The cytokinesis-block micronucleus assay for cryopreserved whole blood

Purpose The cytokinesis-block micronucleus (MN) assay is a widely used technique in basic radiobiology research, human biomonitoring studies and in vitro radiosensitivity testing. Fresh whole blood cultures are commonly used for these purposes, but immediate processing of fresh samples can be logistically challenging. Therefore, we aimed at establishing a protocol for the MN assay on cryopreserved whole blood, followed by a thorough evaluation of the reliability of this assay for use in radiosensitivity assessment in patients. Materials and methods Whole blood samples of 20 healthy donors and 4 patients with a primary immunodeficiency disease (PID) were collected to compare the results obtained with the MN assay performed on fresh versus cryopreserved whole blood samples. MN yields were scored after irradiation with 220 kV X-rays (dose rate 3 Gy/min), with doses ranging from 0.5-2 Gy. Results The application of the MN assay on cryopreserved blood samples was successful in all analyzed samples. The radiation-induced MN and NDI scores in fresh and cryopreserved blood cultures were found to be similar. Acceptable inter-individual and intra-individual variabilities in MN yields were observed. Repeated analysis of cryopreserved blood cultures originating from the same blood sample, thawed at different time points, revealed that MN values remain stable for cryopreservation periods up to one year. Finally, radiosensitive patients were successfully identified using the MN assay on cryopreserved samples. Conclusions To our knowledge, this study is the first report of the successful use of cryopreserved whole blood samples for application of the MN assay. The data presented here demonstrate that the MN assay performed on cryopreserved whole blood is reliable for radiosensitivity testing. Our results also support its wider use in epidemiological, biomonitoring and genotoxicity studies. The presented method of cryopreservation of blood samples might also benefit other assays

Scaffold Free Microtissue Formation for Enhanced Cartilage Repair

Given the low self-healing capacity of fibrocartilage and hyaline cartilage, tissue engineering holds great promise for the development of new regenerative therapies. However, dedifferentiation of cartilage cells during expansion leads to fibrous tissue instead of cartilage. The purpose of our study was to generate 3D microtissues, spheroids, mimicking the characteristics of native fibrocartilage or articular cartilage to use as modular units for implantation in meniscal and articular cartilage lesions, respectively, within the knee joint. A set of parameters was assessed to create spheroids with a geometry compatible with 3D bioprinting for the creation of a biomimetic cartilage construct. Fibrochondrocytes (FC) and articular chondrocytes (AC) spheroids were created using a high-throughput microwell system. Spheroid morphology,  viability, proliferation and extracellular matrix were extensively screened. After 2D expansion, FC and AC dedifferentiated, resulting in a loss of cartilage specific extracellular matrix proteins. Spheroid formation did not result in FC redifferentiation, but did lead to redifferentiation of AC, resulting in microtissues displaying collagen II, aggrecan and glycosaminoglycans. This study demonstrates 3D cartilage mimics that could have a potential application in the next generation of Autologous Chondrocyte Implantation procedures. Moreover, spheroids can be used as building blocks to create cartilage constructs by bioprinting in the future.status: publishe

Scaffold free microtissue formation for enhanced cartilage repair

Given the low self-healing capacity of fibrocartilage and hyaline cartilage, tissue engineering holds great promise for the development of new regenerative therapies. However, dedifferentiation of cartilage cells during expansion leads to fibrous tissue instead of cartilage. The purpose of our study was to generate 3D microtissues, spheroids, mimicking the characteristics of native fibrocartilage or articular cartilage to use as modular units for implantation in meniscal and articular cartilage lesions, respectively, within the knee joint. A set of parameters was assessed to create spheroids with a geometry compatible with 3D bioprinting for the creation of a biomimetic cartilage construct. Fibrochondrocytes (FC) and articular chondrocytes (AC) spheroids were created using a high-throughput microwell system. Spheroid morphology, viability, proliferation and extracellular matrix were extensively screened. After 2D expansion, FC and AC dedifferentiated, resulting in a loss of cartilage specific extracellular matrix proteins. Spheroid formation did not result in FC redifferentiation, but did lead to redifferentiation of AC, resulting in microtissues displaying collagen II, aggrecan and glycosaminoglycans. This study demonstrates 3D cartilage mimics that could have a potential application in the next generation of Autologous Chondrocyte Implantation procedures. Moreover, spheroids can be used as building blocks to create cartilage constructs by bioprinting in the future

The cytokinesis-block micronucleus assay on human cryopreserved whole blood and isolated peripheral blood mononuclear cells

