Optimization of Formaldehyde Fixative Concentration for Individual Blood Cells to Develop a Stabilized Blood Control for Automated Hematology Analyzers.

In a modern haematology laboratory, the complete and differential counts of blood are performed using complex haematology auto analyzers. In order to ensure the accuracy and reliability of test results, various regulatory authorities have prescribed the use of stabilized blood controls. The major pitfalls of these blood controls are their short shelf life. This could be due to the fact that they are prepared by a common cocktail of fixatives which acts on the discrete cells in various ways and would result in either under-fixation or over-fixation of various cells. Thus, in the present study, we have explored and optimized fixative and buffering for individual cells to achieve stable blood control. Blood cells were isolated using the centrifugation technique and were fixed individually with different concentrations of formaldehyde. After fixation, cells were pooled. Analysis of cell count was done till six months. Cells were also analysed morphologically to see the effect of fixation and storage on cell morphology. In the present study we compared the effect of the concentration of formaldehyde fixative for individual cells in the blood and their role in enhancing the shelf life and maintaining the morphology of the cells when suspended in plasma or suitable buffers post-fixation. It was observed that WBCs can be better fixed with 3 and 3.5% formaldehyde in a buffered solution, whereas RBCs and Platelets can be optimally fixed with 2.5% formaldehyde in a buffered solution.

Development and evaluation of a multicomponent bioink consisting of alginate, gelatin, diethylaminoethyl cellulose and collagen peptide for 3D bioprinting of tissue construct for drug screening application.

Three dimensional (3D) bioprinting technology has been making a progressive advancement in the field of tissue engineering to produce tissue constructs that mimic the shape, framework, and microenvironment of an organ. The technology has not only paved the way to organ development but has been widely studied for its application in drug and cosmetic testing using 3D bioprinted constructs. However, not much has been explored on the utilization of bioprinting technology for the development of tumor models to test anti-cancer drug efficacy. The conventional methodology involves a two dimensional (2D) monolayer model to test cellular drug response which has multiple limitations owing to its inability to mimic the natural tissue environment. The choice of bioink for 3D bioprinting is critical as cell morphology and proliferation depend greatly on the property of bioink. In this study, we developed a multicomponent bioink composed of alginate, diethylaminoethyl cellulose, gelatin, and collagen peptide to generate a 3D bioprinted construct. The bioink has been characterised and validated for its printability, shape fidelity and biocompatibility to be used for generating tumor models. Further, a bioprinted tumor model was developed using lung cancer cell line and the efficacy of 3D printed construct for drug screening application was established.

Biofabrication of skin tissue constructs using alginate, gelatin and diethylaminoethyl cellulose bioink.

INTRODUCTION: Biofabrication of skin tissue equivalents using 3D bioprinting technology has gained much attention in recent times due to the simplicity, the versatility of the technology and its ability in bioengineering biomimetic tissue histology. The key component being the bioink, several groups are actively working on the development of various bioink formulations for optimal skin tissue construction. METHODS: Here, we present alginate (ALG), gelatin (GEL) and diethylaminoethyl cellulose (DCEL) based bioink formulation and its application in bioprinting and biofabrication of skin tissue equivalents. Briefly, DEAE cellulose powder was dispersed in alginate solution with constant stirring at 60 °C to obtain a uniform distribution of cellulose fibers; this was then mixed with GEL solution to prepare the bioink. The formulation was systematically characterized for its morphological, physical, chemical, rheological, biodegradation and biocompatibility properties. The printability, shape fidelity and cell-laden printing were assessed using the CellInk bioprinter. RESULTS: The bioink proved to be a good printable, non-cytotoxic and stable hydrogel formulation. The primary human fibroblast and keratinocyte-loaded 3D bioprinted constructs showed excellent cell viability, collagen synthesis, skin-specific marker and biomimetic tissue histology. CONCLUSION: The results demonstrated the successful formulation of ALG-GEL-DCEL bioink and its application in the development of human skin tissue equivalents with distinct epidermal-dermal histological features.

Evaluation of diagnostic accuracy of developed rapid SARS-COV-2 IgG antibody test kit using novel diluent system.

Immunochromatographic assay kits are used in primary diagnostics which is based on the principle of antigen and antibody interaction. These kits play pivotal role in rapid surveillance of infectious diseases at early stages as well as for the surveillance of the contagious diseases. The immunochromatographic test kits lacks sensitivity and specificity with certain diseases. In this study, our intention was to develop a rapid test kit for SARS-COV-2 with a novel diluent system to enhance the efficacy of antigen-antibody binding and thereby the improvement in the sensitivity outlined. Finally, IgG antibodies against SARS-COV-2 virus peptides were analyzed using 25 positive and 25 negative confirmed clinical samples. The sensitivity of the clinical studies showed 91% sensitivity and 100% specificity. Therefore, the authors propose that this assay will be a potential tool for efficient community or sentinel surveillance of SARS-COV-2 infection and additionally, for effective monitoring of convalescent sera therapy.

