Special review series on 3D organotypic culture models: Introduction and historical perspective.

Three dimensional (3D) organ-like (organotypic) culture models are a rapidly advancing area of in vitro biological science. In contrast to monolayer cell culture methods which were developed to achieve proliferation of animal cells in the beginning of in vitro biology, the advancements in 3D culture methods are designed to promote cellular differentiation, and to achieve in vivo-like 3D structure and organotypic functions. This project was conceived through the Society for In Vitro Biology to draw on the expertise of individual scientists with special expertise in organotypic cultures of selected tissues or associated interrogation methods to prepare individual-focused reviews in this series. This introductory manuscript will review the early achievements of animal cell culture in monolayer culture and the limitations of that approach to reproduce functioning organ systems. Among these are the nature and 3D architecture of the substrate on which or in which the cells are grown, physical and mechanical clues from the substrate, cell-cell interactions, and defined biochemical factors that trigger the induction of the 3D organotypic differentiation. The organoid culture requires a source of cells with proliferative capacity (ranging from tissue-derived stem or immortalized cells to the iPSC cultures), a suitable substrate or matrix with the mechanical and stimulatory properties appropriate for the organotypic construct and the necessary stimulation of the culture to drive differentiation of the cell population to form the functioning organotypic construct. Details for each type of organotypic construct will be provided in the following papers.

Demonstrating the persistent antibacterial efficacy of a hand sanitizer containing benzalkonium chloride on human skin at 1, 2, and 4 hours after application.

BACKGROUND: Use of hand sanitizers has become a cornerstone in clinical practice for the prevention of disease transmission between practitioners and patients. Traditionally, these preparations have relied on ethanol (60%-70%) for bactericidal action. METHODS: This study was conducted to measure the persistence of antibacterial activity of 2 preparations. One was a non-alcohol-based formulation using benzalkonium chloride (BK) (0.12%) and the other was an ethanol-based formulation (63%) (comparator product). The persistence of antibacterial activity was measured against Staphylococcus aureus using a technique modification prescribed in American Society for Testing and Materials protocol E2752-10 at up to 4 hours after application. RESULTS: The test product (BK) produced a marked reduction in colony-forming units at each of the 3 time points tested (3.75-4.16-log10 reductions), whereas the comparator produced less than 1-log10 reduction over the same time. The differences were highly significant. DISCUSSION: In the course of patient care or examination, there are instances where opportunities exist for the practitioner's hands to become contaminated (eg, key boards and tables). Persistent antibacterial activity would reduce the chances of transfer to the patient. CONCLUSIONS: These results show a major improvement in persistent antibacterial activity for the BK formulation compared to the comparator ethanol-based formulation.

Best practices for cryopreserving, thawing, recovering, and assessing cells.

Long-term storage of cell stocks insures that cells are available for use whenever needed. Cryopreservation of cells is the method of choice for preservation of important or rare cell stocks. There are several factors to consider when establishing a protocol for freezing, thawing, and recovery of cells after storage. These parameters may include cell concentration, cryoprotectant choice and concentration, and thawing rate among others. Further, the assessment of cell viability and/or function prior to and following cryopreservation is imperative in order to accurately determine downstream utility as well for optimizing the cryopreservation process. This chapter is designed to provide guidance and insight into developing robust and successful protocols for preserving cells that will preserve cell stocks and provide optimal cell yield and viability.

Best practices in cell culture: an overview.

This overview describes a series of articles to provide an unmet need for information on best practices in animal cell culture. The target audience primarily consists of entry-level scientists with minimal experience in cell culture. It also include scientists, journalists, and educators with some experience in cell culture, but in need of a refresher in best practices. The articles will be published in this journal over a six-month period and will emphasize best practices in: (1) media selection; (2) use and evaluation of animal serum as a component of cell culture medium; (3) receipt of new cells into the laboratory; (4) naming cell lines; (5) authenticating cell line identity; (6) detecting and mitigating risk of cell culture contamination; (7) cryopreservation and thawing of cells; and (8) storing and shipping viable cells.

Best practices for the use and evaluation of animal serum as a component of cell culture medium.

Animal serum is a common additive for cell culture medium and is often required at 5 to 10% (v/v) for the attachment and growth of primary and continuous anchorage-dependent (monolayer) cultures. The use of animal serum in cell culture medium confers several advantages and also some risks. This article discusses the use of animal serum as a component of cell culture medium. The best practices associated with the sourcing, storage, thawing, testing, and mitigation of risk associated with the use of animal sera are among the topics described in this article.

Prediction of eye irritation potential of liquid and granular laundry detergent formulas using the bovine corneal opacity and permeability (BCOP) assay.

