove decellularization reagents or by-products should also be considered in that context as part of the decellularization process. Purifications or other isolations of specific ECM components are not considered decellularization and are outside the scope of this document.
1.3 This document describes relevant parameters of decellularization processes used to prepare extracellular matrix materials as medical products.
1.4 This document provides guidance on the measurement of specific and general properties of dECM. This includes both the analysis of cellular material as well as the assessment of the effects of decellularization on dECM properties such as composition, structure, and material properties.
1.5 This document does not provide guidance on the assessment of the host response subsequent to the implantation or other in vivo placement of dECM medical products. Such assessments should instead be conducted as part of biocompatibility studies or other safety and efficacy studies. At a minimum it is recommended that the finished product composed of dECM material shall be assessed in a relevant model that represents the biological responses that the product is expected to experience to ensure that the final material is functioning in accordance with design intentions. An in vivo model will generally be used, but cellular or ex vivo models may also be satisfactory when appropriate.
1.6 This document provides guidance on determining pertinent quality attributes as well as developing and assessing acceptance criteria related to ensuring the consistent evaluation and use of decellularization in manufacturing medical products. Acceptance criteria should address the adequacy of cellular disruption and removal of cellular remnants. Acceptance criteria should define acceptable levels for retention of extracellular matrix components. Acceptance criteria may place limits on damage to retained components. Acceptance criteria should place limits on the persistence of decellularization reagents. This document also provides recommendations on developing process parameters and associated process controls.
1.6.1 This guide recommends attributes as representative measures of decellularization in the direct function of removing cells and cell components. These attributes can also be used to show process consistency, capability, or equivalency. Recommendation of these attributes does not confer additional significance related to product safety and performance.
1.6.2 No consensus has been established regarding decellularization thresholds or classifications. This guide therefore cannot suggest acceptance criteria and instead recommends commonly measured attributes to develop acceptance criteria specific to the design of each unique material and its intended use.
1.7 Decellularized products will require evidence of safety and/or efficacy beyond that related to evaluating the decellularization process. Commonly referenced standards include the ISO 10993 series (see ISO 10993-1) for biocompatibility of medical devices and the ISO 22442 series for medical devices utilizing animal tissues and their derivatives. These assessments are not in the scope of this document, though they may help to identify relevant functional characteristics and test methods as discussed in 5.2.9.
1.8 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.
1.9 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