DNA damage response and repair data with pharmacological modulators of Tousled.

Human Tousled kinase 1 (TLK1) plays an important role in chromatin remodeling, replication, and DNA damage response and repair. TLK1 activity is immediately, but transiently, downregulated after genotoxic insult, and its recovery is important for exit from checkpoint arrest and cell survival after radiation. The data in this article compliments research presented in the paper titled, "Tousled kinase activator, gallic acid, promotes DNA repair and suppresses radiation cytotoxicity in salivary gland cells" [1]. The identification of small molecule activators and inhibitors of TLK1 provided an opportunity to pharmacologically alter the protein׳s activity to elucidate its role in DNA damage response pathways. TLK1 effectors, gallic acid (GA) and thioridazine (THD) activate and inhibit the kinase, respectively, and the data report on the impact of these compounds and the significance of TLK1 to DNA break repair and the survival of human salivary acinar cells.

Contribution of fibroblasts to the mechanical stability of in vitro engineered dermal-like tissue through extracellular matrix deposition.

Tissue-engineered skin with mechanical and biological properties that match the native tissue could be a valuable graft to treat non-healing chronic wounds. Fibroblasts grown on a suitable biodegradable scaffold are a feasible strategy for the development of a dermal substitute above which epithelialization may occur naturally. Cell growth and phenotype maintenance are crucial to ensure the functional status of engineered tissue. In this study, an electrospun biodegradable polymer scaffold composed of a terpolymer PLGC [poly(lactide-glycolide-caprolactone)] with appropriate mechanical strength was used as a scaffold so that undesirable contraction of the wound could be prevented when it was implanted. To enhance cell growth, synthetic PLGC was incorporated with a fibrin-based biomimetic composite. The efficacy of the hybrid scaffold was evaluated by comparing it with bare PLGC in terms of fibroblast growth potential, extracellular matrix (ECM) deposition, polymer degradation, and mechanical strength. A significant increase was observed in fibroblast attachment, proliferation, and deposition of ECM proteins such as collagen and elastin in the hybrid scaffold. After growing fibroblasts for 20 d and 40 d, immunochemical staining of the decellularized scaffolds showed deposition of insoluble collagen and elastin on the hybrid scaffold but not on the bare scaffold. The loss of mechanical strength consequent to in vitro polymer degradation seemed to be balanced owing to the ECM deposition. Thus, tensile strength and elongation were better when cells were grown on the hybrid scaffold rather than the bare samples immersed in culture medium. Similar patterns of in vivo and in vitro degradation were observed during subcutaneous implantation and fibroblast culture, respectively. We therefore postulate that a hybrid scaffold comprising PLGC and fibrin is a potential candidate for the engineering of dermal tissue to be used in the regeneration of chronic wounds.

Identification of p63+ keratinocyte progenitor cells in circulation and their matrix-directed differentiation to epithelial cells.

INTRODUCTION: In the event of chronic diabetes or burn wounds, accomplishing skin regeneration is a major concern. Autologous skin grafting is the most effective remedy, but the tissue harvest may create more nonhealing wounds. Currently available skin substitutes have a limited clinical outcome because of immune reactions arising from the xenobiotic scaffold or allogenous cells. Autologous stem cells that can be collected without an additional injury may be a viable option for skin-tissue engineering. Presence of a low number of keratinocyte progenitor cells (KPCs) within the peripheral blood mononuclear cell (PBMNC) population has been indicated. Identification, isolation, expansion, and differentiation of KPCs is necessary before they are considered for skin regeneration, which is the focus of this study. METHODS: Culture of isolated human PBMNCs on a cell-specific matrix was carried out to induce differentiation of KPCs. Flow cytometry and reverse transcriptase polymerase chain reaction were done for epithelial stem cell marker p63 and lineage markers cytokeratin 5 and cytokeratin 14, to track differentiation. Proliferation was confirmed by quantifying the proliferating cell nuclear antigen-expressing cells. Immunostaining with epithelial cell markers, involucrin and filaggrin, was carried out to establish terminal differentiation. Microscopic analysis confirmed growth and survival of KPCs on the dermal fibroblast monolayer and on a transplantable fibrin sheet. RESULTS: We demonstrated that KPCs are p63(+) and CD34-. The specifically designed composition of the extracellular matrix was found to support selective adhesion, proliferation, and differentiation of p63(+) KPCs. The PBMNC culture for 12 days under controlled conditions resulted in a homogenous population that expressed cytokeratins, and >90% of the cells were found to proliferate. Subculture for 5 days resulted in expression of filaggrin and involucrin, suggesting terminal differentiation. Transfer of matrix-selected KPCs to a dermal fibroblast monolayer or fibrin supported cell proliferation and showed typical hexagonal morphology of keratinocytes within 15 days. CONCLUSIONS: Circulating KPCs were identified with p63, which differentiated into keratinocytes with expression of the cytokeratins, involucrin and filaggrin. Components of the specifically designed matrix favored KPC attachment, directed differentiation, and may turn out to be a potential vehicle for cell transplantation.