Evaluation of eye irritation potential is a routine part of consumer product testing. Increasingly, companies are using in vitro methods to perform these assessments. We have used the bovine corneal opacity and permeability (BCOP) assay for the prediction of eye irritation of liquid and granular laundry detergent formulas. The BCOP assay was selected because it can distinguish between moderate and severe irritants as required to evaluate these classes of formulations. Corneas were maintained in short-term culture and the exposure conditions were optimized using marketed product upper-end benchmark formulas for each product class. The primary endpoint was the loss of epithelium as measured by the change in permeability of the cornea to fluorescein and was complemented by histological evaluation of depth of injury. The opacity endpoint was not used, as the surfactants in these products do not induce opacity in proportion to the depth of injury induced. Liquid laundry detergents were diluted to 25% and exposed to the corneas for 20 min while the granular detergents were diluted to 10% and exposed for 30 min. These conditions were selected for each product type to induce OD490 values in the midrange (between 0.5 and 0.6 absorbance units) and so increased or decreased irritation potential in the test formulas could readily be observed. Seventeen liquid and eleven granular laundry detergents were tested and the OD490 values ranged from 0.278 to 2.193 for the liquid detergents and 0.267 to 0.856 for the granular detergents. Histological changes in the epithelium and stroma were consistent with the OD490 values. These data suggest that the OD490 provides an effective measure of epithelial cell loss (degree of cell lysis) and thus irritation potential for these surfactant-based formulas. The upper-end benchmark set a known upper range for acceptable irritation for the product class. Those formulas inducing lower OD490 values may be considered to fall within the acceptable range while those inducing greater OD490 values should receive further evaluation and perhaps reformulation.

Comparison of in vitro eye irritation potential by bovine corneal opacity and permeability (BCOP) assay to erythema scores in human eye sting test of surfactant-based formulations.

The bovine corneal opacity and permeability (BCOP) assay can be used to predict relative eye irritation potential of surfactant-based personal care formulations relative to a corporate benchmark. The human eye sting test is typically used to evaluate product claims of no tears/no stinging for children's bath products. A preliminary investigation was conducted to test a hypothesis that the BCOP assay could be used as a prediction model for relative ranking of human eye irritation responses under conditions of a standard human eye sting test to surfactant-based formulations. BCOP assays and human eye sting tests were conducted on 4 commercial and 1 prototype body wash (BW) developed specifically for children or as mild bath products. In the human eye sting test, 10 mul of a 10% dosing solution is instilled into one eye of each panelist (n = 20), and the contralateral eye is dosed with sterile water as a control. Bulbar conjunctival erythema responses of each eye are graded at 30 seconds by an ophthalmologist. The BCOP assay permeability values (optical density at 490 nm [OD(490)]) for the 5 BWs ranged from 0.438 to 1.252 (i.e., least to most irritating). By comparison, the number of panelists exhibiting erythema responses (mild to moderately pink) ranged from 3 of 20 panelists for the least irritating BW to 10 of 20 panelists for the most irritating BW tested. The relative ranking of eye irritation potential of the 5 BWs in the BCOP assay compares favorably with the relative ranking of the BWs in the human eye sting test. Based on these findings, the permeability endpoint of the BCOP assay, as described for surfactant-based formulations, showed promise as a prediction model for relative ranking of conjunctival erythema responses in the human eye. Consequently, screening of prototype formulations in the BCOP assay would allow for formula optimization of mild bath products prior to investment in a human eye sting test.

Use of the cytosensor microphysiometer to predict results of a 21-day cumulative irritation patch test in humans.

The cytosensor microphysiometer (mu phi) was investigated as a rapid, relatively inexpensive test to predict performance of skin cleansing wipes on the human 21-day cumulative irritation patch test (21CIPT). It indirectly measures metabolic rate changes in L929 cells as a function of test article dose, by measuring the acidification rate in a low-buffer medium. The dose producing a 50% reduction in metabolic rate (MRD50), relative to the baseline rate, is used as a measure of toxicity. The acute toxicity of the mu phi assay can be compared to the chronic toxicity of the 21CIPT, which is based largely on the exposure of test agents to the epidermal cells, resulting in damage and penetration of the stratum corneum leading to cell toxicity. Two series of surfactant-based cleansing wipe products were tested via the mu phi assay and 21CIPT. The first series, consisting of 20 products, was used to determine a prediction model. The second series of 38 products consisted of routine product development formulas or marketed products. Comparing the results from both tests, samples with an MRD50 greater than 50 mg/ml provided a 21CIPT score consistent with a product that performs satisfactorily in the market. When the MRD50 was greater than 78 mg/ml, the 21CIPT score was usually zero. The mu phi may be more sensitive than the 21CIPT for ranking minimally irritating materials. The mu phi assay is useful as a screen for predicting the performance of a wet wipes formula on the 21CIPT, and concurrently reduces the use of animals for safety testing in a product development program for cleansing wipes.

