Erratum: Complete intra-laboratory validation of a LAL assay for bacterial endotoxin determination in EBV-specific cytotoxic T lymphocytes.

[This corrects the article DOI: 10.1016/j.omtm.2021.05.002.].

Complete intra-laboratory validation of a LAL assay for bacterial endotoxin determination in EBV-specific cytotoxic T lymphocytes.

Endotoxin content is a critical factor that affects the safety of biological pharmaceutical products. International pharmacopoeias describe several reference methods to determine endotoxin levels in advanced therapy medicinal product (ATMP) preparations. Administration of ATMPs must be done as rapidly as possible to ensure complete viability and potency of the cellular product. To evaluate the endotoxin content in the shortest time possible, we chose to validate an alternative method based on the use of the Charles River Portable Testing System (PTS) and FDA-approved cartridges, compliant with the requirements of the European Pharmacopoeia and providing results in <20 min. Here, we describe a unique and complete validation approach for instrument, personnel, and analytical method for assessment of endotoxins in ATMP matrices. The PTS system provides high sensitivity and fast quantitative results and uses less raw material and accessories compared with compendial methods. It is also less time consuming and less prone to operator variability. Our validation approach is suitable for a validated laboratory with trained personnel capable of conducting the ATMP release tests, and with very low intra-laboratory variability, and meets the criteria required for an alternative approach to endotoxin detection for in-process and product-release testing of ATMPs.

Use of 27G needles improves sensitivity and performance of ATCC anaerobe reference microorganism detection in BacT/Alert system.

Effective detection of microbiological contaminations present in medicinal cellular products is a crucial step to ensure patients' safety. In recent decades, several rapid microbiological methods have been developed and validated, but variabilities linked to the use of different resources have led to discordant validation of methods and performance results. Considering this, while developing an in-house BacT/Alert-based method, we evaluated all of the materials used in its validation. Of particular importance, we noticed that the syringe gauge used to inject the samples into the bottles was crucial to obtain robust results. We chose to conduct a comparative test between the BacT/Alert system and the compendial method described in the European Pharmacopoeia, using five dilutions of nine reference microorganism strains and 21G or 27G needles. Our results confirmed that the BacT/Alert system is a valid and faster alternative method to assess sterility of clinical cell therapy products, and that the use of 27G needles increases its sensitivity to detect reference anaerobe microorganisms.

Efficacy of Three Commercial Disinfectants in Reducing Microbial Surfaces' Contaminations of Pharmaceuticals Hospital Facilities.

To evaluate and validate the efficacy of disinfectants used in our cleaning procedure, in order to reduce pharmaceutical hospital surfaces' contaminations, we tested the action of three commercial disinfectants on small representative samples of the surfaces present in our hospital cleanrooms. These samples (or coupons) were contaminated with selected microorganisms for the validation of the disinfectants. The coupons were sampled before and after disinfection and the microbial load was assessed to calculate the Log10 reduction index. Subsequently, we developed and validated a disinfection procedure on real surfaces inside the cleanrooms intentionally contaminated with microorganisms, using approximately 107-108 total colony forming units per coupon. Our results showed a bactericidal, fungicidal, and sporicidal efficacy coherent to the acceptance criteria suggested by United States Pharmacopeia 35 <1072>. The correct implementation of our cleaning and disinfection procedure, respecting stipulated concentrations and contact times, led to a reduction of at least 6 Log10 for all microorganisms used. The proposed disinfection procedure reduced the pharmaceutical hospital surfaces' contaminations, limited the propagation of microorganisms in points adjacent to the disinfected area, and ensured high disinfection and safety levels for operators, patients, and treated surfaces.

Strategy and validation of a consistent and reproducible nucleic acid technique for mycoplasma detection in advanced therapy medicinal products.

