ASTM F2739-19

5.1 The number and distribution of viable and non-viable cells within, or on the surface of, a biomaterial scaffold is one of several important characteristics that may determine 5.2 There are a variety of static and dynamic methods to seed cells on scaffolds, each with different cell seeding efficiencies. In general, static methods such as direct pipetting of cells onto scaffold surfaces have been shown to have lower cell seeding efficiencies than dynamic methods that push cells into the scaffold interior. Dynamic methods include: injection of cells into the scaffold, cell seeding on biomaterials contained in spinner flasks or perfusion chambers, or seeding that is enhanced by the application of centrifugal forces. The methods described in this guide can assist in establishing cell seeding efficiencies as a function of seeding method and for standardizing viable cell numbers within a given methodology.

5.3 As described in Guide F2315, thick scaffolds or scaffolds highly loaded with cells lead to diffusion limitations during culture or implantation that can result in cell death in the center of the construct, leaving only an outer rim of viable cells. Spatial variations of viable cells such as this may be quantified using the tests within this guide. The effectiveness of the culturing method or bioreactor conditions on the viability of the cells throughout the scaffold can also be evaluated with the methods described in this guide.

5.4 These test methods can be used to quantify cells on non-porous or within porous hard or soft 3-D synthetic or natural-based biomaterials, such as ceramics, polymers, hydrogels, and decellularized extracellular matrices. The test methods also apply to cells seeded on porous coatings.

5.5 Test methods described in this guide may also be used to distinguish between proliferating and non-proliferating viable cells. Proliferating cells proceed through the DNA synthesis (S) phase and the mitosis (M) phase to produce two daughter cells. Non-proliferating viable cells are in some phase of the cell cycle, but are not necessarily proceeding through the cell cycle culminating in proliferation.

5.6 Viable cells may be under stress or undergoing apoptosis. Assays for evaluating cell stress or apoptosis are not addressed in this guide.

5.7 While cell viability is an important characteristic of a TEMP, the biological performance of a TEMP is dependent on additional parameters. Additional tests to evaluate and confirm the cell identity, protein expression, genetic profile, lineage progression, extent of differentiation, activation status, and morphology are recommended.

5.8 The main focus of this document is not scaffold toxicity or the toxicity of the scaffold raw materials. This document is meant to address the situation where a scaffold that is thought to be cytocompatible is cultured with cells and the user desires to assess the viability of cells within the construct. Prior to conducting the tests described herein, the raw materials used to make the scaffold should be assessed as described in Practice F748. This testing may include assessment of the release of toxic leachables from the raw materials.

5.9 Methods that remove the cells from a 3-D scaffold may reduce the cell number and viability due to the manipulation required.

5.10 Some scaffold constructs may prevent reliable measurements of cell viability within the scaffolds using the methods described herein. Scaffolds may limit diffusion of assay components into and out of the scaffolds. This is especially problematic for methods that require dyes to penetrate into the scaffold, that require detergents or other cell-lysing agents to diffuse into the construct, that require lysed-cell components to diffuse out of the constructs, or that require assay reactants to diffuse into or out of the scaffold. Diffusion in scaffolds and assay results may also be affected by dense cell populations in scaffolds, the generation of tissue-like structures by the cells within the scaffold, and the presence of cell-generated extracellular matrix (ECM) in the scaffold. The formation of tight junctions between cells and cell-ECM interactions may also limit diffusion, especially in the case of hard tissues such as bone.

5.11 Assay results may be affected by interactions between assay components and the scaffold. Assay components may adsorb to the surface of the scaffold which would affect their participation in the assay and the resulting assay signal. Biochemical interactions between the scaffold and assay components may cause activation or inhibition of the assay chemistries.

5.12 Different cell viability tests may measure different things and may not agree with one another. A large variety of cell viability assays have been developed to measure different aspects of the cell death process. Some of the common measurements include penetration of dyes into the cell, cell metabolic activity, cellular ATP, and leakage of intracellular components out of the cell. Each of these phenomena are related to the state of cell viability in different ways, and may represent different attributes of the cell death process. The mechanism of cell death will also affect the results for these different types of viability measurements. Necrosis, oxygen depravation, starvation, chemical toxicity, apoptosis, anoikis, and mechanical damage represent some of the causes of cell death. Each of these mechanisms may have different effects on the different aspects of cell death that are measured by cell viability assays.

1.1 This guide is a resource of cell viability test methods that can be used to assess the number and distribution of viable and non-viable cells within porous and non-porous, hard or soft biomaterial scaffolds, such as those used in tissue-engineered medical products (TEMPs).

1.2 In addition to providing a compendium of available techniques, this guide describes materials-specific interactions with the cell assays that can interfere with accurate cell viability analysis, and includes guidance on how to avoid or account for, or both, scaffold material/cell viability assay interactions.

1.3 These methods can be used for 3-D scaffolds containing cells that have been cultured in vitro or for scaffold/cell constructs that are retrieved after implantation in living organisms.

1.4 This guide does not propose acceptance criteria based on the application of cell viability test methods.

1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.

1.6 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.

1.7 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.