Prediction of eye irritation potential of surfactant-based rinse-off personal care formulations by the bovine corneal opacity and permeability (BCOP) assay.

Evaluation of eye irritation potential is a routine part of product safety testing. Many companies have elected to use in vitro methods for this evaluation. We used the bovine corneal opacity and permeability (BCOP) assay for the prediction of the eye irritation potential of surfactant-based rinse-off personal care formulations in this study because of its positive performance in previous studies (1), and its potential to measure depth of injury through histological evaluation. The BCOP uses isolated corneas maintained in short-term organ culture and measures changes in opacity and epithelial barrier integrity (as determined by fluorescein passage through the cornea). Surfactants (anionic and nonionic) used in personal care formulations induce only small increases in direct opacity but the degree of epithelial damage is reflected in permeability measurement (OD490). The permeability measurement is the primary endpoint for this class of products. Marketed liquid hand soap was selected as the benchmark due to extensive in vivo and market history data. Using the liquid hand soap, a 25% v/v aqueous dilution with a 30-minute exposure produced the optimal resolution. A series of products with known in vivo eye irritation scores were evaluated to establish a prediction model. Histological evaluation was used to compare the degree of tissue damage to the permeability scores. Based on the permeability scores and histological review, the testing protocol for a surfactant-based rinse-off personal care formulation was developed using a 25% v/v aqueous dilution, a 30-minute exposure, concurrent testing of the benchmark control, and use of permeability measurements as the endpoint for the evaluation of eye irritation potential.

Evaluation of some in vitro tests to reduce and replace the sub-acute animal toxicity studies.

The toxicologic and carcinogenic potential of chemicals is usually determined through a sequence of acute, sub-acute (14-day), sub-chronic (90-day) and chronic (two-year) studies in rats and mice of both sexes. The US National Toxicology Program (NTP) does not conduct acute toxicity studies. Dose levels for 14-day toxicity studies are typically estimated from information in the literature, if available. The toxicology information obtained from 14-day studies is used in the selection of doses for 90-day studies. The protocol for 14-day studies consists of five doses and control groups and five animals per group of each sex and species, resulting in the use of 120 animals per study. At present, in addition to refining the current testing protocols, the NTP is evaluating the potential for in vitro test methods to partially or completely avoid the need for 14-day toxicity studies, especially for chemicals where the dermal route of exposure is used. The in vitro assays used were the EpiDerm bioassay to estimate dermal irritation, the neutral red uptake (NRU) bioassay to estimate systemic toxicity and the primary rat hepatocyte cytotoxicity (PRHC) assay to estimate hepatotoxicity. The purpose of using these assays was to assess their potential for predicting relative in vivo toxicity and to support dose selection decisions for 90-day studies. In general, based on these limited number of studies, the EpiDerm and NRU tests were predictive of the responses observed in in vivo studies. However, a larger comparative database is needed to derive definitive conclusions regarding the value of in vitro tests in the prediction of in vivo effects.

Quality assurance for in vitro alternative test methods: quality control issues in test kit production.

In vitro toxicology methods are being adopted by regulatory agencies worldwide. Many of these methods have been validated by using proprietary materials, often in the form of test kits. Guidelines for the use of Good Laboratory Practice methods for in vitro methods have been proposed. However, users of the data from these methods also need to be reassured that the proprietary materials and the test kits will provide consistent, good quality data over time, not just during the validation process. This paper presents an overview of the methods currently used by representatives of kit manufacturers and contract testing laboratories to ensure that the results from methods that utilise test kits are reproducible over time and across different types of test materials. This information will be valuable as a basis for future discussion on the need for formalised oversight of the quality of these materials.

Ocular safety: a silent (in vitro) success story.

Ocular irritation testing has been one of the animal test methods most criticised by animal welfare advocates. Additional criticism has arisen from within the scientific community, based on the variability of the animal test results and the questionable relevance of the extremely high dose levels employed. As a result, the Draize eye irritation test has been one of the main targets for in vitro replacement. Despite extensive efforts, however, there is still no in vitro method that is fully validated as a regulatory replacement. In spite of this, many individual companies are using diverse in vitro ocular irritation tests to gain important safety and efficacy information about their products and raw materials, eliminating the need for animal testing in the process. This is done in a safe fashion by applying intelligent testing paradigms. ECVAM has played a major role in this success, through its many programmes that have emphasised the importance of understanding the true toxicological need, and then using in vitro tests to provide that information. Thus, even in the absence of a successfully validated regulatory assay, the desired result of reducing animal testing is being met.