Advanced therapy medicinal products (ATMP) are required to maintain their quality and safety throughout the production cycle, and they must be free of microbial contaminations. Among them, mycoplasma contaminations are difficult to detect and undesirable in ATMP, especially for immunosuppressed patients. Mycoplasma detection tests suggested by European Pharmacopoeia are the "culture method" and "indicator cell culture method" which, despite their effectiveness, are time consuming and laborious. Alternative methods are accepted, provided they are adequate and their results are comparable with those of the standard methods. To validate a novel in-house method, we performed and optimized, a real time PCR protocol, using a commercial kit and an automatic extraction system, in which we tested different volumes of matrix, maximizing the detection sensitivity. The results were compared with those obtained with the gold standard methods. From a volume of 10 ml, we were able to recognize all the mycoplasmas specified by the European Pharmacopoeia, defined as genomic copies per colony forming unit ratio (GC/CFU). Our strategy allows to achieve faster and reproducible results when compared with conventional methods and meets the sensitivity and robustness criteria required for an alternative approach to mycoplasmas detection for in-process and product-release testing of ATMP.

A quality risk management model approach for cell therapy manufacturing.

International regulatory authorities view risk management as an essential production need for the development of innovative, somatic cell-based therapies in regenerative medicine. The available risk management guidelines, however, provide little guidance on specific risk analysis approaches and procedures applicable in clinical cell therapy manufacturing. This raises a number of problems. Cell manufacturing is a poorly automated process, prone to operator-introduced variations, and affected by heterogeneity of the processed organs/tissues and lot-dependent variability of reagent (e.g., collagenase) efficiency. In this study, the principal challenges faced in a cell-based product manufacturing context (i.e., high dependence on human intervention and absence of reference standards for acceptable risk levels) are identified and addressed, and a risk management model approach applicable to manufacturing of cells for clinical use is described for the first time. The use of the heuristic and pseudo-quantitative failure mode and effect analysis/failure mode and critical effect analysis risk analysis technique associated with direct estimation of severity, occurrence, and detection is, in this specific context, as effective as, but more efficient than, the analytic hierarchy process. Moreover, a severity/occurrence matrix and Pareto analysis can be successfully adopted to identify priority failure modes on which to act to mitigate risks. The application of this approach to clinical cell therapy manufacturing in regenerative medicine is also discussed.

Autologous in vivo adipose tissue engineering in hyaluronan-based gels--a pilot study.

BACKGROUND: There is a major clinical need for strategies for adequately reconstructing the soft tissue defects found after deep burns, tumor resection, or trauma. A promising solution is adipose tissue engineering with preadipocytes, stem-cell derived precursors of the adipose tissue, implanted within biomaterials. This pilot study evaluated hyaluronan gels mixed with autologous undifferentiated preadipocytes in a pig model for their potency to generate new fat. MATERIALS AND METHODS: Preadipocytes were isolated from intra-abdominal pig fat by collagenase digestion, plated on fibronectin-coated culture dishes in Dulbecco's modified Eagle medium/Ham's F12 (Biochrom, Berlin, Germany) combined with 10% pig serum, expanded, and mixed with hyaluronan gel. Two types of gels with varying degrees of amidation of the carboxyl groups were tested (HYADD3, HYADD4). Cell-loaded gels and unseeded controls were injected subcutaneously into the ears of three pigs, explanted at 6 wk, and analyzed histologically. RESULTS: Both cell-loaded specimens were detected macroscopically. They demonstrated a slight volume effect with limited stability after 6 wk. Unloaded HYADD3 and HYADD4 controls could not be identified at the time of explantation. Histology of HYADD3 revealed islets of mature adipocytes and vessels embedded in fat tissue surrounded by gel. In contrast, no fat formation was found in HYADD4 gels when implanted in the ear. CONCLUSIONS: Histological findings demonstrate that HYADD3 is a promising gel for generating adipose tissue. Even though HYADD3 might be a potential material for the reconstruction of small tissue defects, the question remains as to whether the adipose tissue within the gel is attributable to preadipocyte maturation or ingrowth from neighboring tissue.

Structural and rheological characterization of hyaluronic acid-based scaffolds for adipose tissue engineering.