Purpose: The cytokinesis-block micronucleus (MN) assay is a widely used technique in human biodosimetry studies, occupational genotoxicity studies and in vitro radiosensitivity testing. Fresh whole blood cultures (WBC) are commonly used for these purposes, but the requirement for immediate processing can be logistically challenging. Therefore, we aimed at establishing two novel protocols for the MN assay on cryopreserved whole blood and fresh or cryopreserved isolated peripheral blood mononuclear cells (PBMCs). Additionally, a thorough evaluation of the reliability of these assays for use in biological dosimetry and radiosensitivity assessment was performed. Materials and methods: The G0 MN assay was performed on fresh and cryopreserved whole blood, freshly isolated and cryopreserved PBMCs from healthy human blood samples. MN yields were scored after irradiation with 220 kV X rays, with doses ranging from 0,5–2 Gy. Additionally, blood samples of 4 patients with a suspected radiosensitive phenotype were analyzed with both protocols. Results: The optimized MN assays on PBMCs and cryopreserved whole blood cultures showed adequate inter-individual and intra-individual variabilities. MN values were significantly lower for fresh PBMCs than for fresh whole blood. Cryopreservation of PBMCs resulted in significant higher MN values compared to fresh PBMCs, while cryopreserved whole blood cultures showed no significant differences in MN yields compared to fresh whole blood cultures. Importantly, after different cryopreservation periods, MN values remained stable for both cryopreserved PBMCs (6 months) and whole blood (1 year). The radiosensitive patients, included in this study, were correctly identified using fresh as well as cryopreserved PBMCs and cryopreserved whole blood. Conclusions: Our new MN assay protocols on fresh PBMCs, cryopreserved PBMCs and cryopreserved whole blood demonstrate to be reliable tools for biodosimetry and radiosensitivity studies

Cryopreserved whole blood and isolated peripheral blood mononuclear cells as alternative sample types for the cytokinesis-block micronucleus assay

Background The cytokinesis-block micronucleus (MN) assay is widely used in basic radiobiological research, environmental biomonitoring studies, biological dosimetry and in vitro radiosensitivity testing. Fresh whole blood cultures are generally used for the MN assay, limiting the application of this assay in studies where immediate processing of fresh samples is logistically challenging. Therefore, we standardized the MN assay on cryopreserved whole blood samples and isolated peripheral blood mononuclear cells (PBMCs). The reliability of both MN assays was thoroughly evaluated for use in radiosensitivity assessment. Materials and methods Blood of healthy donors and radiosensitive patients was collected and the MN assay was performed on fresh whole blood, cryopreserved whole blood and isolated PBMCs. MN yields were scored after irradiation with 220 kV X-rays, with doses ranging from 0.5 - 2 Gy. Results and conclusion Fresh whole blood samples showed the highest MN values, while both cryopreserved PBMCs and cryopreserved whole blood cultures showed similar but slightly lower MN values. Inter-individual and intra-individual variabilities were acceptable for all three sample types. The cryopreservation period of PBMCs or whole blood has no significant impact on MN yields, analyzed up to six months and one year, respectively. In addition, radiosensitive patients could successfully be identified using these alternative sample types. Here, we demonstrate the possibility of using cryopreserved isolated PBMCs or whole blood for the MN assay. Depending on the application, the optimal sample type can be selected. PBMCs are highly advantageous when multiple other assays need to be performed on a single blood sample, while cryopreserved whole blood is favorable when a quick and simple sample storing procedure is required in large-scale multicenter studies. References Sioen S, Cloet K, Vral A, Baeyens A. The Cytokinesis-Block Micronucleus Assay on Human Isolated Fresh and Cryopreserved Peripheral Blood Mononuclear Cells. J Pers Med. 2020 Sep 14;10(3):125. doi: 10.3390/jpm10030125. Beyls E, Baeyens A, Vral A. The cytokinesis-block micronucleus assay for cryopreserved whole blood. Int J Radiat Biol. 2021 Jul 1:1-9. doi: 10.1080/09553002.2021.1941378

Fibroblast-based radiosensitivity assessment for primary immunodeficiency patients

Background & aim Several human syndromes with a genetic defect in one of the DNA double strand break (DSB) recognition and repair proteins have been described. These patients are defined by their immunological defects, cancer susceptibility, neurological abnormalities and sensitivity to ionizing radiation (IR). Malfunction of the patient’s immune system may be the most overt clinical feature and initially present with a primary immunodeficiency disease (PID). As DNA damaging agents are often applied in diagnostic and therapeutic procedures, identification of radiosensitive individuals in this patient population is essential to optimize their clinical care. As an alternative for the well-established lymphocyte-based assays, which are not feasible for certain PID patients, reliable fibroblast-based radiosensitivity (RS) analysis is proposed in this study. Methods Fibroblasts derived from PID patients with a confirmed or suspected DNA repair defect (mutations in ATM, Artemis, XLF, LIGIV, NBS1, RAG1/2) were irradiated with X-rays (0,5 and 1 Gy) in the G0 phase of the cell cycle. RS was assessed with the γ-H2AX foci test and an optimized cytokinesis-block micronucleus (MN) assay. Using fibroblasts from healthy individuals, the cut-off value for RS was determined. Results & conclusion Patients with an Artemis and RAG1/2 mutation could respectively be identified as radiosensitive and not radiosensitive with both the γ-H2AX foci and G0 MN assay. The MN test was not feasible for XLF and LIG4 mutated fibroblasts, but their high γ-H2AX foci levels post-irradiation clearly indicated a radiosensitive phenotype. For these patients, the observed in vitro RS correlated with the expected clinical response to IR. However, ATM defective fibroblasts, known for their extreme RS, show high MN yields following irradiation, while these cells were not considered radiosensitive with the γ-H2AX foci assay. Also for NBS1 mutated cells, residual foci levels did not reach higher than the RS cut-off value. Although limitations in the use of a single RS assay were observed, the combination of the fibroblast MN and γ-H2AX foci test clearly improved the RS assessment for PID patients