In this study the attention has been focused on the ester derivative of hyaluronic acid (HA), HYAFF11, as a potential three-dimensional scaffold in adipose tissue engineering. Different HYAFF11 sponges having different pore sizes, coated or not coated with HA, have been studied from a rheological and morphological point of view in order to correlate their structure to the macroscopic and degradation properties both in vitro and in vivo, using rat model. The in vitro results indicate that the HYAFF11 sponges possess proper structural and mechanical properties to be used as scaffolds for adipose tissue engineering and, among all the analysed samples, uncoated HYAFF11 large-pore sponges showed a longer lasting mechanical stability. From the in vivo results, it was observed that the elastic modulus of scaffolds seeded with preadipocytes, the biohybrid constructs, and explanted after 3 months of implantation in autologous rat model are over one order of magnitude higher than the corresponding values for the native tissue. These results could suggest that the implanted scaffolds can be invaded and populated by different cells, not only adipocytes, that can produce new matrix having different properties from that of adipose tissue.

ACP gel: a new hyaluronic acid-based injectable for facial rejuvenation. Preclinical data in a rabbit model.

BACKGROUND: Facial aging results from reduced biosynthetic activity of dermal fibroblasts and decreased deposition of extracellular matrix components, such as hyaluronic acid, a glycosaminoglycan responsible for skin hydration and turgidity. Exogenous hyaluronic acid injections provide a safe though short-term solution for facial rejuvenation. Using a rabbit model, the authors investigated residence time and tolerability of ACP gel, a new hyaluronic acid cross-linked derivative, compared with high-molecular-weight native hyaluronic acid currently used for facial rejuvenation (Ial System). METHODS: ACP gel 1% and 2%, Ial System, and saline were intradermally injected into the backs of 12 New Zealand rabbits: six animals were used to follow volume maintenance and redness up to 10 days and the other six animals were euthanized at days 2, 6, 8, 10, 14, and 21 (one animal per time point) to histologically assess biocompatibility. RESULTS: ACP gel 2% had the longest residence time, showing a significantly better volume maintenance than the other samples, especially in the initial study period (71 percent of original volume versus 23 percent and 21 percent of ACP gel 1% and Ial System, respectively, at day 2). Macroscopically, no adverse events were observed in the treated animals. Histologic examination confirmed the absence of adverse events, persistent inflammation, tissue degeneration, or necrosis. ACP gel macrophage-mediated phagocytosis was more persistent with respect to the Ial System, consistent with its longer residence time. CONCLUSION: ACP gel 2% is a promising dermal biorevitalizer, characterized by a high safety profile and prolonged residence time in relation to native high-molecular-weight hyaluronic acid.

Implantation of preadipocyte-loaded hyaluronic acid-based scaffolds into nude mice to evaluate potential for soft tissue engineering.

The reconstruction of soft tissue defects following extensive deep burns or tumor resections remains an unresolved problem in plastic and reconstructive surgery since adequate implant materials are still not available. Preadipocytes, immature precursor cells found between mature adipocytes in adipose tissue, are a potential material for soft tissue engineering since they can proliferate and differentiate into adipose tissue after transplantation. In previous studies, we identified hyaluronan benzyl ester (HYAFF 11) sponges to be promising carrier matrices. This study now evaluates, in vitro and in vivo, a new sponge architecture with pores of 400 microm either made of plain HYAFF 11 or HYAFF 11 coated with the extracellular matrix glycosaminoglycan hyaluronic acid. Human preadipocytes were isolated, seeded onto carriers and implanted into nude athymic mice. Explants harvested after 3, 8, and 12 weeks were examined for macroscopical appearance, thickness, weight, pore structure, histology, and immunohistochemistry. Compared to previous studies, we found better penetration of cells into both types of scaffolds, with more extensive formation of new vessels throughout the construct but with only minor adipose tissue. Our encouraging results contribute towards a better seeded and vascularised scaffold but also show that the enhancement of adipogenic conversion of preadipocytes remains a major task for further in vivo experiments.